NMR spectroscopy and chemometric models to detect a specific non-porcine ruminant contaminant in pharmaceutical heparin.

Heparin has been used successfully as a clinical antithrombotic for almost one century. Its isolation from animal sources (mostly porcine intestinal mucosa) involves multistep purification processes starting from the slaughterhouse (as mucosa) to the pharmaceutical plant (as the API). This complex supply chain increases the risk of contamination and adulteration, mainly with non-porcine ruminant material. The structural similarity of heparins from different origins, the natural variability of the heparin within samples from each source as well as the structural changes induced by manufacturing processes, require increasingly sophisticated methods capable of detecting low levels of contamination. The application of suitable multivariate classification approaches on API 1H NMRspectra serve as rapid and reliable tools for product authentication and the detection of contaminants. Soft Independent Modeling of Class Analogies (SIMCA), Discriminant Analysis (DA), Partial Least Square Discriminant Analysis (PLS-DA) and local classification methods (kNN, BNN and N3) were tested on about one hundred certified heparin samples produced by 14 different manufacturers revealing that Partial Least Squares Discriminant Analysis (PLS-DA) provided the best discrimination of contaminated batches, with a balanced accuracy of 97%.

Structural analysis of glycosaminoglycans from Oviductus ranae.

Oviductus ranae (O.ran.) has been widely used as a tonic and a traditional animal-based Chinese medicine. O.ran. extracts have been reported to have numerous biological activities, including activities that are often associated with mammalian glycosaminoglycans such as anti-inflammatory, antiosteoperotic, and anti-asthmatic. Glycosaminoglycans are complex linear polysaccharides ubiquitous in mammals that possess a wide range of biological activities. However, their presence and possible structural characteristics within O.ran. were previously unknown. In this study, glycosaminoglycans were isolated from O.ran. and their disaccharide compositions were analyzed by liquid chromatography-ion trap/time-of-flight mass spectrometry (LC-MS-ITTOF). Heparan sulfate (HS)/heparin (HP), chondroitin sulfate (CS)/dermatan sulfate (DS) and hyaluronic acid (HA) were detected in O.ran. with varied disaccharide compositions. HS species contain highly acetylated disaccharides, and have various structures in their constituent chains. CS/DS chains also possess a heterogeneous structure with different sulfation patterns and densities. This novel structural information could help clarify the possible involvement of these polysaccharides in the biological activities of O.ran..

Resonance light scattering technique for sensitive detection of heparin using plasmonic Cu2-xSe nanoparticles.

Heparin (Hep) is a mucopolysaccharide sulfate anticoagulant drug widely used in clinic and thus the quantification of Hep concentration is of great significance. In this work, a simple resonance light scattering (RLS) method for the determination of Hep concentration using plasmonic copper selenide nanoparticles (Cu2-xSe NPs) was developed. The positively-charged Cu2-xSe NPs capped by cetyltrimethyl ammonium bromide (CTAB) were aggregated by negatively-charged Hep, consequently inducing a significant enhancement in the RLS signals of plasmonic Cu2-xSe NPs. Under the optimal reaction conditions, the increased RLS intensity is linearly correlated with the concentrations of Hep in the range of 0.01-0.60 µg mL-1 (R2 = 0.999), with a low detection limit (LOD) of 4.0 ng mL-1 (3σ). This method offered a simple, sensitive and selective strategy for determining Hep, which can be further successfully applied to quantify Hep in the heparin sodium injection.

Analysis of Heparan sulfate/heparin from Colla corii asini by liquid chromatography-electrospray ion trap mass spectrometry.

Colla corii asini (CCA) made from donkey-hide has been widely used as a health-care food and an ingredient of traditional Chinese medicine. Heparan sulfate (HS)/heparin is a structurally complex class of glycosaminoglycans (GAGs) that have been implicated in a wide range of biological activities. However, their presence within CCA, and their possible structural characteristics, were previously unknown. In this study, GAG fractions containing HS/heparin were isolated from CCA and their disaccharide compositions were analyzed by high sensitivity liquid chromatography-ion trap/time-of-flight mass spectrometry (LC-MS-ITTOF). This revealed that, in addition to the eight commonly seen HS disaccharides, the four rare N-unsubstituted disaccharides were also detected in significant quantities. The disaccharide compositions varied significantly between HS/heparin fractions indicating chains with differing domain structures. This novel structural information may lead to a better understanding of the biological activities (i.e. anticoagulation and antitumor action) of CCA.

In Vitro Assay for Studying the Aggregation of Tau Protein and Drug Screening.

Aggregation of tau protein and formation of paired helical filaments is a hallmark of Alzheimer's disease and other tauopathies. Compared to other proteins associated with neurodegenerative diseases, the reported in vitro aggregation kinetics for tau protein are less consistent presenting a relatively high variability. Here we describe the development of an in vitro aggregation assay that mimics the expected steps associated with tau misfolding and aggregation in vivo. The assay uses the longest tau isoform (huTau441) which contains both N-terminal acidic inserts as well as four microtubule binding domains (MBD). The in vitro aggregation is triggered by addition of heparin and followed continuously by thioflavin T fluorescence in a 96 well microplate format. The tau aggregation assay is highly reproducible between different wells, experimental runs and batches of the protein. The aggregation leads to tau PHF-like morphology which is very efficient in seeding the formation of de novo fibrillar structures. In addition to its application in studying the mechanism of tau misfolding and aggregation, the current assay is a robust tool for screening drugs that could interfere with the pathogenesis of tau.

Heparin supplement counteracts the prohemostatic effect of prothrombin complex concentrate and factor IX concentrate: An in vitro evaluation.

INTRODUCTION: Coagulation factor concentrates like factor IX (FIX) and prothrombin complex concentrate (PCC) can contain anticoagulant substances that may hamper their procoagulant effectiveness in the treatment of hemophilia B or reversal of oral anticoagulation, as well as the laboratory assessment thereof. The aim of the present study was to evaluate the influence of anticoagulant heparin supplement on the prohemostatic potential of different PCCs and FIX concentrates. MATERIALS AND METHODS: Prohemostatic potential was evaluated in vitro employing PT/aPTT, thrombography (TGA) and thromboelastography (TEG) with FIX deficient plasma, vitamin K antagonist (VKA)-anticoagulated plasma and plasma anticoagulated with rivaroxaban. RESULTS: Most PCCs contained heparin, while heparin was detected in 1 out of 4 examined FIX concentrates. All heparin-containing clotting factor concentrates showed severely hampered prohemostatic effects when therapeutic doses were added to anticoagulated plasmas. Upon heparin removal, comparable prohemostatic effects were observed. Of importance is the notion that the anticoagulant effect of heparin was enhanced by rivaroxaban, resulting in a 7 fold increased PT sensitivity towards heparin in the presence of 500µg/L rivaroxaban. CONCLUSIONS: Compositional differences between clotting factor concentrates should be taken into account. Therapeutic levels of heparin may be co-infused when treating emergency bleeds with high prohemostatic drug doses, particularly those recommended in the reversal of non-VKA anticoagulants such as rivaroxaban by PCC. Given the relative short half-life of heparin compared to vitamin K-dependent clotting factors, an anticoagulant heparin effect shortly after concentrate infusion should be considered clinically and while interpreting laboratory coagulation parameters.

Luminescence response of an osmium(II) complex to macromolecular polyanions for the detection of heparin and chondroitin sulfate in biomedical preparations.

Heparin, dextran sulfate (DS), chondroitin sulfate (CS), and carrageenan are found to enhance the luminescence intensity of an osmium(II) carbonyl complex with phenanthroline (phen) and 4-phenylpyridine (4-phpy) ligands in aqueous and ethanol solutions. The enhancing effect of the polyanions on the luminescence of the complex is heavily dependent on the sulfate content and other factors such as structure, solubility, and counter ions of the polyanion. The highly sulfated dextran and ι-carrageenan have the most profound effect, while the low charged κ-carrageenan and CS have the least response in aqueous solution. All polyanions exhibited enhanced luminescence intensity of the complex in ethanol solutions, and even the low charged CS and κ-carrageenan enhanced the luminescence more than 4 times. DS contamination of the sodium heparin at 5% can show a significant increase in luminescence response. The osmium complex is found to be highly successful in the fast and sensitive detection of heparin in commercial injectable samples with various backgrounds as well as the detection of CS in over the counter food supplement tablets.

Identification of a simple and sensitive microplate method for the detection of oversulfated chondroitin sulfate in heparin products.

Heparin is a commonly implemented anticoagulant used to treat critically ill patients. Recently, a number of commercial lots of heparin products were found to be contaminated with an oversulfated chondroitin sulfate (OSCS) derivative that could elicit a hypotensive response in pigs following a single high-dose infusion. Using both contaminated heparin products and the synthetically produced derivative, we showed that the OSCS produces dose-dependent hypotension in pigs. The no observed effect level (NOEL) for this contaminant appears to be approximately 1mg/kg, corresponding to a contamination level of approximately 3%. We also demonstrated that OSCS can be identified in heparin products using a simple, inexpensive, commercially available heparin enzyme immunoassay (EIA) kit that has a limit of detection of approximately 0.1%, well below the NOEL. This kit may provide a useful method to test heparin products for contamination with oversulfated GAG derivatives.

Improved impurity fingerprinting of heparin by high resolution (1)H NMR spectroscopy.

For improving the identification of potential heparin impurities such as oversulfated chondroitin sulfate (OSCS) the standard 2D (1)H-(1)H NMR NOESY was applied. Taking advantage of spin diffusion and adjusting the experimental parameters accordingly additional contaminant-specific signals of the corresponding sugar ring protons can easily be detected. These are usually hidden by the more intense heparin signals. Compared to the current 1D (1)H procedure proposed for screening commercial unfractionated heparin samples and focusing on the contaminants acetyl signals more informative and unique fingerprints may be obtained. Correspondingly measured (1)H fingerprints of a few potential impurities are given and their identification in two contaminated commercial heparin samples is demonstrated. The proposed 2D NOESY method is not intended to replace the current 1D method for detecting and quantifying heparin impurities but may be regarded as a valuable supplement for an improved and more reliable identification of these contaminants.

Oversulfated chondroitin sulfate is a contaminant in heparin associated with adverse clinical events.

Recently, certain lots of heparin have been associated with an acute, rapid onset of serious side effects indicative of an allergic-type reaction. To identify potential causes for this sudden rise in side effects, we examined lots of heparin that correlated with adverse events using orthogonal high-resolution analytical techniques. Through detailed structural analysis, the contaminant was found to contain a disaccharide repeat unit of glucuronic acid linked beta1-->3 to a beta-N-acetylgalactosamine. The disaccharide unit has an unusual sulfation pattern and is sulfated at the 2-O and 3-O positions of the glucuronic acid as well as at the 4-O and 6-O positions of the galactosamine. Given the nature of this contaminant, traditional screening tests cannot differentiate between affected and unaffected lots. Our analysis suggests effective screening methods that can be used to determine whether or not heparin lots contain the contaminant reported here.

Colorimetric heparinase assay for alternative anti-metastatic activity.

Heparanase has been previously associated with the metastatic potential, inflammation, and angiogenesis of tumor cells. Heparanase activity has been detected by means of UV absorption, radiolabeled substrates, electrophoretic migration, and heparan sulfate affinity assays. However, those methods have proven to be somewhat problematic with regards to application to actual biological samples, the accessibility of the immobilized substrates, experimental sensitivity, and the separation of degraded products. Rather than focusing on heparanase activity, then, we have developed a rapid, alternative colorimetric heparinase assay, on the basis of the recent finding that sulfated disaccharides generated from heparin by bacterial heparinase exhibit biological properties comparable to those from heparan sulfate by mammalian heparanase. In this study, the concentrations of porcine heparin and bacterial heparinase I were determined using a Sigma Diagnostics Kit. Morus alba was selected as a candidate through this assay system, and an inhibitor, resveratrol, was purified from its methanol extract. Its anti-metastatic effects on the pulmonary metastasis of murine B16 melanoma cells were also evaluated. Our findings suggest that this assay may prove useful as a diagnostic tool for heparinase inhibition, as an alternative anti-metastatic target.

Stability and in vitro efficacy of antibiotic-heparin lock solutions potentially useful for treatment of central venous catheter-related sepsis.

OBJECTIVES: Increasing numbers of patients for whom infection is a major risk are dependent on central venous catheters. Antibiotic-anticoagulant locks may have a role in preventing or treating catheter-related infections. The aim of this study was to determine the in vitro stability and efficacy of antibiotic-heparin lock solutions. METHODS: Candidate antibiotics (amikacin, ciprofloxacin, flucloxacillin, gentamicin, linezolid, teicoplanin) were investigated in vitro, either individually or in combination, in solution with heparin. The solutions were initially tested for visual precipitation. The efficacy of stable solutions and taurolidine was then tested in a catheter model bioassay system against microorganisms commonly encountered in catheter-related septicaemia. RESULTS: In general, lower concentrations of heparin (

Induction of dog sperm capacitation by glycosaminoglycans and glycosaminoglycan amounts of oviductal and uterine fluids in bitches.

Ejaculated sperm collected from 12 beagle dogs were incubated in canine capacitation medium (CCM), supplemented with 5 microg/ml chondroitin sulfate A (CS), 5 microg/ml hyaluronic acid (HA), or 5 microg/ml heparin (HP) for 7 hr at 38 degrees C in a 5% CO2 in air atmosphere to investigate the effects of glycosaminoglycans (GAGs) on dog sperm capacitation. The percentages of motile sperm, hyperactivated sperm (%HY), and acrosome-reacted sperm (%AR) in all media were examined after 4 hr and 7 hr of incubation. The oviducts and uteri of 9 anestrous and 18 estrous beagle bitches were removed under halothane inhalation anesthesia to measure the total GAG amounts in oviductal and uterine fluids. The lumens of the ampulla of the oviducts, isthmus of the oviducts, and the uterine horns were each flushed with 1 ml HEPES-EDTA fluid. Total GAG amounts in the flush fluids obtained were measured with a spectrophotometer. Sperm motility (51-59%), %HY (79-86%), and %AR (31-36%) in CCM supplemented with CS, HA, or HP were significantly higher after 7 hr of incubation than when incubated in CCM without GAGs (P<0.01 or 0.05). The mean total GAG amounts in the fluids from the ampulla and isthmus of the oviducts and the uterine horns in the estrous bitches were higher than in the anestrous bitches. These results indicate that GAGs in the oviductal and uterine fluids in estrous bitches are associated with in vivo sperm capacitation.

Quality assessment of intraoperative blood salvage and autotransfusion.

Intraoperative blood salvage and autotransfusion are commonly used to minimize exposure to banked blood. Although this technique has been used widely for years, data vary regarding the quality of autotransfused blood. Salvaged blood may contain plasma, residual heparin, and free hemoglobin released from damaged cells. All of these factors may contribute to the adverse sequelae sometimes seen with autotransfusion. For these reasons, we have monitored autotransfused blood to assess its quality. Intraoperative blood salvage was used during most cardiac procedures and at the discretion of the surgeon in other specialties. Blood was collected through a double lumen catheter that was anticoagulated with heparin, filtered, centrifuged, and washed with saline. A sample of the blood was removed for analysis, which included hematocrit, heparin assay, fibrinogen, and free hemoglobin levels. Over a 6-year period, 1593 patients had intraoperative blood salvage with quality assessment. The majority of patients underwent cardiac operations (941 patients, 59%), whereas 243 had orthopedic (15%) and 208 had vascular (13%) procedures. Additionally, there were 127 pediatric patients (8%) and 74 miscellaneous procedures (5%). The highest average yield of salvaged blood was during vascular procedures (1073 +/- 76 mL), whereas orthopedic cases had the lowest yield (378 +/- 19 mL) and hematocrit (39%). There was minimal residual heparin activity, even in patients requiring systemic anticoagulation (0.3 to 0.5 units/mL). Patients undergoing pediatric procedures had the lowest concentration of free hemoglobin (476 mg/L), whereas all adult patients had higher free hemoglobin levels, especially vascular operations (990 mg/L). Intraoperative salvaged blood has minimal heparin activity, even in procedures requiring systemic anticoagulation. Fibrinogen, a marker of residual plasma, was undetectable in the majority of cases. These data indicate that intraoperative blood salvage generally results in a high-quality product (good hematocrit, low heparin, minimal plasma), although there are significant differences in free hemoglobin levels depending on the operative procedure.

[The influence of semen preparation and culture medium on the success of IVF in cattle]. / Der Einfluss von Samenaufbereitung und Kulturmedium auf den IVF-Erfolg beim Rind.

The effect of two semen-preparation methods and the presence and absence of capacitation-inducing supplements in the fertilization medium on IVF-results were examined in a 2 x 2 factorial design. In vitro matured oocytes were fertilized either with transmigrated (TM) or with swim-up (SU) prepared native semen. In the first group, the fertilization medium contained 0.8 microgram/ml heparin, 1.2 micrograms/ml hypotaurine and 0.2 microgram/ml epinephrine. However, in the second group, no capacitation-inducing or motility-enhancing substances were used. After SU-treatment, the supplements were necessary to obtain sufficient fertilization results. In the second group (non-supplemented medium), the percentages of penetrated and fertilized oocytes were decreased significantly (P < 0.001). By contrast, transmigrated semen required no additional supplementation of the fertilization medium. The penetration and fertilization rates of this semen-preparation method were equal in both groups.

Heparin washout in the pediatric Cell Saver bowl.

The possibility of residual heparin in washed red cells transfused to neonatal or pediatric cardiac patients following bypass prompted a measurement of heparin concentrations. Samples were taken during 10 adult and 10 neonatal and pediatric bypass cases. Sample A was from the bypass circuit, Sample B from the Haemonetics Cell Saver bowl inlet before washing, Sample C from the Cell Saver bowl outlet after washing, and Sample D from the patient ten minutes after protamine. Heparin concentrations were measured by a chromogenic assay using activated Factor X. There was no significant difference between the adult and pediatric groups in the levels of heparin concentration on bypass, pre-washing and post-washing, and in the patients following protamine. In the pediatric group, only .002% of the pre-washed heparin remained after washing. This extremely low level of heparin (.0027 units/ml) is only 0.34 units in a 125 ml pediatric unit of Cell Saver blood. Based on post bypass patient samples, this has no clinical significance. Therefore, the Cell Saver can be used safely with neonates and pediatric patients without concern regarding residual heparin when properly processed.

[Heparin from the meadowsweet (Filipendula ulmaria) and its properties]. / Geparin iz tavolgi viazolistnoi, ego svoistva.

In the present work, the nature of an anticoagulant from Philipendula ulmaria was studied. A method for purification of this anticoagulant was developed. Using diverse methods it was shown that the molecular weight, data on element (sulphur, nitrogen, and hydrogen) content, spectral characteristics in the infrared region of the spectrum, and electrophoretic properties of the product indicate its similarity to heparin of animal origin.

[The content of a heparin-like anticoagulant in the flowers of the meadowsweet (Filipendula ulmaria)]. / Soderzhanie geparinopodobnogo antikoagulianta v tsvetkakh tavolgi viazolistnoi.

The flowers of Filipendula ulmaria were found to contain heparin bound to the plant proteins in the form of a complex. This complex enhances the anticoagulant and fibrinolytic properties of the nonenzymatic nature at its administration to animals both intramuscularly and intravenously. The neutralizing effect of protaminesulphate on the anticoagulant activity of the plant heparin was shown. The identity of the action on the hemostasis system of heparin of animal and plant origin was found.

Cell receptors to sulphated polysaccharides in the acute and chronically inflamed synovial joint.

Receptors to sulphated polysaccharides have recently been discovered on "free" joint fluid cells and synovial membrane cells in the normal joint. A search for these receptors on cells was made in rabbits with acute and chronic adjuvant inflammatory arthritis in an attempt to further elucidate their role in joint homeostasis. These experiments demonstrated a significant increase in cell numbers within the joint. Receptor activity was most marked on macrophages found free within the synovial fluid. It is postulated that exogenous cells may be important in the process of joint destruction and are outside the control of the normal joint regulatory mechanisms. The endogenous cell population, which exhibits receptor activity, may be responding to the process of joint destruction by proliferation as a secondary phenomenon.