Strong Anion-exchange High-performance Liquid Chromatography Research Articles

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.

Quantitative Analysis of Impurities in Unfractionated Heparin of Bovine Origin.

The USP heparin sodium monograph lists impurities with specifications developed for porcine derived products. Most of these impurities are of biological origin and are present in porcine intestinal mucosa, the tissue source used in the production of porcine heparin. One of the specified impurities, oversulfated chondroitin sulfate (OSCS), has been introduced in the monograph to detect intended adulteration of heparin products with this impurity. The evaluation of bovine intestinal heparin as an alternative source of pharmaceutical heparin included an evaluation of bovine heparin with the current USP heparin sodium monograph methods. This evaluation included a comparison of impurity quantities observed in multiple bovine intestinal heparin samples against the specifications found in the USP heparin sodium monograph. The impurities investigated in this study were protein, galactosamine, nucleotidic impurities, and OSCS. Bovine intestinal heparin met the requirements in the tests for protein, galactosamine, and nucleotidic impurities. A potential issue was observed with the strong anion exchange high performance liquid chromatography (SAX-HPLC) used to analyze for the presence of OSCS. While the OSCS was well-resolved from the bovine heparin peak, the resolution of dermatan sulfate from heparin did not consistently meet system suitability requirements in the current USP Heparin sodium monograph. The overall levels of impurities observed in bovine intestinal mucosal heparin were comparable to those observed in porcine intestinal mucosal heparin. Bovine intestinal mucosal heparin can be produced with acceptable impurity levels that align with these important quality attributes found in porcine heparin.

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.

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.

The use of SAX-HPLC–CD as a heparin screening strategy

Heparin, a heterogeneous polysaccharide, has been widely used as an anticoagulant for decades. Recently, however, international events involving the sudden onset of allergic-type reactions following heparin administration led to numerous fatalities, and demanded the use of multiple laborious, time consuming techniques to identify an economically motivated adulterant. Using these methods cooperatively, the semi-synthetic molecule known as oversulfated chondroitin sulfate (OSCS), was found to be present at significant concentrations. Since the discovery of this adulterant, several analytical methods have been put forth or updated to advance the process of screening pharmaceutical heparins; of these, strong anion exchange high performance liquid chromatography (SAX-HPLC) methods have now become routine. In this preliminary work, we report the use of circular dichroism (CD) detection in conjunction with existing SAX-HPLC methods to quantitate various sulfated polysaccharides. The proposed strategy exploits the selectivity associated with CD detection of heparin and heparin-like polysaccharides, while taking advantage of the method's insensitivity to the use of mobile phase additives and programmed gradients. The limit of detection of heparin by CD was found to be ∼0.22 mg/mL, whereas traditional UV/Vis detection yielded a detection limit of ∼1.09 mg/mL. The success of CD detection varied for other polymers, however no significant modifications were made to the separations method to capitalize on the advantages of CD detection.

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.

Analysis of crude heparin by 1H NMR, capillary electrophoresis, and strong-anion-exchange-HPLC for contamination by over sulfated chondroitin sulfate

We previously published a strong-anion-exchange-high performance liquid chromatography (SAX-HPLC) method for the detection of the contaminant over sulfated chondroitin sulfate (OSCS) in heparin sodium active pharmaceutical ingredient (API). While APIs have been processed to remove impurities, crude heparins contain insoluble material, chondroitin sulfates, heparan sulfate, and proteins that may interfere with the recovery and measurement of OSCS. We examined 500 MHz 1H NMR, capillary electrophoresis (CE), and SAX-HPLC to quantify OSCS in crude heparin. Using our standard API protocol on OSCS spiked crude heparin samples; we observed a weight percent LOD and LOQ for the NMR approach of 0.1% and 0.3%, respectively, while the SAX-HPLC method gave values of 0.03% and 0.09%, respectively. CE data was not amenable to quantitative measurement of OSCS in crude heparin. We developed a modified HPLC sample preparation protocol using crude dissolved at the 100 mg/mL level with a 2.5 M NaCl solution. This SAX-HPLC approach gave a weight percent LOD of 0.02% and a LOQ of 0.07% and had better performance characteristics than that of the protocol used for APIs.

Direct determination of dialkyl phosphates by strong anion‐exchange liquid chromatography/atmospheric pressure chemical ionization mass spectrometry using a quadrupole ion trap instrument

The direct determination of dialkyl phosphates (DAPs) in water by strong anion-exchange (SAX) liquid chromatography/atmospheric pressure chemical ionization (APCI) mass spectrometry was investigated. The SAX high-performance liquid chromatography (HPLC) column was eluted with methanol/water gradients containing ammonium formate (AF) separating the DAPs which included six dimethyl- and diethyl-substituted phosphates, thiophosphates, and dithiophosphates. The high buffer concentrations required for separation were compatible with -ve APCI, but in +ve APCI the DAPs were unstable giving anomalous ions such as [M+15]+ and [M+29]+. These ions are believed to result from ion molecule reactions with CH3OH2+ in the plasma. DAPs are very stable in -ve APCI being detected as abundant [M-H](-) ions, even with 200 mM AF. At higher AF concentration formate clusters ([M+45](-) and [M+91](-)) were seen. Fragmentation by collision-activated dissociation (CAD) was more efficient for deprotonated ethyl-substituted DAPs which lost ethylene followed by ethanol. APCI instrument detection limits were in the low ng/mL range and the response was highly linear. Isotope dilution quantitation using d10-diethyl dithiophosphate (DEDTP) as an internal standard produced an instrument detection limit of 2 ng DEDTP/mL and method detection limit (MDL) of 9.3 ng/mL with accuracy of 99% (spike concentration, 25 ng/mL). DAP mixtures required storage in cold, dry conditions and alcohol solvents should be avoided because of solvolysis reactions.

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.

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.

Biotransformation of Arsenate to Arsenosugars by Chlorella vulgaris.

Chlorella vulgaris was cultivated in a growth medium containing arsenate concentration of <0.01, 10, 100 and 1000 mg l−1. Illumination was carried out in 12 h cycles for 5 days. The health status of the culture was monitored by continuous pH and dissolved oxygen (DO) readings. Destructive sampling was used for the determination of biomass, chlorophyll, total arsenic and arsenic species. The chlorophyll a content, the DO and pH cycles were not significantly different for the different arsenate concentrations in the culture. In contrast, biomass production was significantly (p < 0.05) increased for the arsenic(V) treatment at 1000 mg l−1 compared with 100 mg l−1. The arsenic concentration in the algae increased with the arsenate concentration in the culture. However, the bioconcentration factor decreased a hundred-fold with increase of arsenate from the background level to 1000 mg l−1. The arsenic species were identified by using strong anion-exchange high-performance liquid chromatography–inductively coupled plasma mass spectrometry analysis after methanol/water (1 : 1) extraction. The majority (87–100%) of the extractable arsenic was still arsenate; arsenite was found to be between 1 and 6% of total extractable arsenic in the algae. In addition to dimethylarsinic acid, one unknown arsenical (almost co-eluting with methylarsonic acid) and three different arsenosugars have been identified for the first time in C. vulgaris growing in a culture containing a mixture of antibiotics and believed to be axenic. The transformation to arsenosugars in the algae is not dependent on the arsenate concentration in the culture and varies between 0.2 and 5% of total accumulated arsenic. Although no microbiological tests for bacterial contamination were made, this study supports the hypothesis that algae, and not associated bacteria, produce the arsenosugars. Copyright © 2003 John Wiley & Sons, Ltd.

Stability of colistin and colistin methanesulfonate in aqueous media and plasma as determined by high-performance liquid chromatography.

The stabilities of colistin and colistin methanesulfonate (CMS) in different aqueous media were studied by specific high-performance liquid chromatography (HPLC) methods. Colistin was stable in water at 4 and 37 degrees C for up to 60 days and 120 h, respectively. However, degradation was observed when colistin was stored in isotonic phosphate buffer (0.067 M, pH 7.4) and human plasma at 37 degrees C. The stability of CMS from three different sources in water was explored by strong-anion-exchange (SAX) HPLC for CMS and by measuring the concentrations of colistin formed from the hydrolysis of CMS. The peaks of CMS in SAX HPLC disappeared almost completely after 12 h at 37 degrees C, but appeared to remain intact for up to 2 days at 4 degrees C. Over the same period, there was no formation of colistin at 4 degrees C. In water, phosphate buffer, and plasma, there was rapid formation of colistin within 24 to 48 h at 37 degrees C from the three sources of CMS. The hydrolysis products were assumed to be a complex mixture of many different sulfomethyl derivatives, including colistin. The stability of a fourth source of CMS in Mueller-Hinton broth examined during 30 min at 37 degrees C revealed no formation of colistin. Along with previous microbiological studies, this suggested that different sulfomethyl CMSs possess intrinsic antibacterial activity. These results will be helpful for understanding the pharmacokinetics and pharmacodynamics of colistin and CMS in humans and animals.

Separation of capsular polysaccharide K4 and defructosylated K4 derived disaccharides by high‐performance capillary electrophoresis and high‐performance liquid chromatography

A rapid, highly sensitive and reproducible high-performance capillary electrophoresis (HPCE) method (electrokinetic chromatography with sodium dodecyl sulfate) is described for the determination of disaccharides present in the polysaccharide from the uropathogenic Escherichia coli K4 bacteria (05:K4:H4) and its defructosylated product. Following chondroitinase digestion of K4 and its derivative, the two disaccharides, DeltaHexAFrc-GalNAc for K4 and deltaHexA-GalNAc for defructosylated K4, are separated and readily determined within 20 min on an uncoated fused-silica capillary using normal polarity at 20 kV and detection at 230 nm. Comparison was made by separation of these two disaccharides in isocratic strong-anion exchange HPLC. A linear relationship was found for the two unsaturated disaccharides over a wide range of concentrations, from approximately 0.5 to 5 micro g for high-performance liquid chromatography (HPLC) and from approximately 0.06 to 0.3 micro g for HPCE. The HPCE separation produced a greater detection sensitivity (about 10 times greater) than HPLC. The described methods were used to evaluate the defructosylation process of K4 under drastic acid conditions. Good correspondence was found for the amount of unsaturated disaccharides for the two techniques.

Isolation and characterization of proteoglycan derived from human placenta and its biological activities.

Chondroitin sulfates proteoglycans were isolated from human placenta. For the identification of enzymatic digestion products of isolated proteoglycan, strong anion, exchange-high performance liquid chromatography (SAX-HPLC) was performed. By the action of chondroitin ABC and chondroitin B lyase, three unsaturated disaccharides 2-acetamide-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-galactose (deltaDi-OS), 2-acetamide-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose (deltaDi-6S) and 2-acetamide-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose (deltaDi-4S) were produced from the human placenta proteoglycan. The anticoagulant activity of chondroitin sulfate proteoglycan was evaluated by activated partial thromboplastin time (aPTT) assay and thrombin time (TT) assay. The clotting times of aPTT and TT were increased from 72 to 144 sec and 19 to 27 sec, respectively. The immuno-modulating activity of chondroitin sulfate proteoglycan was examined by cell proliferation assay and these results suggest that it may play a role in suppression of the function of immune-related cells.

Isolation and identification of chondroitin sulfates from the mud snail.

Chondroitin sulfates were isolated from the mud snail. For the quantitative analysis of enzymatic digestion products of isolated chondroitin sulfates, strong anion exchange-high performance liquid chromatography (SAX-HPLC) was performed. By the action of chondroitinase ABC, three unsaturated disaccharides 2-acetamide-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-galactose (delta Di-OS), 2-acetamide-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose (delta Di-6S) and 2-acetamide-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose (delta Di-4S) were produced from the mud snail chondroitin sulfates. The analysis showed that relative proportion of delta Di-OS/delta Di-6S/delta Di-4S was 58.7/3.1/38.2. The immunomodulating activity of chondroitin sulfate was examined by cell proliferation assay and these results suggest that it might be a immunosuppressant.