Strong Anion-exchange High-performance Liquid Research Articles

Knockout of the intellectual disability-linked gene Hs6st2 in mice decreases heparan sulfate 6-O-sulfation, impairs dendritic spines of hippocampal neurons, and affects memory.

Heparan sulfate (HS) is a linear polysaccharide that plays a key role in cellular signaling networks. HS functions are regulated by its 6-O-sulfation, which is catalyzed by three HS 6-O-sulfotransferases (HS6STs). Notably, HS6ST2 is mainly expressed in the brain and HS6ST2 mutations are linked to brain disorders, but the underlying mechanisms remain poorly understood. To determine the role of Hs6st2 in the brain, we carried out a series of molecular and behavioral assessments on Hs6st2 knockout mice. We first carried out strong anion exchange-high performance liquid chromatography and found that knockout of Hs6st2 moderately decreases HS 6-O-sulfation levels in the brain. We then assessed body weights and found that Hs6st2 knockout mice exhibit increased body weight, which is associated with abnormal metabolic pathways. We also performed behavioral tests and found that Hs6st2 knockout mice showed memory deficits, which recapitulate patient clinical symptoms. To determine the molecular mechanisms underlying the memory deficits, we used RNA sequencing to examine transcriptomes in two memory-related brain regions, the hippocampus and cerebral cortex. We found that knockout of Hs6st2 impairs transcriptome in the hippocampus, but only mildly in the cerebral cortex. Furthermore, the transcriptome changes in the hippocampus are enriched in dendrite and synapse pathways. We also found that knockout of Hs6st2 decreases HS levels and impairs dendritic spines in hippocampal CA1 pyramidal neurons. Taken together, our study provides novel molecular and behavioral insights into the role of Hs6st2 in the brain, which facilitates a better understanding of HS6ST2 and HS-linked brain disorders.

A rapid method for extraction, purification and structure analysis of chondroitin sulfate from six marine tissues

Abstract This study was designed to establish a strategy for the extraction, purification, and structure analysis of chondroitin sulfate (CS) in milligram amounts. Crude acidic polysaccharides were extracted from six kinds of marine animals by enzymatic hydrolysis and hexadecylpyridinium chloride precipitation and purified by Q Sepharose Fast Flow strong anion exchange column. The purification of each crude polysaccharide was completed within 1 h. The structure of the polysaccharides, i.e. their chemical characterization, functional group, molecular weight and monosaccharide composition, were analyzed by colorimetry, nuclear magnetic resonace and high-performance liquid chromatogrpahy, respectively. All polysaccharides were identified as CS. The oligosaccharide profile produced by enzyme hydrolysis of polysaccharides was determined by strong anion-exchange high-performance liquid chromatorgraphy. This method can be widely applied to the extraction and characterization of chondroitin sulfate from unknown raw materials, screening high-quality sources of functional polysaccharides, and laying a good foundation for the following study of the structure–function relationship of polysaccharides.

Combination of strong anion exchange liquid chromatography with microchip capillary electrophoresis sodium dodecyl sulfate for rapid two-dimensional separations of complex protein mixtures

Two-dimensional separations provide a simple way to increase the resolution and peak capacity of complex protein separations. The feasibility of a recently developed instrumental approach for two-dimensional separations of proteins was evaluated. The approach is based on the general principle of two-dimensional gel electrophoresis. In the first dimension, semi-preparative strong anion exchange high-performance liquid chromatography is utilized and fractions are collected by means of a fraction collector. They are subsequently analyzed in the second dimension with microchip capillary electrophoresis sodium dodecyl sulfate. Microchip capillary electrophoresis provides the necessary speed (approximately 1 min/fraction) for short analysis. In this study, three different samples were investigated. Different constructs of soluble guanylyl cyclase were expressed in Sf9-cells using the baculovirus expression system. Cell lysates were analyzed and the resulting separations were compared. In our experimental setup, the soluble guanylyl cyclase was identified among hundreds of other proteins in these cell lysates, indicating its potential for screening, process control, or analysis. The results were validated by immunoblotting. Samples from Chinese hamster ovary cell culture before and after a purification step were investigated and approximately 9% less impurities could be observed. The separation patterns obtained for human plasma are closely similar to patterns obtained with two-dimensional gel electrophoresis and a total of 218 peaks could be observed. Overall, the approach was well applicable to all samples and, based on these results, further directions for improvements were identified.Graphical abstract.

Extraction and Quantification of Soluble, Radiolabeled Inositol Polyphosphates from Different Plant Species using SAX-HPLC.

The phosphate esters of myo-inositol, also termed inositol phosphates (InsPs), are a class of cellular regulators playing important roles in plant physiology. Due to their negative charge, low abundance and susceptibility to hydrolytic activities, the detection and quantification of these molecules is challenging. This is particularly the case for highly phosphorylated forms containing 'high-energy' diphospho bonds, also termed inositol pyrophosphates (PP-InsPs). Due to its high sensitivity, strong anion exchange high-performance liquid chromatography (SAX-HPLC) of plants labeled with [3H]-myo-inositol is currently the method of choice to analyze these molecules. By using [3H]-myo-inositol to radiolabel plant seedlings, various InsP species including several non-enantiomeric isomers can be detected and discriminated with high sensitivity. Here, the setup of a suitable SAX-HPLC system is described, as well as the complete workflow from plant cultivation, radiolabeling and InsP extraction to the SAX-HPLC run and subsequent data analysis. The protocol presented here allows the discrimination and quantification of various InsP species, including several non-enantiomeric isomers and of the PP-InsPs, InsP7 and InsP8, and can be easily adapted to other plant species. As examples, SAX-HPLC analyses of Arabidopsis thaliana and Lotus japonicus seedlings are performed and complete InsP profiles are presented and discussed. The method described here represents a promising tool to better understand the biological roles of InsPs in plants.

Extraction and Quantification of Soluble, Radiolabeled Inositol Polyphosphates from Different Plant Species using SAX-HPLC

The phosphate esters of myo-inositol, also termed inositol phosphates (InsPs), are a class of cellular regulators playing important roles in plant physiology. Due to their negative charge, low abundance and susceptibility to hydrolytic activities, the detection and quantification of these molecules is challenging. This is particularly the case for highly phosphorylated forms containing ‘high-energy’ diphospho bonds, also termed inositol pyrophosphates (PP-InsPs). Due to its high sensitivity, strong anion exchange high-performance liquid chromatography (SAX-HPLC) of plants labeled with [3H]-myo-inositol is currently the method of choice to analyze these molecules. By using [3H]-myo-inositol to radiolabel plant seedlings, various InsP species including several non-enantiomeric isomers can be detected and discriminated with high sensitivity. Here, the setup of a suitable SAX-HPLC system is described, as well as the complete workflow from plant cultivation, radiolabeling and InsP extraction to the SAX-HPLC run and subsequent data analysis. The protocol presented here allows the discrimination and quantification of various InsP species, including several non-enantiomeric isomers and of the PP-InsPs, InsP7 and InsP8, and can be easily adapted to other plant species. As examples, SAX-HPLC analyses of Arabidopsis thaliana and Lotus japonicus seedlings are performed and complete InsP profiles are presented and discussed. The method described here represents a promising tool to better understand the biological roles of InsPs in plants.

Purification, characterisation and antioxidant activities of chondroitin sulphate extracted from Raja porosa cartilage

This study was designed to isolate (isolation yield of 36.51 %) and characterise chondroitin sulphate (CS) from skate (Raja porosa) cartilage. Gel permeation chromatography demonstrated that the Raja porosa chondroitin sulphate (RPCS) obtained was a relatively uniform polysaccharide with a molecular weight of 40,752 Da and a purity of 94.0 %. Fourier-transform infrared, nuclear magnetic resonance spectroscopy and strong anion-exchange high-performance liquid chromatography indicated that the content of the predominant GlcA-GalNAc6S unit in RPCS was 65.84 %, which was higher than that in shark CS (53.93 %). Furthermore, RPCS displayed more effective free radical scavenging effects than did shark CS, indicating the potential of RPCS to promote oxidative stress resistance and to act as an antioxidant agent. Skate cartilage could be exploited as a sufficient and sustainable source for the preparation of CS with higher 6S-sulphation, which in turn could be scaled up for use in the pharmaceutical industry.

The threshold effect between the soil bioavailable molar Se:Cd ratio and the accumulation of Cd in corn (Zea mays L.) from natural Se-Cd rich soils

There is little available information about the important interactions between selenium and cadmium (Se-Cd) in crops grown on natural Se-Cd rich soils. We investigated their interactive effects on the translocation and uptake of Se and Cd from soils to crops. Corn (Zea mays L.) roots, stems, leaves, and grains, and their corresponding rhizosphere soils were collected from naturally Se-Cd rich areas in Wumeng Mountain, Guizhou, China. The Se and Cd levels were determined in the soils, roots, stems, leaves, and grains. Soil bioavailable Se and Cd were also determined. The low soil bioavailable molar ratios for Se and Cd (Se:Cd) (≤0.7) improved Cd accumulation in the plants. However, relatively high Se:Cd molar ratios (>0.7) in the soils prevented Cd from entering the plants, but the effect of the soil Se:Cd on Se accumulation in corn was not significant. The strong anion exchange-high performance liquid chromatography-inductively coupled plasma mass spectroscopy (SAX-HPLC-ICP-MS) chromatograms showed that Se-Cd complexes occurred in the leaves, which likely indicated that direct interactions between Se and Cd happened there. The results suggested that thresholds for soil bioavailable Se:Cd molar ratios played a role in the interaction between Se and Cd in corn under natural conditions.

Structural characterization and in vitro antioxidant activities of chondroitin sulfate purified from Andrias davidianus cartilage

Origin and manufacturing process are key factors affecting the biological activities of chondroitin sulfate (CS), which can be utilized as a nutraceutical in dietary supplements. Herein, we extracted and purified CS from the cartilage of artificially breeding Andrias davidianus (ADCS), i.e., Chinese giant salamander (CGS), one of the prospective functional food source materials in China. Low molecular weight CS (LMWADCS) was then prepared by free radical depolymerization of ADCS. High-performance gel permeation chromatography (HPGPC) analysis showed that the average molecular weight (Mw) of ADCS was 49.2 kDa, while the Mw of LMWADCS was 6.4 kDa. After the eliminative degradation of ADCS by chondroitinase ABC, strong anion-exchange high-performance liquid chromatography (SAX-HPLC) analysis showed that the disaccharide composition of ADCS was 14.6% ΔDi0S, 60.9% ΔDi6S and 24.5% ΔDi4S. Then, in vitro antioxidant assays were performed with ADCS, LMWADCS and CS from a commercial source. Our results showed that LMWADCS exerted the highest total antioxidant activity out of the total antioxidant capacity, including the capacity of scavenging DPPH radicals, hydroxyl radicals and superoxide anion radicals. From the results of this study, we can conclude that the Mw and composition of ADCS are different from those reported for bovine and shark CS, and LMWADCS can be utilized as a valuable and potential nutraceutical for the functional food industry.

Heparan Sulfate Identification and Characterisation: Method II. Enzymatic Depolymerisation and SAX-HPLC Analysis to Determine Disaccharide Composition.

Heparan sulfate (HS) is purified from complex matrices and often not fully characterised to validate its assignment. The characterisation of heparins and heparan sulfates through enzymatic depolymerisation and subsequent strong anion-exchange high performance liquid chromatography (SAX-HPLC) analysis and quantitation of the resulting disaccharides is a critical tool for assessing the structural composition of this class of compound. This protocol details a methodology to reproducibly determine the disaccharide composition of heparan sulfate by enzymatic depolymerisation and SAX-HPLC analysis. A complementary method for identification and characterisation of heparan sulfate can be found at Carnachan and Hinkley (2017).

Establishment of chondroitin B lyase-based analytical methods for sensitive and quantitative detection of dermatan sulfate in heparin

Dermatan sulfate (DS) is one of the hardest impurities to remove from heparin products due to their high structural similarity. The development of a sensitive and feasible method for quantitative detection of DS in heparin is essential to ensure the clinical safety of heparin pharmaceuticals. In the current study, based on the substrate specificity of chondroitin B lyase, ultraviolet spectrophotometric and strong anion-exchange high-performance liquid chromatographic methods were established for detection of DS in heparin. The former method facilitated analysis in heparin with DS concentrations greater than 0.1mgmL−1 at 232nm, with good linearity, precision and recovery. The latter method allowed sensitive and accurate detection of DS at concentrations lower than 0.1mgmL−1, exhibiting good linearity, precision and recovery. The linear range of DS detection using the latter method was between 0.01 and 0.5mgmL−1.

Heparinase Digestion-Based Disaccharide Composition Analysis of Heparin and Heparinoid Drugs

Abstract Heparin and low molecular weight heparin have been used widely in clinical therapy as anticoagulants in cardiovascular disease and in hemodialysis. Crude heparin is usually prepared from porcine intestinal mucosa. Purified heparin is a mixture of polysaccharides consisting mainly of repeating GlcNS(6S)-IdoA2S disaccharides and other disaccharides with different GlcNAc/GlcNS±3S±6S-GlcA/IdoA±2S residues. Heparin injections are prepared by purification and then sterilization of ungraded heparin. Low molecular weight heparins are dominant heparin-based drugs used clinically, which are prepared by degrading heparin into smaller molecules. As a result, low molecular weight heparins share the same major disaccharides with heparin, but may have different reducing and non-reducing ends due to different preparation methods. In the current study, we analyzed the disaccharide composition of clinically used heparin and heparin-based drugs. Heparinase I, II and III were used to degrade all heparin and heparin-based drugs including heparin sodium injection, Enoxaparin sodium injection, Nadroparin calcium injection, Dalteparin sodium injection, and Fondaparinux sodium injection into disaccharides. All the degraded products were analyzed by strong anion exchange-high performance liquid chromatography (SAX-HPLC) equipped with an online UV-detector. Commercially available unsaturated disaccharide standards were then used for structural identification. Furthermore, unusual disaccharides present in Nadroparin, Dalteparin and Fondaparinux were confirmed by reversed-phase ion-pair HPLC coupled with mass spectrometry analysis. The developed method produced detailed structural information, which should be useful for quality control of heparin and heparin-based drugs.

Determination of deoxyribonucleoside triphosphate concentrations in yeast cells by strong anion-exchange high-performance liquid chromatography coupled with ultraviolet detection.

DNA polymerase assays are commonly used for the detection of deoxyribonucleoside triphosphates (dNTPs) in biological samples. For better specificity and accuracy, high-performance liquid chromatography (HPLC) methods have been developed for the analysis of the four dNTPs in complex samples. Here we describe a simple method using isocratic strong anion-exchange (SAX) chromatographic separation coupled with ultraviolet detection (UV) for the analysis of the four dNTPs in budding yeast Saccharomyces cerevisiae. This method can be applied to other species of yeast or bacteria.

Identification of heparin samples that contain impurities or contaminants by chemometric pattern recognition analysis of proton NMR spectral data

Chemometric analysis of a set of one-dimensional (1D) (1)H nuclear magnetic resonance (NMR) spectral data for heparin sodium active pharmaceutical ingredient (API) samples was employed to distinguish USP-grade heparin samples from those containing oversulfated chondroitin sulfate (OSCS) contaminant and/or unacceptable levels of dermatan sulfate (DS) impurity. Three chemometric pattern recognition approaches were implemented: classification and regression tree (CART), artificial neural network (ANN), and support vector machine (SVM). Heparin sodium samples from various manufacturers were analyzed in 2008 and 2009 by 1D (1)H NMR, strong anion-exchange high-performance liquid chromatography, and percent galactosamine in total hexosamine tests. Based on these data, the samples were divided into three groups: Heparin, DS ≤ 1.0% and OSCS = 0%; DS, DS > 1.0% and OSCS = 0%; and OSCS, OSCS > 0% with any content of DS. Three data sets corresponding to different chemical shift regions (1.95-2.20, 3.10-5.70, and 1.95-5.70 ppm) were evaluated. While all three chemometric approaches were able to effectively model the data in the 1.95-2.20 ppm region, SVM was found to substantially outperform CART and ANN for data in the 3.10-5.70 ppm region in terms of classification success rate. A 100% prediction rate was frequently achieved for discrimination between heparin and OSCS samples. The majority of classification errors between heparin and DS involved cases where the DS content was close to the 1.0% DS borderline between the two classes. When these borderline samples were removed, nearly perfect classification results were attained. Satisfactory results were achieved when the resulting models were challenged by test samples containing blends of heparin APIs spiked with non-, partially, or fully oversulfated chondroitin sulfate A, heparan sulfate, or DS at the 1.0%, 5.0%, and 10.0% (w/w) levels. This study demonstrated that the combination of 1D (1)H NMR spectroscopy with multivariate chemometric methods is a nonsubjective, statistics-based approach for heparin quality control and purity assessment that, once standardized, minimizes the need for expert analysts.

Highly Sensitive Potentiometric Strip Test for Detecting High Charge Density Impurities in Heparin

Contamination of heparin with oversulfated chondroitin sulfate (OSCS) became a matter of grave concern in the medical field after many fatal responses to OSCS tainted heparin products occurred during the 2007-2008 period. Even though standard lab-based analytical techniques such as nuclear magnetic resonance (NMR) and strong anion-exchange high performance liquid chromatography (SAX-HPLC) have proven useful for monitoring the OSCS content in heparin products, an easy-to-use, quick, portable, and cost-efficient method is still needed for on-site monitoring during and after the heparin production. In this report, a disposable strip-type electrochemical polyion sensor is described for detection of low levels of OSCS contamination in heparin. A magnetic actuator is incorporated into this simple electrode-based microfluidic device in order to create the mixing effect necessary to achieve equilibrium potential changes of the sensor within a microfluidic channel. The planar membrane electrode detector within the sample channel is prepared with a tridodecylmethylammonium chloride (TDMAC)-doped poly(vinyl chloride) (PVC) membrane essentially equivalent to previously reported polyanion-sensitive electrodes. When the concentration of heparin applied to the single-use strip device is 57 mg/mL (in only 20 μL of sample), the same concentration recommended in the NMR analysis protocol for detecting OSCS in heparin, the detection limit is 0.005 wt % of OSCS, which is ca. 20 times lower than the reported detection limit of the NMR method.

Heparinase 1 selectivity for the 3,6-di-O-sulfo-2-deoxy-2-sulfamido-α-D-glucopyranose (1,4) 2-O-sulfo-α-L-idopyranosyluronic acid (GlcNS3S6S-IdoA2S) linkages

Porcine intestinal mucosa heparin was partially depolymerized by recombinant heparinase 1 (heparin lyase 1, originating from Flavobacterium heparinum and expressed in Escherichia coli) and then fractionated, leading to the isolation of 22 homogeneous oligosaccharides with sizes ranging from disaccharide to hexadecasaccharide. The purity of these oligosaccharides was determined by gel electrophoresis, strong anion exchange and reversed-phase ion-pairing high-performance liquid chromatography. The molecular mass of oligosaccharides was determined using electrospray ionization-mass spectrometry and their structures were elucidated using one- and two-dimensional nuclear magnetic resonance spectroscopy at 600MHz. Five of the characterized oligosaccharides represent new compounds. The most prominent oligosaccharide comprises the common repeating unit of heparin, ΔUA2S-[-GlcNS6S-IdoA2S-](n)-GlcNS6S, where ΔUA is 4-deoxy-α-l-threo-hex-4-eno-pyranosyluronic acid, GlcN is 2-deoxy-2-amino-d-glucopyranose, IdoA is l-idopyranosyluronic acid, S is sulfate and n=0-7. A second prominent heparin oligosaccharide motif corresponds to ΔUA2S-[GlcNS6S-IdoA2S](n)-GlcNS6S-IdoA-GlcNAc6S-GlcA-GlcNS3S6S (where n=0-5 and GlcA is d-glucopyranosyluronic acid), a fragment of the antithrombin III binding site in heparin. The prominence of this second set of oligosaccharides and the absence of intact antithrombin III binding sites suggest that the -GlcNS3S6S-IdoA2S- linkage is particularly susceptible to heparinase 1.

Evidence That Heparin Saccharides Promote FGF2 Mitogenesis through Two Distinct Mechanisms

Heparin-like saccharides play an essential role in binding to both fibroblast growth factors (FGF) and their receptors at the cell surface. In this study we prepared a series of heparin oligosaccharides according to their size and sulfation level. We then investigated their affinity for FGF2 and their ability to support FGF2 mitogenesis of heparan sulfate-deficient cells expressing FGFR1c. Tetra- and hexasaccharides bound FGF2, but failed to dimerize the growth factor. Nevertheless, these saccharides promoted FGF2-mediated cell growth. Furthermore, whereas enzymatic removal of the non-reducing end 2-O-sulfate group had little effect on the 1:1 interaction with FGF2, it eliminated the mitogenic activity of these saccharides. This evidence supports the symmetric two-end model of ternary complex formation. In contrast, even at very low concentrations, octasaccharide and larger heparin fragments conferred a potent mitogenic activity that was independent of terminal 2-O-sulfation. This correlated with the ability to dimerize FGF2 in an apparently cooperative manner. This data suggests that potent mitogenic signaling results from heparin-mediated trans-dimerization of FGF2, consistent with the asymmetric model of ternary complex formation. We propose that, depending on saccharide structure, there are different architectures and modes of ternary complex assembly that differ in stability and/or efficiency of transmembrane signaling.

Quantitative and Qualitative Evaluation of Chondroitin Sulfate in Dietary Supplements

We propose to evaluate the amount and quality of chondroitin sulfate (CS) from several food supplement preparations in the form of tablets, caplets, or capsules containing CS in varying contents and formulations in the presence of various additives and ingredients, with no other pretreatment than centrifugation to remove insoluble material. To quantify CS, two different analytical approaches were applied after their validation: specific and sensitive agarose-gel electrophoresis and strong-anion exchange–high-performance liquid chromatography (SAX-HPLC) determination of the constituent disaccharides after treatment with specific chondroitin lyases. The CS content in finished products evaluated using the two specific validated methods were found to conform to the label specifications. It is worth mentioning that the quantitative determinations have been performed using a very high pure European Pharmacopeia CS reference standard having substantially the same properties as the food supplement CS samples. Furthermore, by means of the specific agarose-gel elctrophoresis approach, we can exclude the presence in the nutraceuticals of other sulfated polysaccharides produced by extraction from tissues, in particular heparin, heparan sulfate, and dermatan sulfate. The SAX-HPLC separation of unsaturated disaccharides for the nutraceutical CS was also used to evaluate its quality and the possible origin. No disulfated disaccharides typical of CS from cartilaginous fishes, making the charge density lower than 1.0, were found, thus confirming the bovine or porcine origin, the most common sources of this “terrestrial” polysaccharide. Finally, high-performance size-exclusion chromatography (HPSEC) analysis was applied to evaluate the CS molecular mass in food supplements also in the presence of additives and other ingredients. This analytical approach confirmed that the nutraceutical CS samples are of high molecular mass and are not degraded during the food supplement preparations. In conclusion, this multianalytical approach can be used in the direct quantitation and evaluation of the quality and the possible origin of CS contained in food supplement formulations.

Acceptor Specificity of the Pasteurella Hyaluronan and Chondroitin Synthases and Production of Chimeric Glycosaminoglycans

The hyaluronan (HA) synthase, PmHAS, and the chondroitin synthase, PmCS, from the Gram-negative bacterium Pasteurella multocida polymerize the glycosaminoglycan (GAG) sugar chains HA or chondroitin, respectively. The recombinant Escherichia coli-derived enzymes were shown previously to elongate exogenously supplied oligosaccharides of their cognate GAG (e.g. HA elongated by PmHAS). Here we show that oligosaccharides and polysaccharides of certain noncognate GAGs (including sulfated and iduronic acid-containing forms) are elongated by PmHAS (e.g. chondroitin elongated by PmHAS) or PmCS. Various acceptors were tested in assays where the synthase extended the molecule with either a single monosaccharide or a long chain (approximately 10(2-4) sugars). Certain GAGs were very poor acceptors in comparison to the cognate molecules, but elongated products were detected nonetheless. Overall, these findings suggest that for the interaction between the acceptor and the enzyme (a) the orientation of the hydroxyl at the C-4 position of the hexosamine is not critical, (b) the conformation of C-5 of the hexuronic acid (glucuronic versus iduronic) is not crucial, and (c) additional negative sulfate groups are well tolerated in certain cases, such as on C-6 of the hexosamine, but others, including C-4 sulfates, were not or were poorly tolerated. In vivo, the bacterial enzymes only process unsulfated polymers; thus it is not expected that the PmCS and PmHAS catalysts would exhibit such relative relaxed sugar specificity by acting on a variety of animal-derived sulfated or epimerized GAGs. However, this feature allows the chemoenzymatic synthesis of a variety of chimeric GAG polymers, including mimics of proteoglycan complexes.

Heparin Octasaccharides Inhibit Angiogenesis In vivo

In previous experiments, we showed that heparin oligosaccharides inhibit the angiogenic cytokine fibroblast growth factor-2. Here, we present the first in vivo study of size-fractionated heparin oligosaccharides in four models of angiogenesis that are progressively less dependent on fibroblast growth factor-2. Heparin oligosaccharides were prepared using size-exclusion gel filtration chromatography and characterized through depolymerization and strong anion exchange high-performance liquid chromatography. Size-defined oligosaccharides (20 mg/kg/d) were given to mice bearing s.c. sponges that were injected with fibroblast growth factor-2 (100 ng/d). After 14 days, octasaccharides and decasaccharides reduced the microvessel density to levels below control. In a second experiment, HEC-FGF2 human endometrial cancer cells that overexpress fibroblast growth factor-2 were implanted in a hollow fiber placed s.c. in vivo. Oligosaccharides were given at 20 mg/kg/d for 2 weeks and the data again showed that octasaccharides significantly reduced microvessel density around the fiber (P = 0.03). In a more complex model, where angiogenesis was induced by a broad spectrum of growth factors, including vascular endothelial growth factor, we implanted H460 lung carcinoma cells in hollow fibers and treated the animals with oligosaccharides at 20 mg/kg/d over 3 weeks. Octasaccharides reduced the microvessel density to that of control. Preliminary investigation of 6-O-desulfated heparins showed that these also had antiangiogenic activity. Finally, we examined the inhibitory potential of hexasaccharides and octasaccharides given at 20 mg/kg/d and these inhibited the growth of H460 lung carcinoma in vivo. At clinically attainable concentrations, significant anticoagulation (activated partial thromboplastin time, anti-factor Xa, and anti-factor IIa) was not observed in vitro unless species containing > or =16 saccharide residues were investigated. Thus, our preclinical data show that heparin octasaccharides represent novel antiangiogenic compounds that can be given without the anticoagulant effects of low molecular weight heparin.

Quantitative analysis of chondroitin sulfate in raw materials, ophthalmic solutions, soft capsules and liquid preparations

We performed the quantitative analysis of chondroitin sulfate (CS) obtained from raw materials and various pharmaceutical preparations. To quantify CS content in raw materials and in an ophthalmic solution, each test sample and the authentic CS were first digested by chondroitinase ABC. The CS disaccharides produced were analyzed by strong anion-exchange high-performance liquid chromatography (SAX-HPLC) and CS content was quantified by calculating the total peak areas of the disaccharides derived from a CS calibration curve. In the case of soft capsules, CS was first extracted with hexane followed by phenol–chloroform to remove oil and protein ingredients. The extracted CS samples were depolymerized by chondroitinase ABC and CS content was determined. Quantitative analysis of the disaccharides derived from raw materials and an ophthalmic solution showed the CS contents (%) were 39.5 ± 0.1 to 105.6 ± 0.1 and 103.3 ± 1.2, respectively. In case of CS analysis in soft capsules and liquid preparations, the overall recovery (%) of the spiked CS was 96.79 ± 0.53–103.54 ± 1.78 and 97.10 ± 1.82 to 103.17 ± 2.34, respectively. In conclusion, the quantitative analysis of the disaccharides produced by enzymatic digestion can be used in the direct quantitation of CS containing pharmaceutical formulations.