High-performance liquid chromatographic determinations of disaccharides resulting from enzymatic degradation of isomeric chondroitin sulfates.

The use of Whatman Partisil-10 PAC and Altex LiChrosorb NH₂ bonded phase columns for the high-performance liquid chromatographic separation of the disaccharides obtained from digestion of isomeric chondroitin sulfates with chondroitinases has been investigated. The substituted unsaturated disaccharides in the enzymatic degradation mixtures undergo rapid isocratic separations on both bonded stationary phases. A complete separation can be established within 10 min. The development of these procedures has expedited enzymatic studies of isomeric chondroitin sulfates.The rate of depolymerization of chondroitin sulfates by the action of chondroitinases based on quantitative high-performance liquid chromatographic separations and detection of the disaccharide products was studied.     

The application of high-performance liquid chromatography in enzymatic assays of chondroitin sulfate isomers in normal human urine

A study of the urinary excretion of isomeric chondroitin sulfates in normal individuals by high-performance liquid chromatographic (HPLC) determinations of the unsaturated disaccharides produced by digestion with chondroitinases is described. The composition of the HPLC mobile phase was systematically varied in order to select the optimal conditions for separation.The data show that chondroitin 4-sulfate is the major component of the chondroitin sulfate isomers in normal urine, and that chondroitin 6-sulfate is a lesser component. It is also evident that dermatan sulfate is present in small quantities in normal urine.     

Hyaluronic acid and chondroitin sulfate unsaturated disaccharides analysis by high-performance liquid chromatography and fluorimetric detection with dansylhydrazine

A system capable of resolving all the known unsaturated nonsulfated, mono- and disulfated disaccharides derived from chondroitin sulfate samples, dermatan sulfate, and hyaluronic acid after their derivatization with dansylhydrazine and separation by HPLC and fluorimetric detection is reported. This method was found superior to others in that unsaturated disaccharides can be separated with good resolution in about 50 min in an isocratic solvent with a sensitivity greater than about 50 pmol (approx 20-30 ng) and linearity from 50 to 500 pmol. The system was applied to the analysis of various chondroitin sulfate samples, including highly sulfated species and dermatan sulfate, and also to a defructosylated polysaccharide with a chondroitin backbone purified from Escherichia coli U1-41. Excellent agreement was obtained with traditional compositional analysis performed by anion-exchange HPLC separation and UV absorption at 230 nm.     

Pretreatment procedure for the microdetermination of chondroitin sulfate in plasma and urine

A new, simple, and rapid pretreatment method for the determination of chondroitin sulfate, dermatan sulfate, and hyaluronan from urine and blood plasma samples has been developed. Plasma proteins were first converted into small peptides by digestion using a nonspecific protease, actinase E, and the resulting small peptides were removed by centrifugal filtration. The retained, residual crude glycosaminoglycans, including chondroitin/dermatan sulfates and hyaluronan, were converted into unsaturated disaccharides through the action of chondroitin sulfate lyses. Next, these disaccharides were recovered and purified using centrifugal filtration together with DeltaDi-UA2S, added as an internal standard. The filtered disaccharide mixture was analyzed by HPLC with fluorometric postcolumn derivatization using 2-cyanoacetamide as a fluorogenic reagent. This method was applied to a pharmacokinetic study of chondroitin sulfate administered intravenously to mice. The half-life of the administered chondroitin sulfates, having molecular masses from 6 to 50 kDa, varied depending on their molecular sizes. This new method should be useful for studies on the metabolic fate of exogenously administered glycosaminoglycans in small experimental animals.     

A high-performance liquid chromatography for constituent disaccharides of chondroitin sulfate and dermatan sulfate isomers

An improved high-performance liquid chromatography for unsaturated disaccharides prepared from chondroitin sulfate and dermatan sulfate isomers was developed using an ion-exchange resin made from a sulfonized styrene-divinylbenzene copolymer. By this newly devised method, it was found that the retention times of representative unsaturated disaccharides are very unique and appear in the following order: unsaturated 6-sulfated, nonsulfated, and 4-sulfated disaccharides. The content of the individual unsaturated disaccharides could be measured at similar sensitivities with ultraviolet absorbance. Sensitive and unique retention times as well as good resolution were found for various unsaturated disulfated disaccharides. The new microassay method by HPLC can be used to determine chondroitin sulfate and dermatan sulfate isomers in amounts as small as 100 ng to 8 micrograms. The practicality of this method was verified by application to the separation and quantitation of chondroitin sulfate and dermatan sulfate isomers from human coronary arteries.     

Isolation and structural studies of heparan sulfates and chondroitin sulfates from three species of molluscs

The isolation and some structural features of heparan sulfates and chondroitin sulfates from three species of molluscs (Pomacea sp., Tagelus gibbus, and Anomalocardia brasiliana) are reported. It is shown that heparan sulfates with structural similarities to the ones found in mammals are present in the three molluscs analyzed. All the heparan sulfates were degraded by heparitinases I and II to four distinct unsaturated disaccharides with the same properties as the ones present in heparan sulfates of mammalian origin. This suggests that these four disaccharide units are maintained through the evolution. Furthermore, the proportion of these units varied in the heparan sulfates according to the species of origin. The chondroitin sulfates, on the other hand, exhibit different structural features according to the species of origin. For instance, by the action of chondroitinase AC, 4- and nonsulfated disaccharides are produced from Pomacea chondroitin, whereas 4- and 6-sulfated disaccharides are formed from Tagelus and Anomalocardia. The possible role of these compounds in cell recognition and/or adhesiveness is discussed in view of the present findings.     

High-performance liquid chromatography of pyridylamino derivatives of unsaturated disaccharides produced from chondroitin sulfate isomers by chondroitinases

A sensitive method was developed for the separation and quantitation of four unsaturated disaccharides (delta Di-0S, delta Di-4S, delta Di-6S, and delta Di-diS) by high performance liquid chromatography. The unsaturated disaccharides were coupled with a fluorescent compound, 2-aminopyridine. Complete separation of the resulting pyridylamino derivatives was achieved on a column of muBondapak-C18 with 8 mM KH2PO4-Na2HPO4 (pH 6.0)/methanol (30/l, by volume) as a mobile phase. There was a linear relationship between the fluorescence emission (peak height), and the amount of each authentic disaccharide used for the coupling reaction. This method was applied to analyze commercially available chondroitin sulfates A and C, dermatan sulfate, and urinary glycosaminoglycans obtained from patients with mucopolysaccharidosis after digestion with chondroitinases. The data indicated that the present method is useful for the separation and quantitation of nmol-pmol levels of the unsaturated disaccharides produced from chondroitin sulfate isomers by chondroitinases and can be used for their structural characterization.     

An automated version of the periodate-thiobarbituric acid assay for analysis of delta-4,5 unsaturated uronic acids and its application to the assay of hyaluronic acids and chondroitin sulfates

An automated periodate-thiobarbituric acid assay of Δ-4,5 unsaturated uronic acids which avoids extraction of chromogen has been developed and applied to the analysis of hyaluronic acid and chondroitin sulfates in standard glycosaminoglycan mixtures and in biological samples following digestion with eliminase enzymes. Assay of hyaluronic acid was linear between 0.1 and 2.5 μg of uronic acid, when digested with hyaluronidase from S. hyalurolyticus and use directly in the automated procedure. The measurement of unsaturated disaccharide standards (25–100 μm) derived from chondroitin sulfates was also linear although the color yields were different. The proportions of chondroitin sulfate isomers were estimated by assay of the unsaturated chondroitin disaccharides which has been separated by thin-layer chromatography.     

Isolation and characterization of an induced chondroitinase ABC from Flavobacterium heparinum

During the investigation of alternative methods for the large scale preparation of chondroitinases AC, B and C from Flavobacterium heparinum, a new chondroitinase activity was observed. This new enzyme, like the other chondroitinases, acts as an eliminase, forming unsaturated sulfated disaccharides from dermatan and chondroitin sulfates. In contrast to the chondroitinases previously described, which are endoglycosidases, this chondroitinase ABC cleaves the glycosidic linkages in an exolytic fashion, beginning at the reducing end of the substrate molecules. The oligosaccharides formed as transient products by the action of either chondroitinases or testicular hyaluronidase upon dermatan and chondroitin sulfates are also rapidly degraded by the chondroitinase ABC, regardless of their size or the presence of delta-4,5 unsaturation in the terminal uronic acid residue. The maximum activity of the chondroitinase ABC occurs at 30 degrees C and at pH 6.0-7.5. Only 15% of the activity was observed at 37 degrees C, indicating that the enzyme is very sensitive to thermal denaturation. It is strongly inhibited by phosphate ions and is also inhibited by the unsaturated disaccharides formed.     

Capillary electrophoresis for the analysis of chondroitin sulfate- and dermatan sulfate-derived disaccharides

High-voltage capillary zone electrophoresis (CZE) has been used for the first time in the analysis of non-, mono-, di-, and trisulfated disaccharides derived from chondroitin sulfate, dermatan sulfate, and hyaluronic acid. These glycosaminoglycans are first depolymerized using polysaccharide lyases. The resulting unsaturated disaccharide products can be detected by their ultraviolet absorbance at 232 nm. Different retention times were obtained for each unsaturated disaccharide analyzed by CZE. The application of a constant voltage across a 70-cm fused silica capillary using a single, simple buffer system resolved an eight-component mixture within 40 min. Quantitation of disaccharides derived from chondroitin sulfate using chondroitin ABC lyase (EC 4.2.2.4) and mixtures of unsaturated disaccharide standards was possible requiring only picogram quantities of sample. The disaccharides examined had a net charge of from -1 to -4 and were resolved primarily on the basis of net charge and secondarily on the basis of charge distribution. Two unsulfated disaccharides both containing the same unsaturated uronic acid residue were analyzed. One was from chondroitin having an N-acetylgalactosyl residue and one from hyaluronate having an N-acetylglycosyl residue. Despite the fact that they differed only by the chirality at one center, these disaccharides were resolved by CZE. CZE is a fast and simple method that represents a powerful new tool for analysis and separation of acidic disaccharide components of glycosaminoglycans.     

A new electrophoretic method for the separation of disaccharides obtained by digestion of chondroitin sulfates and dermatan sulfate with chondroitinases

A new rapid and simple method has been developed for the separation of disaccharides obtained by chondroitinase digestion of chondroitin sulfates and dermatan sulfate using electrophoresis on cellulose acetate plates (Titan III cellulose acetate plates). Three disaccharides are completely separated by electrophoresis in barium acetate or calcium acetate in a short time, and less than 50 μg of glycosaminoglycan samples can be analyzed within 2 h.     

Two squid skin proteoglycans each containing chondroitin sulfates with different sulfation patterns.

Two chondroitin sulfate containing proteoglycans, amounting to approximately 6% of the tissue proteoglycans, were isolated from the skin of the squid. They were almost completely extracted by 4 M guanidine hydrochloride in the presence of proteinase inhibitors, and then they were separated by DEAE-Sephacel chromatography and isolated by further chromatography on Sepharose CL-4B. Each proteoglycan contained two types of chondroitin sulfates that differed in their sulfation patterns. One proteoglycan (molecular mass (M(r)) 5.6 x 10(5)) contained, on the average, four chondroitins (M(r) 8.4 x 10(4)) and five chondroitin sulfates (M(r) 3.4 x 10(4)), whereas the other proteoglycan (M(r) 5.2 x 10(5)) contained three chondroitin sulfates (M(r) 1.1 x 10(5)) and five oversulfated chondroitin sulfates (M(r) 4.3 x 10(4)). The glycosaminoglycans were released from the proteoglycans by treatment with alkaline borohydride, separated from the oligosaccharides by chromatography on Bio-Gel P-30, and isolated by chromatography on DEAE-Sephacel and Sepharose CL-6B. Chondroitin sulfates were degraded by chondroitinase AC to an extent of 70% and consisted of significant amounts of disaccharides sulfated at C-4 of the galactosamine, disulfated disaccharides, and small amounts of nonsulfated disaccharides, as well as disaccharides that bore sulfates at C-6. Oversulfated chondroitin sulfate was degraded by chondroitinase AC to only 40% and contained appreciable amounts of disulfated and trisulfated disaccharides. The glycosaminoglycans also contained neutral monosaccharides; glucose was the predominant neutral sugar. A part of the oligosaccharides of both proteoglycans was of identical structure to that of chondroitin sulfate.     

Analysis of unsaturated disaccharides from glycosaminoglycuronan by high-performance liquid chromatography

A rapid and simple analytical method for unsaturated disaccharide isomers formed by enzymatic digestion from hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate, and heparin by high-performance liquid chromatography using an amine-bound silica column with a linear gradient of sodium dihydrogen phosphate was developed. The analyses were performed on isomers of two groups belonging to the chondroitin sulfate family and the heparin sulfate family. In both families, disaccharide isomers eluted in the order non-, mono-, di-, and trisulfated disaccharides by elevating salt concentrations. The method was applied to the analysis of constituent disaccharides of representative sulfated glycosaminoglycans, which proved that most constituents could be quantified separately. This method is advantageous in that enzymatic digests can be applied directly on a column without any pretreatment and good resolution of several disaccharides can be obtained by one chromatography.     

Analysis of glycosaminoglycan-derived disaccharides by capillary electrophoresis using laser-induced fluorescence detection

A quantitative and highly sensitive method for the analysis of glycosaminoglycan (GAG)-derived disaccharides that relies on capillary electrophoresis (CE) with laser-induced fluorescence detection is presented. This method enables complete separation of 17 GAG-derived disaccharides in a single run. Unsaturated disaccharides were derivatized with 2-aminoacridone to improve sensitivity. The limit of detection was at the attomole level and approximately 100-fold more sensitive than traditional CE-ultraviolet detection. A CE separation timetable was developed to achieve complete resolution and shorten analysis time. The relative standard deviations of migration time and peak areas at both low and high concentrations of unsaturated disaccharides are all less than 2.7 and 3.2%, respectively, demonstrating that this is a reproducible method. This analysis was successfully applied to cultured Chinese hamster ovary cell samples for determination of GAG disaccharides. The current method simplifies GAG extraction steps and reduces inaccuracy in calculating ratios of heparin/heparan sulfate to chondroitin sulfate/dermatan sulfate resulting from the separate analyses of a single sample.     

High-performance liquid chromatography and on-line mass spectrometry detection for the analysis of chondroitin sulfates/hyaluronan disaccharides derivatized with 2-aminoacridone

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 GalNAcSO₄ 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.     

ATP synthase of yeast: structural insight into the different inhibitory potencies of two regulatory peptides and identification of a new potential regulator

Quantitative and qualitative alterations of renal oversulfated chondroitin/dermatan sulfates (C/DSs) accompanied by the development of tubulointerstitial nephritis were examined. The rat model with unilateral ureteral obstruction (UUO) is a suitable model for study of renal interstitial fibrosis, and was utilized in the present study. Cortical regions of serial sections of UUO kidney and sham-operated kidney on glass slides were processed using a small surgical knife under dark field microscopy. Oversulfated C/DSs in tissue sections on a glass slide were degraded to unsaturated disaccharides using chondroitinase ABC and ACII digestion in the presence of bacterial collagenase. The resulting unsaturated disaccharides were subsequently determined by HPLC. These in situ investigations yielded the following results: (1) marked increases in oversulfated C/DSs content and decreases in the oversulfation degree of C/DSs were observed in fibrous lesions, compared to non-fibrous lesions, and (2) iduronic acid content in C/DSs in fibrous lesions was significantly lower than that in non-fibrous lesions. These findings indicate that oversulfated C/DSs with low-iduronic acid content represent a potential marker for tubulointerstitial nephritis.     

Preparation of unsaturated disaccharides by eliminative cleavage of heparin and heparan sulfate with heparitinases.

1. Six kinds of unsaturated disaccharides were prepared by enzymatic digestion of heparin and heparan sulfate with heparitinases I0 and IV, and subsequent column chromatography. They were identified by HPLC showing good separation from each other. 2. The content of each unsaturated disaccharide fraction was determined colorimetrically, and found to range from 130.7 to 722.3 mumol. 3. Molecular extinction coefficient of each unsaturated disaccharide was calculated from absorbance at a wavelength of around 230 nm where a peak appeared on the ultraviolet spectrum of each disaccharide solution at pH 2. The values varied from 6000 to 6600.     

Electrophoretic and enzymatic analysis of glycosaminoglycans in the blood serum of patients with hyperthyroidism

An effect of hyperthyroidism on the composition and levels of glycosaminoglycans in the blood serum was studied. Glycosaminoglycans isolated from 1-ml blood samples were assayed with the following techniques: carbazole, electrophoretic and enzymatic. Separation and assay of particular GAG were made with bidirectional electrophoresis. Isomers of the remaining chondroitin sulphates were assayed enzymatically. Electrophoretograms of GAG in blood serum of healthy women have shown two fractions: low sulphate chondroitin sulphate and chondroitin-4-sulphate. The same fractions of GAG were found in blood serum of the female patients with hyperthyroidism. Mean concentration of GAG in the blood serum of hyperthyroid patients increased by 51%: low sulphate chondroitin sulphate and chondroitin-4-sulphate concentrations increased by 22% and 190% respectively. Chondroitin sulphates in the blood serum of both groups were degraded to unsaturated disaccharides not containing sulphur and unsaturated 4-sulphate disaccharides. Concentrations of unsaturated 4-sulphate and unsaturated sulphur-free disaccharides increased by 71% and 17% in hyperthyroidism. Observed changes in the blood serum GAG concentrations reflect changes in the connective tissue metabolism in hyperthyroidism.     

High performance liquid chromatography of unsaturated disaccharides produced from chondroitin sulfates by chondroitinase.

High performance liquid chromatography was performed by the ion pair method on unsaturated disaccharides produced from chondroitin sulfates by the action of chondroitinase. Completely separated peaks corresponding to deltaDi-0S, deltaDi-4S, and deltaDi-6S were obtained on a column of mu-bondapak-C18 with 0.035 M tetra-butylammonium phosphate (pH 7.54) as a mobile phase. There was a linear relationship between the ratio of the peak area and the amount of each standard tested.     

Isolation and characterization of an induced chondroitinase ABC from Flavobacterium heparium

During the investigation of alternative methods for the large sclae preparation of chondroitinases AC, B and C from Flavobacterium heparinum, a new chondroitinase activity was observed. This new enzyme, like the other chondroitinases, acts as an eliminase, forming unsaturated sulfated disaccharides from dermatan and chondroitin sulfates. In contrast tot he chondroitinases previously described, which are endoglycosidases, this chondroitinase ABC cleaves the glycosidic linkages in an exolytic fashiom, beginning at the reducing end of the substrate molecules. The oligosaccharides formed as transient products by the action of either chondroitinases or testicular hyaluronidase upon dermatan and chontroitin sulfates are also rapidly degraded by the chondroitinase ABC, regardless of their size or the presence of Δ-4,5 unsaturation in the terminal uronic acid residue. The maximum activity of the chondroitinase ABC occurs at 30°C and at pH 6.0–7.5. Only 15% of the activity was observed at 37°C, indicating that the enzyme is very sensitive to thermal denaturation. It is stronly inhibited by phosphate ions and is also inhibited by the unsaturated disaccharides formed.