chondroitin-sulfates and 2-aminoacridone

Capillary electrophoresis is a separation technique with high resolving power and sensitivity with applications in glycosaminoglycan analysis. In this chapter, we present validated protocols for determining the variously sulfated chondroitin or dermatan sulfate-derived disaccharides. These approaches involve degradation of the polysaccharides with specific chondro/dermato-lyases and electrophoretic analysis with capillary zone electrophoresis in a low pH operating buffer and reversed polarity. This methodology has been applied to drug/nutraceutical formulations or to biologic samples (blood serum, lens capsule) and has been validated. Analysis of biologic tissue samples is often more demanding in terms of detection sensitivity, and thus concentration pretreatment steps and/or a derivatization step with 2-aminoacridone are often advisable.

Topics: Aminoacridines; Carbohydrate Conformation; Chondroitin Sulfates; Chondroitinases and Chondroitin Lyases; Dermatan Sulfate; Dietary Supplements; Disaccharides; Electrophoresis, Capillary; Humans; Lens Capsule, Crystalline; Pharmaceutical Preparations; Polysaccharides; Reproducibility of Results

Hyaluronan (HA), chondroitin sulfate (CS), and heparan sulfate (HS) are glycosaminoglycans (GAGs) with a great importance in biological processes as they participate in functional cell properties, such as migration, adhesion, and proliferation. A perturbation of the quantity and/or the sulfation of GAGs is often associated with pathological conditions. In this chapter, we present valuable and validated protocols for the analysis of HA-, CS-, and HS-derived disaccharides after derivatization with 2-aminoacridone and by using the fluorophore-assisted carbohydrate electrophoresis (FACE). FACE is a well-known technique and a reliable tool for a fast screening of GAGs, as it is possible to analyze 16 samples at the same time with one electrophoretic apparatus. The protocols for the gel preparation are based on the variations of the acrylamide/bisacrylamide and buffer concentrations. Different approaches for the extraction and purification of the disaccharides of various biologic samples and pharmaceutical preparations are also stressed.

Topics: Aminoacridines; Animals; Buffers; Chemistry, Pharmaceutical; Chondroitin Sulfates; Chromatography, High Pressure Liquid; Disaccharides; Electrophoresis; Electrophoresis, Polyacrylamide Gel; Fluorescent Dyes; Glycosaminoglycans; Heparin; Heparitin Sulfate; Humans; Hyaluronic Acid; Lens Capsule, Crystalline; Mice; Pharmaceutical Preparations; Rats

In this study, we developed an on-line reverse-phase high-performance liquid chromatography-electrospray ionization-mass spectrometry (RP-HPLC-ESI-MS) separation and structural characterization of hyaluronan (HA)/chondroitin sulfate (CS)/dermatan sulfate (DS) disaccharides released by enzymatic treatment and derivatized with 2-aminoacridone (AMAC), providing a high-resolution system also applicable by using a further fluorimetric detector (Fp) before ESI-MS spectral acquisition. Isomeric nonsulfated HA and CS/DS disaccharides, isomeric monosulfated and isomeric disulfated CS/DS disaccharides, and the trisulfated species were distinctly separated and unambiguously identified by their retention times and mass spectra in negative ionization mode. In general, no multiply charged ions were detected even for highly charged disaccharides, but the presence of desulfonated products for highly sulfated species due to the relative instability of sulfo groups was observed. RP-HPLC-ESI-MS of each AMAC disaccharide was found to be linear from 3 to 500 ng with very high coefficient of correlation values due to the high efficiency of separation and the sharp outline of the peaks. Various CS/DS samples were characterized for disaccharide composition, and minor oligomer species identified as GalNAcSO(4) at the nonreducing end of chains was observed as a common component of these macromolecules. Furthermore, purified endogenous normal human plasma CS disaccharides were also evaluated by means of RP-HPLC-(Fp)-ESI-MS.

Topics: Aminoacridines; Chondroitin ABC Lyase; Chondroitin Sulfates; Chromatography, High Pressure Liquid; Dermatan Sulfate; Disaccharides; Humans; Hyaluronic Acid; Spectrometry, Mass, Electrospray Ionization

This work describes improved workup and instrumental conditions to enable robust, sensitive glycosaminoglycan (GAG) disaccharide analysis from complex biological samples. In the process of applying CE with LIF to GAG disaccharide analysis in biological samples, we have made improvements to existing methods. These include (i) optimization of reductive amination conditions, (ii) improvement in sensitivity through the use of a cellulose cleanup procedure for the derivatization, and (iii) optimization of separation conditions for robustness and reproducibility. The improved method enables analysis of disaccharide quantities as low as 1 pmol prior to derivatization. Biological GAG samples were exhaustively digested using lyase enzymes, the disaccharide products and standards were derivatized with the fluorophore 2-aminoacridone and subjected to reversed polarity CE-LIF detection. These conditions resolved all known chondroitin sulfate (CS) disaccharides or 11 of 12 standard heparin/heparan sulfate disaccharides, using 50 mM phosphate buffer, pH 3.5, and reversed polarity at 30 kV with 0.3 psi pressure. Relative standard deviation in migration times of CS ranged from 0.1 to 2.0% over 60 days, and the relative standard deviations of peak areas were less than 3.2%, suggesting that the method is reproducible and precise. The CS disaccharide compositions are similar to those obtained by our group using tandem MS. The reversed polarity CE-LIF disaccharide analysis protocol yields baseline resolution and quantification of heparin/heparan sulfate and CS/dermatan sulfate disaccharides from both standard preparations and biologically relevant proteoglycan samples. The improved CE-LIF method enables disaccharide quantification of biologically relevant proteoglycans from small samples of intact tissue.

Topics: Aminoacridines; Animals; Cartilage; Cattle; Cellulose; Chondroitin Sulfates; Dermatan Sulfate; Disaccharides; Electrophoresis, Capillary; Fluorescence; Glycosaminoglycans; Heparin; Heparitin Sulfate; Humans; Lyases; Reproducibility of Results; Sensitivity and Specificity

Urinary trypsin inhibitor (ulinastatin) is a characteristic protein pharmaceutical which contains both glycosaminoglycans and N-linked glycans in its molecule and has been used for treatment of acute pancreatitis. The comparability of ulinastatin preparations of different lots or from different companies was studied by using conventional analytical approaches such as SDS-PAGE, cellulose acetate membrane electrophoresis, and HP size-exclusion chromatography (SEC) and also by using newly developed techniques such as CE and MALDI-TOF MS. The methods using SEC and SDS-PAGE according to The Japanese Pharmacopoeia showed similar molecular masses for two different preparations, and the estimated molecular masses were significantly different from those observed with MALDI-TOF MS. We also showed that the electrophoretic methods using cellulose acetate membrane electrophoresis and CE can be used for comparability assessments of ulinastatin preparations. In addition, we analyzed the unsaturated disaccharides derived from glycosaminoglycan (chondroitin 4-sulfate chain) and N-linked oligosaccharides attached to ulinastatin by CE after releasing them by enzymatic digestion followed by fluorescent labeling with 2-aminoacridone and 2-aminobenzoic acid, respectively. The results indicated that carbohydrate chains are important as markers for comparability assessments of ulinastatin pharmaceutical preparations.

Topics: Amino Acid Sequence; Aminoacridines; Chondroitin Sulfates; Electrophoresis; Fluorescent Dyes; Glycoproteins; Molecular Sequence Data; Oligosaccharides; ortho-Aminobenzoates; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Hyaluronan (HA) and chondroitin sulfate (CS) are glycosaminoglycans (GAGs) with great importance in biological events, since they participate in and regulate cell adhesion, migration and proliferation. Quantitation and analysis of the fine structure of GAGs are increasingly important for understanding many biological processes, among which are many critical aspects of pathology development and specific phenotype descriptions. Human umbilical cord and human synovial fluid are connective tissues containing high amounts of GAGs and change in the quantity and structure of these macromolecules is described in tissue development and is commonly associated with disease. Moreover, also in Xenopus laevis embryo development and chondrocyte cultures, the GAG content and structure play a critical role. A rapid analysis of hyaluronan and chondroitin sulfate Delta-disaccharides derived from the above human and animal samples, derivatized with 2-aminoacridone and analyzed by polyacrylamide gel electrophoresis, is described in this report. Qualitative and quantitative analysis were performed by comparing their migration and the pixel density with standard Delta-disaccharides, running in the same gel. Since this method allows the analysis of large numbers of samples simultaneously in one gel and has a relatively high sensitivity (less than 25 pmol), it is suggested as a cost-effective and useful tool for the fast screening of small amounts of hyaluronan and chondroitin sulfate disaccharides.

Topics: Aminoacridines; Animals; Cells, Cultured; Chondroitin Sulfates; Culture Media; Disaccharides; Down Syndrome; Electrophoresis, Polyacrylamide Gel; Fetal Blood; Humans; Hyaluronic Acid; Sensitivity and Specificity; Synovial Fluid; Xenopus laevis

Various combinations of fluorescent dyes, polyacrylamide gels, and electrophoresis buffers were tested by fluorophore-assisted carbohydrate electrophoresis (FACE) for the purpose of analyzing sulfated and nonsulfated glycosaminoglycan (GAG) oligosaccharides in which disaccharides and low-molecular weight oligosaccharides were included. A nonionic fluorescent dye was found to be suitable for analyzing sulfated disaccharides derived from sulfated GAGs (e.g., chondroitin sulfate, dermatan sulfate) because sulfated disaccharides themselves had enough anionic potential for electrophoresis. The migration rates of chondroitin sulfate (CS) disaccharides in polyacrylamide gels were affected by the number of sulfate residues and the conformation of each disaccharide. When an anionic fluorescent dye, 8-aminonaphthalene-1,3,6-trisulfonic acid disodium salt (ANTS), was coupled with sulfated GAG oligosaccharides, nearly all of the conjugates migrated at the electrophoretic front due to the added anionic potential. Nonsulfated hyaluronan (HA) oligosaccharides (2-16 saccharides) were subjected to electrophoresis by coupling with a nonionic fluorescent dye, 2-aminoacridone (AMAC), but did not migrate in the order of their molecular size. Especially di-, tetra-, hexa-, and octasaccharides of HA migrated in the reverse order of their molecular size. HA/CS oligosaccharides were able to migrate in the order of their chain lengths by coupling with an anionic fluorescent dye in a nonborate condition.

Topics: Aminoacridines; Animals; Borates; Chondroitin Sulfates; Decapodiformes; Disaccharides; Electrophoresis, Polyacrylamide Gel; Fluorescent Dyes; Glycosaminoglycans; Hyaluronic Acid; Molecular Structure; Molecular Weight; Naphthalenes; Oligosaccharides; Whales

In this report, we describe a polyacrylamide gel electrophoresis for the analysis of fluorophore-labelled hyaluronan (HA) and chondroitin sulfate (CS) Delta-disaccharides. The method utilizes derivatization of reducing ends of hyaluronan and the variously sulfated chondroitin sulfate Delta-disaccharides with 2-aminoacridone (AMAC), followed by electrophoresis in Tris-HCl buffer (pH 8.8), and polyacrylamide gel (T 25%/C 3.75%). The method was applied to the analysis of GAGs secreted into the culture medium of human aortic smooth muscle cells and the obtained results were compared with those analysed by fluorophore assisted carbohydrate electrophoresis (FACE). The described method is a useful and sensitive tool for the rapid monitoring of GAGs in high number of biologic samples.

Topics: Aminoacridines; Aorta; Cells, Cultured; Chondroitin Sulfates; Disaccharides; Electrophoresis, Polyacrylamide Gel; Fluorescence; Fluorescent Dyes; Glycosaminoglycans; Humans; Hyaluronic Acid; Italy

Microchip electrophoresis is a powerful tool for fast analysis of nucleic acids and has expanded its applicability to the analysis of various biological materials including proteins and carbohydrates. Glycosaminoglycans have intrinsic negative charges, and are good targets for electrophoretic analysis. In the present paper, we developed a method to analyze oligosaccharides and unsaturated disaccharides derived from some glycosaminoglycans after digestion with specific enzymes followed by derivatization with 2-aminoacrydone (AMAC) by reductive amination. The method described here allowed rapid analysis of oligosaccharides derived from glycosaminoglycans within 150 s with high sensitivity. We show an application of the present technique to the glycosaminoglycan analysis in cultured HeLa cells.

Topics: Aminoacridines; Chondroitin Sulfates; Disaccharides; Electrophoresis, Microchip; Fluorescent Dyes; Glycosaminoglycans; HeLa Cells; Humans; Hyaluronic Acid; Oligosaccharides; Sensitivity and Specificity

An ultrasensitive capillary electrophoretic method for separating the variously sulfated chondroitin/dermatan sulfate-derived delta-disaccharides after digestion with chondro/dermatolyases and derivatization with the fluorophore 2-aminoacridone is described. All known mono-, di- and tri-sulfated delta-disaccharides were completely separated using 15 mM orthophosphate buffer (pH 3.0) at 20 kV without any interference of the excess derivatizing reagent. They were detected at the anode (reversed polarity) using either an Ar-ion laser-induced fluorescence (LIF) detector (excitation wavelength 488 nm) or a UV detector. The sensitivity obtained by LIF (0.51 pmol/l) was at least 100 and 10 times higher as compared to those obtained by UV detection at 232 nm of underivatized delta-disaccharides and at 254 nm of those derivatized with aminoacridone, respectively. The method has been easily applied to the analysis of chondroitin/dermatan sulfates from various tissues at the attogram level, including chondrotin/dermatan sulfates from normal and aneurysmal human abdominal aortas.

Topics: Aminoacridines; Chondroitin Sulfates; Dermatan Sulfate; Disaccharides; Electrophoresis, Capillary; Humans; Indicators and Reagents; Lasers; Sensitivity and Specificity; Spectrometry, Fluorescence

A series of disaccharides derived from chondroitin sulfate and heparin/heparan sulfate were derivatized at their reducing ends with the fluorophore 2-aminoacridone. The resulting labeled compounds derived from chondroitin sulfate or heparin/heparan sulfate were well separated and could be quantified by capillary electrophoresis and HPLC with a fluorescence detector. This method was successfully applied to the analysis of the disaccharide composition of sulfated tetra- and hexasaccharides derived from chondroitin sulfate and heparin/heparan sulfate. Remarkably, the saturated and the corresponding unsaturated chondrodisaccharides, the structures of which differ only in their nonreducing terminal uronic acid residues, were also well separated from each other by capillary electrophoresis. This facilitated disaccharide composition analysis of saturated chondrooligosaccharides prepared by testicular hyaluronidase digestion as well as unsaturated chondro- and heparin/heparan sulfate-oligosaccharides prepared by digestion with chondroitinases and heparitinases. The developed method employing the derivatization by 2-aminoacridone in conjunction with capillary electrophoresis or HPLC with a fluorescence detector allows a simple, sensitive, and quantitative disaccharide composition analysis of the glycosaminoglycan-derived oligosaccharides.

Topics: Aminoacridines; Chondroitin Lyases; Chondroitin Sulfates; Chromatography, High Pressure Liquid; Cystine; Disaccharides; Electrophoresis, Capillary; Fluorescent Dyes; Fluorometry; Glycosaminoglycans; Heparin; Heparitin Sulfate; Oligosaccharides; Polysaccharide-Lyases