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PubMed2002

Identification, structural analysis and function of hyaluronan in developing fish larvae (leptocephali).

Hyaluronan (HA) has been identified as the principal glycosaminoglycan (CAG) in the highly hydrated, extracellular body matrix of the larval stage (leptocephalus) of seven species of true eels (Teleostei: Elopomorpha: Anguilliformes) and the ladyfish Elops saurus (Elopiformes), and was found as a minor GAG component in the bonefish Albula sp. (Albuliformes). Identification was based on: (1) HPLC separation of unsaturated disaccharides derived from chondroitinase ABC digests of whole-body GAG extracts; (2) 1H NMR analyses of native GAG polymers; and (3) degradation of GAG extracts by Streptomyces hyaluronan lyase. The unsaturated disaccharide 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-glucose (DeltaDi-HA) accounted for 92.4-99.8% of the total disaccharides in chondroitinase digests. Trace amounts of unsaturated disaccharides of chondroitin sulfate were also present. Two-dimensional gCOSY spectra of the native HA polymer were similar for all species. Proton assignments for the HA disaccharide repeat (GlcAbeta1-3GlcNAcbeta1-4) in D(2)O, based on gCOSY, DQF-COSY and TOCSY analyses for the eel Ahlia egmontis, were concordant with published chemical shifts for HA oligosaccharides. In addition to its presumed role in maintaining the structural integrity and hydration of the gelatinous body of the leptocephalus, HA is postulated to function as a storage polysaccharide in those species in which it is the predominant GAG.

Animals↗
PubMed2001

Evidence of block and randomly sequenced chondroitin polysaccharides: sequential enzymatic digestion and quantification using ion trap tandem mass spectrometry.

A method for determining the sequence type of the disaccharide repeat region of cartilage samples is introduced. The samples are sequentially subjected to selective and nonselective enzymatic digestion, and the isomeric products from each step are quantified using tandem mass spectrometry. The two-step digestion/quantification protocol identifies whether the global makeup of the polymer is "alternating", "random", or "blocked" with respect to the two main components of the cartilage, 4- and 6-sulfated disaccharides. Using this procedure, the sequence type of two biologically isolated chondroitin polysaccharides was identified. The results for chondroitin sulfate A, isolated from bovine trachea, are consistent with the 4- and 6-sulfated disaccharides randomly distributed throughout the repeat region of the polysaccharide. For chondroitin sulfate C, shark cartilage, the 6-sulfated disaccharides are adjacent to each other to a larger extent than one would expect for a randomly distributed polymer, indicating that "blocks" of repeating disaccharides with the same sulfation site are present.

Animals↗
PubMedDate not supplied

Glycosaminoglycan composition of the large freshwater mollusc bivalve Anodonta anodonta.

In this paper, glycosaminoglycans from the body of the large freshwater mollusc bivalve Anodonta anodonta were recovered at about 0.6 mg/g of dry tissue, composed of chondroitin sulfate (approximately 38%), nonsulfated chondroitin (about 21%), and heparin (41%). This last polysaccharide was found to consist of a large percentage (approximately 88%) of a fast-moving species possessing a lower molecular mass and sulfate group amount and about 12% of a more sulfated, slow-moving component having a greater molecular mass. The chondroitin sulfate was composed of approximately 28% of the 6-sulfated disaccharide, 46% of the 4-sulfated disaccharide, and about 26% of the nonsulfated disaccharide, with a charge density value of 0.74. Heparin was subjected to the oligosaccharide mapping after treatment with heparinase and then separation of the resulting unsaturated oligosaccharides by SAX-HPLC. A heparin sample from Anodonta anodonta showed a degree of sulfation similar to that of bovine mucosal heparin because of the presence of approximately the same mol % of the trisulfated disaccharide (DeltaUA2S(1-->4)-alpha-D-GlcN2S6S), a slight modification of the other oligosaccharides, and a significant increase of the disaccharide bearing the sulfate group in position 3 of the N-sulfoglucosamine 6-sulfate (-->4)-beta-D-GlcA(1-->4)-alpha-D-GlcN2S3S6S(1-->) part of the ATIII-binding region. However, the anticoagulant activity of mollusc heparin was quite similar to that of pharmaceutical grade heparin. The data obtained again emphasize the heterogeneity of GAGs from molluscs.

Animals↗
PubMed2002

Oral bioavailability of chondroitin sulfate (Condrosulf) and its constituents in healthy male volunteers.

OBJECTIVE: Drug treatment of osteoarthritis (OA) includes symptomatic slow-acting drugs (SYSADOA). This class of compounds have a slow onset of action and improve OA symptoms. Among the SYSADOA, Condrosulf) (manufactured by IBSA), whose active ingredient is chondroitin sulfate, has proven to be a valuable therapeutic tool for the symptomatic treatment of OA after oral administration. The aim of this study was to assess the bioavailability of chondroitin sulfate and its constituents after oral administration of Condrosulf) to 20 healthy male volunteers. Pharmacokinetic parameters and the structure and properties of plasma chondroitin sulfate were determined after administration of Condrosulf). The possible physiological regulation of plasma levels of endogenous chondroitin sulfate during the day was also assessed. DESIGN: Condrosulf) (composed of bovine origin chondroitin sulfate, 4 g) was orally administered to 20 healthy human volunteers, and chondroitin sulfate derivatives were extracted and purified from plasma over a 48 h period. Polysaccharide fractions absorbed by oral route were characterized and quantified by agarose-gel electrophoretic technique, and densitometric scanning. In addition, the percentage of constituent disaccharides and charge density were measured in an effort to physico-chemically characterize chondroitin sulfate fractions absorbed per os. RESULTS: Plasma levels of endogenous chondroitin sulfate were detectable in all subjects, and the mean values calculated on six subjects varied during the day from 0.3 to 5.3 microg/ml. After administration of Condrosulf), chondroitin sulfate plasma levels increased (more than 200%) in all subjects with a peak concentration after 2h, with the increase reaching significance from 2 to 6h. Absorption of exogenous chondroitin sulfate was also proved by the change in the composition of disaccharides in plasma after drug administration with respect to baseline. A significant decrease in the relative amount of non-sulfated disaccharide was measured (reaching the minimum relative percentage of 22.96+/-11.68% at 4h). At the same time 4-sulfated disaccharide increased to a maximum of 60.50+/-10.45% after 4h and 6-sulfated disaccharide appeared in blood, reaching a maximum concentration of 17.33+/-6.52% after 2h. Concomitantly the mean charge density increased from 0.40+/-0.09 at pre-dose to a maximum of 0.78+/-0.11 4h after Condrosulf) administration. As for safety, the treatment was well tolerated and did not determine any relevant change in vital signs nor ECG. CONCLUSIONS: From this study and literature data, it appears that exogenous chondroitin sulfate (Condrosulf) is absorbed as a high molecular mass polysaccharide together with derivatives resulting from a partial depolymerization and/or desulfation.

Administration, Oral↗
PubMed1998

Heparan sulfate oligosaccharides require 6-O-sulfation for promotion of basic fibroblast growth factor mitogenic activity.

The interaction of heparan sulfate (HS) with basic fibroblast growth factor (bFGF) is influential in enabling the growth factor to bind to its cell surface tyrosine kinase receptor. In this study, we have investigated further the structural properties of HS required to mediate the activity of bFGF in a mitogenic assay. We have prepared a library of heparinase III-generated HS oligosaccharides fractionated by both their size (dp6-dp12) and sulfate content. The ability of these oligosaccharides to activate bFGF in a mitogenic assay was then correlated with their length and disaccharide composition. All octa- and hexasaccharide fractions tested were unable to activate bFGF. Dodeca- and decasaccharide fractions were found to contain both activating and non-activating oligosaccharides, and showed a clear correlation between total sulfate content and the level of activatory activity. Disaccharide analysis of a range of dodeca- and decasaccharide fractions showed that both activating and non-activating oligosaccharides were composed mainly of N-sulfated and IdoA(2S)-containing disaccharides. The only significant difference between activating and non-activating oligosaccharides was the content of 6-O-sulfated disaccharides, in particular the disaccharide IdoA(2S)alpha1,4GlcNSO3(6S). These results show that there is a requirement for 6-O-sulfation of N-sulfated glucosamine residues, in addition to the 2-O-sulfation of IdoA, for the promotion of bFGF mitogenic activity by naturally occurring HS oligosaccharides. Analysis of the structure-activity relationships in the dodecasaccharide fractions in particular, suggests that a minimum bFGF activation sequence exists which is dependent on the positioning of at least one 6-O-sulfate group.

Cell Line↗
PubMed1996

Ion exchange HPLC microanalysis of chondroitin sulfate: quantitative derivatization of chondroitin lyase digestion products with 2-aminopyridine.

Sulfated glycosaminoglycans such as chondroitin sulfate are composed of three structural domains, a linkage oligosaccharide, connecting the chain to the core protein, a variably sulfated disaccharide repeat structure within the chain and a nonreducing terminal, and these domains may confer specific functions on particular chain populations. We report here a new and highly sensitive method for the detection and quantitation of all nonreducing terminal residues and internal disaccharides obtained by chondroitinase ABC or ACII digestion of aggrecan chondroitin sulfate. The procedure involves a quantitative reductive animation of the reducing ends of sulfated mono- and disaccharide chondroitinase products with 2-aminopyridine and boranedimethylamine. All derivatized saccharides can be separated and quantitated by fluorescence in a single chromatographic step on an AS4A anion exchange column, eluted with a gradient (0-500 nM) of sodium trifluoroacetate. The reproducibility and stability of the derivatisation, together with the sensitivity of the chromatography system, allowed for routine quantitation in the range of 3-500 pmol of reducing group (corresponding to about 1.5-250 ng of disaccharide or 0.75-125 ng of monosaccharide). Moreover, the fluorescence yield (fluorescence area units per pmol of reducing group) was virtually identical for all saccharides analyzed. Application of this method to an analysis of aggrecan purified from calf epiphyseal cartilage and from rat chondrosarcoma chondrocyte cultures allowed a precise identification and quantitation of the internal disaccharides and the nonreducing terminal structures, together with an estimation of the number average molecular weight of CS chains in these aggrecan preparations.

Aminopyridines↗
PubMed2004

Structural characterization of the bovine tracheal chondroitin sulfate chains and binding of Plasmodium falciparum-infected erythrocytes.

Sequestration of Plasmodium falciparum-infected red blood cells (IRBCs) in the human placenta is mediated by chondroitin 4-sulfate (C4S). A cytoadherence assay using chondroitin sulfate proteoglycans (CSPGs) is widely used for studying C4S-IRBC interactions. Bovine tracheal chondroitin sulfate A (CSA) preparation lacking a major portion of core protein has been frequently used for the assay. Here the CSPG purified from bovine trachea and CSA were assessed for IRBC binding and the CS chains studied in detail for structure-activity relationship. The IRBCs bound at significantly higher density to the CSPG than CSA. The CS chains of CSPG/CSA are heterogeneous with varying levels of 4- and 6-sulfates, which are distributed such that approximately 80% of the 4-sulfated disaccharides are present as single and blocks of two or three separated by one to three 6-sulfated disaccharides. The remainder of the 4-sulfated disaccharides is present in blocks composed of 4-12 units, separated by 6-sulfated disaccharides. In the IRBC adherence inhibition analysis, CSA fragments with 88%-92% 4-sulfate were significantly less inhibitory than the intact CSA, indicating that the regions consisting of shorter 4-sulfated blocks efficiently bind IRBCs despite the presence of relatively high levels of 6-sulfate. This is because the 6-sulfated disaccharides have unsubstituted C-4 hydroxyls that are crucial for IRBC binding. The presence of high levels of 6-sulfate, however, significantly interfere with the IRBC binding activity of CSA, which otherwise would more efficiently bind IRBCs. Thus our study revealed the distribution pattern of 4- and 6-sulfate in bovine tracheal CSA and structural basis for IRBC binding.

Animals↗
PubMed2002

Enhancement of t-PA-mediated plasminogen activation by partially defucosylated glycosaminoglycans from the sea cucumber Stichopus japonicus.

Sea cucumber glycosaminoglycan (SC-GAG) was isolated from the body wall of the sea cucumber Stichopus japonicus. The SC-GAG consists of a chondroitin sulfate E-type core polymer with sulfated fucose branches attaching glycosidically to almost every disaccharide unit of the core polymer at the C-3 position of the GlcA or at C-4 and/or C-6 position(s) of GalNAc. SC-GAG was subjected to mild acid-hydrolysis, which cleaved selectively the glycosidic linkages between the core polymer and the fucose branches, resulting in two types of partially defucosylated SC-GAG derivatives. One type (type A), obtained by 3 h-hydrolysis, contained 33% of the fucose branches and the other type (type B), obtained by 6-h hydrolysis, contained 10% of the fucose branches. The molecular masses of types A and B were determined to be 8 and 4 kDa, respectively, by gel permeation HPLC. A chondroitinase ABC (Chase ABC)-digestion demonstrated that types A and B contained 46 and 66% of digestable disaccharide units, respectively, and both types contained 29% of E-type unsaturated disaccharide units bearing no fucose branches. Intact SC-GAG and types A and B were compared for t-PA-mediated plasminogen activation by an in vitro assay system. Although intact SC-GAG and type B exhibited rather weak activity at 6.25 microg/ml, type A exhibited 5 to 10-fold higher activity than intact SC-GAG and type B at the same concentration. The activity of type A was almost one-third that of purified chondroitin sulfate E (127 kDa containing 64.5% E-type disaccharide units) from squid cartilage at 6.25 microg/ml concentration. These results suggest that t-PA-mediated plasminogen activation requires the presence of E-type disaccharide units bearing no fucose branches and a molecular mass larger than 7.5 kDa in terms of the chondroitin sulfate E structure with or without fucose branching.

Animals↗
PubMed1983

Structure of teichoic-acid--glycopeptide complexes from cell walls of Bacillus cereus AHU 1030.

From lysozyme digests of N-acetylated cell walls of Bacillus cereus AHU 1030, two acidic polymer fractions with molecular weights of about 24000 and 45000 were isolated by ion-exchange chromatography and gel chromatography. These polymer fractions, containing glycerol, phosphorus and glucose in a molar ratio of 1.00:1.00:0.85 together with small amounts of glycopeptide components and mannosamine, were characterized as teichoic-acid-glycopeptide complexes with one and two teichoic acid chains made of 60-65 repeating glycerol phosphate units that were mostly glucosylated. Mild alkali treatment of the complexes yielded a disaccharide-linked glycopeptide. The disaccharide was liberated from the glycopeptide by mild acid treatment and identified as N-acetylmannosaminyl(beta 1 leads to 4)N-acetylglucosamine. On the other hand, the same disaccharide linked to the teichoic acid chain was obtained by direct heating of the cell walls at pH 2.5. These results lead to a conclusion that in the cell walls of this strain the glycerol teichoic acid chain is attached to the glycan chain of peptidoglycan through this disaccharide unit. The disaccharide is linked at its reducing and nonreducing ends to the glycan chain and the teichoic acid chain, respectively, through phosphodiester bridges.

Bacillus cereus↗
PubMed1993

4-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-3-deoxy-D-manno-2-octulosonic acid, a constituent of lipopolysaccharides of the genus Pectinatus.

A disaccharide containing GlcN and 3-deoxy-D-manno-2-octulosonic acid (Kdo) was detected after acidic methanolysis and peracetylation of hydrofluoric-acid-treated smooth-type and rough-type lipopolysaccharide of Pectinatus cerevisiiphilus and Pectinatus frisingensis, which are strictly anaerobic bacteria capable of growing in packaged beer. The disaccharide was also found in alkali-treated lipopolysaccharide, but was not directly detectable from intact lipopolysaccharide. This suggested that the disaccharide carried a phosphate residue. The position of this phosphate was shown, by GLC/MS of appropriately degraded and derivatized samples, to be O6 of the GlcN. Methylation analysis of the purified disaccharide revealed that GlcN was linked to position 4 of Kdo. The acetylated derivative of the disaccharide was isolated in pure form, and, by 1H-NMR and 13C-NMR spectroscopy, it was confirmed to possess the structure alpha-D-GlcpN-(1'-->4)-Kdo. In the lipopolysaccharide the amino group of GlcN is free.

Carbohydrate Sequence↗
PubMed2004

Trehalose synthase of Mycobacterium smegmatis: purification, cloning, expression, and properties of the enzyme.

Trehalose synthase (TreS) catalyzes the reversible interconversion of trehalose (glucosyl-alpha,alpha-1,1-glucose) and maltose (glucosyl-alpha1-4-glucose). TreS was purified from the cytosol of Mycobacterium smegmatis to give a single protein band on SDS gels with a molecular mass of approximately 68 kDa. However, active enzyme exhibited a molecular mass of approximately 390 kDa by gel filtration suggesting that TreS is a hexamer of six identical subunits. Based on amino acid compositions of several peptides, the treS gene was identified in the M. smegmatis genome sequence, and was cloned and expressed in active form in Escherichia coli. The recombinant protein was synthesized with a (His)(6) tag at the amino terminus. The interconversion of trehalose and maltose by the purified TreS was studied at various concentrations of maltose or trehalose. At a maltose concentration of 0.5 mm, an equilibrium mixture containing equal amounts of trehalose and maltose (42-45% of each) was reached during an incubation of about 6 h, whereas at 2 mm maltose, it took about 22 h to reach the same equilibrium. However, when trehalose was the substrate at either 0.5 or 2 mm, only about 30% of the trehalose was converted to maltose in >or= 12 h, indicating that maltose is the preferred substrate. These incubations also produced up to 8-10% free glucose. The K(m) for maltose was approximately 10 mm, whereas for trehalose it was approximately 90 mm. While beta,beta-trehalose, isomaltose (alpha1,6-glucose disaccharide), kojibiose (alpha1,2) or cellobiose (beta1,4) were not substrates for TreS, nigerose (alpha1,3-glucose disaccharide) and alpha,beta-trehalose were utilized at 20 and 15%, respectively, as compared to maltose. The enzyme has a pH optimum of about 7 and is inhibited in a competitive manner by Tris buffer. [(3)H]Trehalose is converted to [(3)H]maltose even in the presence of a 100-fold or more excess of unlabeled maltose, and [(14)C]maltose produces [(14)C]trehalose in excess unlabeled trehalose, suggesting the possibility of separate binding sites for maltose and trehalose. The catalytic mechanism may involve scission of the incoming disaccharide and transfer of a glucose to an enzyme-bound glucose, as [(3)H]glucose incubated with TreS and either unlabeled maltose or trehalose results in formation of [(3)H]disaccharide. TreS also catalyzes production of a glucosamine disaccharide from maltose and glucosamine, suggesting that this enzyme may be valuable in carbohydrate synthetic chemistry.

Amino Acid Sequence↗
PubMed1976

Problems in anlysis of faecal sugar.

Significant amounts of sugar were found in 22% of 180 faecal samples from 135 children with acute or chronic diarrhoea. The methods used were the Clinitest method and paper chromatography. There was very good correlation between the results of these methods. Screening by Ph was less reliable. Various di- and monosaccharides were found. However, a disaccharide was never found without the simultaneous finding of its component monosaccharides. In vitro studies showed that the faecal flora has the ability to split disaccharides very rapidly. Within a few minutes much of the disaccharide had been split and no traces could be found after 30 minutes. Since the same process is assumed to take place in the lower gut, children with disacchardase deficency cannot be expected to excrete disaccharide alone in their faeces without the corresponding monosaccharides. The lack of a disaccharide in the faeces does not exclude the possibility of disaccharidase deficiency. Acid hydrolysis of faecal samples in cases of suspected sucrase deficiency seems not to be necessary.

Carbohydrates↗
PubMed1986

Intestinal mucosal mast cells from rats infected with Nippostrongylus brasiliensis contain protease-resistant chondroitin sulfate di-B proteoglycans.

Rats infected with the helminth Nippostrongylus brasiliensis were injected i.p. with 2 mCi of [35S] sulfate on days 13, 15, 17, and 19 after infection. The intestines were removed from animals on day 20 or 21 after infection, the intestinal cells were obtained by collagenase treatment and mechanical dispersion of the tissue, and the 35S-labeled mucosal mast cells (MMC) were enriched to 60 to 65% purity by Percoll centrifugation. The cell-associated 35S-labeled proteoglycans were extracted from the MMC-enriched cell preparation by the addition of detergent and 4 M guanidine HCl and were partially purified by density gradient centrifugation. The isolated proteoglycans were of approximately 150,000 m.w., were resistant to pronase degradation, and contained highly sulfated chondroitin sulfate side chains. Analysis by high-performance liquid chromatography of chondroitinase ABC-treated 35S-labeled proteoglycans from these rat MMC revealed that the chondroitin sulfate chains consisted predominantly of disaccharides with the disulfated di-B structure (IdUA-2SO4----GalNAc-4SO4) and disaccharides with the monosulfated A structure (G1cUA----GalNAc-4SO4). The ratio of disaccharides of the di-B to A structure ranged from 0.4 to 1.6 in three experiments. Small amounts of chondroitin sulfate E disaccharides (GlcUA----GalNAc-4,6-diSO4) were also detected in the chondroitinase ABC digests of the purified rat MMC proteoglycans, but no nitrous acid-susceptible heparin/heparan sulfate glycosaminoglycans were detected. The presence in normal mammalian cells of chondroitin sulfate proteoglycans that contain such a high percentage of the unusual disulfated di-B disaccharide has not been previously reported. The rat intestinal MMC proteoglycans are the first chondroitin sulfate proteoglycans that have been isolated from an enriched population of normal mast cells. They are homologous to the chondroitin sulfate-rich proteoglycans of the transformed rat basophilic leukemia-1 cell and the cultured interleukin 3-dependent mouse bone marrow-derived mast cell, in that these chondroitin sulfate proteoglycans as well as rat serosal mast cell heparin proteoglycans are all highly sulfated, protease-resistant proteoglycans.

Animals↗
PubMed1995

Specificity of glycopeptide-specific T cells.

We examined the specificity of glycopeptide-specific CD4 T cells following procedures similar to those previously reported by us. The disaccharide galabiose (Gal alpha 1-4Gal) was attached to the middle of the 52-61 peptide of hen egg lysozyme. This peptide is well known to bind to I-Ak molecules. CBA/J mice were immunized and T cell hybridomas were derived from the popliteal lymph node T cells. For this study, we selected hybridomas that recognized galabiose conjugated to 52-61 at residue Ser 56. We demonstrate here that these hybridomas showed specificity for galabiose and not cellobiose (Glc beta 1-4Glc). Peptides containing galabiose at residue 53 did not stimulate the T cell hybridomas and neither did galabiose conjugated to the 34-45 peptide of HEL. Acetylation of the hydroxyl groups of the disaccharide resulted in loss of T cell reactivity. These results need to be contrasted with those in which the T cells were directed to galabiose, attached to the amino terminus of 52-61 or to Ser at residue 53. With these results, the fine specificity of recognition of the disaccharide was not apparent. Our results indicate two sets of glycopeptide-specific T cells. One is probably induced by a conformational change induced by the disaccharide on the peptide bound to class II MHC molecules. The second set contains elements of specificity for both the disaccharide and the peptide.

Amino Acid Sequence↗
PubMed2002

The bioavailability and pharmacokinetics of glucosamine hydrochloride and low molecular weight chondroitin sulfate after single and multiple doses to beagle dogs.

OBJECTIVE: The purpose of this study was to determine the oral bioavailability and pharmacokinetics of a glucosamine (GL) and the disaccharides of chondroitin sulfate (CS) after single and multiple-dosing of a GL/CS combination (Cosamin, Cosequin). METHODS: Male beagle dogs (n = 8, 12 kg) received the following treatments: (1) IV GL (500 mg)/CS (400 mg), (2) p.o. GL (1500 mg)/CS (1200 mg), (3) p.o. GL (2000 mg)/CS (1600 mg), (4) p.o. GL (1500 mg)/CS (1200 mg) QD for days 1-7 and p.o. GL (3000 mg)/CS (2400 mg) from days 8 to 14. Blood samples were collected over 24 h and glucosamine and the disaccharides of chondroitin sulfate were determined. Pharmacokinetic analysis was performed on glucosamine and total chondroitin sulfate disaccharides and parameters were compared across treatments using ANOVA with post hoc analysis. RESULT: After the IV administration, glucosamine declined rapidly in a bi-exponential fashion with a mean (+/- S.D.) elimination t(1/2) of 0.52 (0.25) h. GL absorption was relatively fast (C(max) = 8.95 microg/ml, and T(max) 1.5 h after 1500 mg dose) and the mean bioavailability of glucosamine after single dosing was approximately 12%. The extent of absorption of chondroitin sulfate as indicated by the mean C(max) (21.5 microg/ml) and mean AUC (187 microg/ml h) of total disaccharides after dosing (1600 mg) provides evidence that chondroitin sulfate is absorbed orally. The bioavailability of CS ranged from 4.8 to 5.0% after single dosing and 200-278% upon multiple dosing. CONCLUSION: The results of this study show that both glucosamine and chondroitin sulfate (measured as total disaccharides) are bioavailable after oral dosing. In addition, the low molecular weight chondroitin sulfate used in this study displays significant accumulation upon multiple dosing.

Administration, Oral↗
PubMed2001

Analytical method of chondroitin/dermatan sulfates using high performance liquid chromatography/turbo ionspray ionization mass spectrometry: application to analyses of the tumor tissue sections on glass slides.

We established a highly sensitive quantitative analytical method for chondroitin/dermatan sulfates by LC/MS method. By this method, the unsaturated disaccharides produced after the enzymatic digestion of chondroitin/dermatan sulfates can be determined in the amounts as low as 0.5 pmol levels. The use of tetrabutylammonium hydroxide as an ion-pair reagent for LC/MS allowed us to separate unsaturated 4-sulfated disaccharide and unsaturated 6-sulfated disaccharide. Furthermore, the peak areas of unsaturated disaccharides were increased almost 10 times by the postcolumn addition of acetonitrile. We applied this LC/MS method to the analyses of unsaturated disaccharides from chondroitin/dermatan sulfates in the tissues sections on glass slides, which were prepared from MethA tumor-bearing mice. This method brought about considerable reduction in the time distance from sample collection to preparation of analytical results.

Chondroitin Sulfates↗
PubMed1980

Enzymatic studies of urinary isomeric chondroitin sulfates from patients with mucopolysaccharidoses. The application of high performance liquid chromatography.

The high-performance liquid chromatographic (HPLC) method for the determination of unsaturated sulfated disaccharides is a comprehensive and reliable method which expedites ensymatic studies of isomeric chondroitin sulfates. Responses for these unsaturated disaccharides derived from urinary chondroitin sulfates were linear from 100 ng to 10 micrograms injected and good quantitation was obtained for 25 microliters or less of samples placed on the column. This method which is more sensitive and accurate than methods now being used has considerable potential for the chemical diagnosis of patients with mucopolysaccharidoses and for the clarification of glycosaminoglycan structure. The isomeric chondroitin sulfates in urines from patients with mucopolysaccharidoses were studied by enzyme digestion with chondroitinases followed by HPLC determination of the sulfated unsaturated disaccharides produced. Evaluation by HPLC of the unsaturated 4-sulfated disaccharide produced by digestion of the urinary GAG with chondroitinases ABC and AC revealed rapidly and quantitatively the large amounts of dermatan sulfate present in Hurler, Hunter, and Maroteaux-Lamy urines. Chondroitin 4-sulfate predominated in Sanfilippo urinary isomeric chondroitin sulfates whereas chondroitin 6-sulfate and chondroitin 4-sulfate were shown to be present in nearly equal amounts in Morquio urine. An oversulfated chondroitin sulfate was detected in small amounts in some of these urines. This was demonstrated by the detection of an unsaturated disulfated disaccharide after digestion with chondroitinase ABC but not with chondroitinase AC.

Chondroitin↗
PubMed1981

Radiolabelled oligosaccharides as substrates for the estimation of sulfamidase and the detection of the Sanfilippo type A syndrome.

(1) A series of tritiated oligosaccharides, 2-sulfamino-2-deoxy-D-[1-14C]glucose (GlcNS) and [sulfamino-34S]heparin were evaluated as substrates for sulfamidase present in cultured human skin fibroblasts. (2) The following radiolabelled disaccharides were prepared from heparin: O-(alpha-2-sulfamino-2-deoxy-D-glucopyranosyl)-(1 leads to 3)-L-[6,3H]idonic acid (GlcNS-IdOA) and O-(alpha-2-sulfamino-2-deoxy-D-glucopyranosyl)-(1 leads to 3)-2,5 anhydro-L-[6,3H]idonic acid (HlcNS-anIdOA). Other radiolabelled oligosaccharides evaluated as sulfamidase substrates were the disaccharides O-(alpha-2-sulfamino-2-deoxy-D-glucopyranosyl)-(1 leads to 4)-L-[6,3H]idose (GlcNS-Ido) and O-(alpha-2-sulfamino-2-deoxy-D-glucopyranosyl)-)1 leads to 4)-L-[6,3H]idose 2-sulfate (GlcNS-Ido(OS)) and a preparation containing the tetrasaccharide GlcNS-UA-GlcNS-l-idonic acid, GlcNS-UA-GlcNS-anhydro-L-idonic acid and GlcNS-UA-GlcNS-L-gulonic acid. (3) Sulfamidase activity assessed with GlcNS-IdOA and GlcNS-anIdOA were approximately equal and up to 4, 8 and 800 times higher than the value obtained using [sulfamino-35S]heparin, GlcNS-Ido(OS) and GlcNS-Ido respectively. Under the assay conditions used GlcNS was not de-N-sulfated. These results demonstrate that C6 carboxyl and C2 sulfate ester groups on the adjacent residue to the sulfaminoglucosamine moiety are important structural requirements in the mechanism of action or binding of sulfamidase toward N-sulfated disaccharides. The results obtained for a partially characterized mixture of tetrasaccharides suggest that they are degraded four times faster than their disaccharide structural counterparts. (4) No detectable sulfamidase activity toward [sulfamino-35S]heparin, monosaccharide, disaccharide or tetrasaccharide substrates could be detected using homogenates of fibroblast cultures from Sanfilippo A patients (sulfamidase deficient). (5)sulfamidase activity measured with GlcNS-IdOA exhibited a pH optimum at 4.5 to 5.5, an apparent Km of approximately 220 mumol/l and potent inhibition by sulfate ions.

Cell Line↗