Characterization of a heparan sulfate and a peculiar chondroitin 4-sulfate proteoglycan from platelets. Inhibition of the aggregation process by platelet chondroitin sulfate proteoglycan

A high molecular weight chondroitin sulfate proteoglycan (Mr 240,000) is released from platelet surface during aggregation induced by several pharmacological agents. Some details on the structure of this compound are reported. beta-Elimination with alkali and borohydride produces chondroitin sulfate chains with a molecular weight of 40,000. The combined results indicate a proteoglycan molecule containing 5-6 chondroitin sulfate chains and a protein core rich in serine and glycine residues. Degradation with chondroitinase AC shows that a 4-sulfated disaccharide is the only disaccharide released from this chondroitin sulfate, characterizing it as a chondroitin 4-sulfate homopolymer. It is shown that this proteoglycan inhibits the aggregation of platelets induced by ADP. Analysis of the sulfated glycosaminoglycans not released during aggregation revealed the presence of a heparan sulfate in the platelets. Degradation by heparitinases I and II yielded the four disaccharide units of heparan sulfates: N,O-disulfated disaccharide, N-sulfated disaccharide, N-acetylated 6-sulfated disaccharide, and N-acetylated disaccharide. The possible role of the sulfated glycosaminoglycans on cell-cell interaction is discussed in view of the present findings.     

Distribution of chondroitin sulfate in cartilage proteoglycans under associative conditions.

Proteoglycan aggregates and proteoglycan subunits were extracted from bovine articular cartilage with guanidine-HC1 folowed by fractionation by equilibrium centrifugation in cesium chloride density gradients. The distribution of chondroitin sulfates (CS) in the cartilage proteoglycans was studied at the disaccharide level by digestion with chondroitinases. In the proteoglycan aggregate fraction, it was observed that the proportion of 4-sulfated disaccharide units to total CS increased from the bottom to the top fractions, whereas that of 6-sulfated disaccharide units was in the reverse order. Thus, the ratio of 4-sulfated disaccharide units to 6-sulfated disaccharide units increased significantly with decreasing density. The proportion of non-sulfated disaccharide units to total CS tended to increase with increasing density. These data indicate a polydisperse distribution of CS chains, under the conditions used here, in proteoglycan aggregates from bovine articular cartilage.     

Structure of chondroitin sulfates analyses of the products formed from chondroitin sulfates A and C by the action of the chondroitinases C and AC from Flavobacterium heparinum

The structures of chondroitin sulfate A from whale cartilage and chondroitin sulfate C from shark cartilage have been examined with the aid of the chondroitinases AC and C from Flavobacterium heparinum. The analyses of the products formed from the chondroitin sulfates by the action of the chondroitinases have shown that three types of oligosaccharides compose the structure of chondroitin sulfate A, namely, a dodeca-, hexa- and a tetra-saccharide, containing five, two and one 4-sulfated disaccharides per 6-sulfated disaccharide residue, respectively. The polymer contains an average of 3 mol of each oligosaccharide per mol of chondroitin sulfate A. Each mol of chondroitin sulfate C contains an average of 5 mol of 4-sulfated disaccharide units. A tetra-saccharide containing one 4-sulfated disaccharide and one 6-sulfated disaccharide was isolated from this mucopolysaccharide by the action of the chondroitinase C, indicating that the 4-sulfated disaccharides are not linked together in one specific region but spaced in the molecule.     

The Engelbreth-Holm-Swarm mouse tumor produces undersulfated heparan sulfate and oversulfated galactosaminoglycans

Glycosaminoglycans were prepared from the Engelbreth-Holm-Swarm mouse tumor. Enzymatic analysis demonstrated heparan sulfate (95.8%) and chondroitinase ABC-sensitive galactosaminoglycans (4.2%). HPLC analysis of the disaccharide units showed that heparan sulfate chains were undersulfated on average, comprising approximately 30% nonsulfated and 60% N-sulfated disaccharide units with small proportions of other monosulfated and disulfated disaccharide units. In contrast, galactosaminoglycan chains were oversulfated, containing an appreciable proportion (15%) of a 4,6-disulfated (so-called E-type) disaccharide unit in addition to 51% of a 4-sulfated, 22% of a 6-sulfated, and 11% of a nonsulfated disaccharide unit. The significance of the oversulfated disaccharide structure is discussed in relation to the possible regulation of functions of hybrid proteoglycans from which the galactosaminoglycan chains are derived.     

The low sulfated chondroitin sulfate proteoglycans of human placenta have sulfate group-clustered domains that can efficiently bind Plasmodium falciparum-infected erythrocytes

Plasmodium falciparum infection in pregnant women results in the chondroitin 4-sulfate-mediated adherence of the parasite-infected red blood cells (IRBCs) in the placenta, adversely affecting the health of the fetus and mother. We have previously shown that unusually low sulfated chondroitin sulfate proteoglycans (CSPGs) in the intervillous spaces of the placenta are the receptors for IRBC adhesion, which involves a chondroitin 4-sulfate motif consisting of six disaccharide moieties with approximately 30% 4-sulfated residues. However, it was puzzling how the placental CSPGs, which have only approximately 8% of the disaccharide 4-sulfated, could efficiently bind IRBCs. Thus, we undertook to determine the precise structural features of the CS chains of placental CSPGs that interact with IRBCs. We show that the placental CSPGs are a mixture of two major populations, which are similar by all criteria except differing in their sulfate contents; 2-3% and 9-14% of the disaccharide units of the CS chains are 4-sulfated, and the remainder are nonsulfated. The majority of the sulfate groups in the CSPGs are clustered in CS chain domains consisting of 6-14 repeating disaccharide units. While the sulfate-rich regions of the CS chains contain 20-28% 4-sulfated disaccharides, the other regions have little or no sulfate. Further, we find that the placental CSPGs are able to efficiently bind IRBCs due to the presence of 4-sulfated disaccharide clusters. The oligosaccharides corresponding to the sulfate-rich domains of the CS chains efficiently inhibited IRBC adhesion. Thus, our data demonstrate, for the first time, the unique distribution of sulfate groups in the CS chains of placental CSPGs and that these sulfate-clustered domains have the necessary structural elements for the efficient adhesion of IRBCs, although the CS chains have an overall low degree of sulfation.     

Age-dependent constitution of chondroitin sulfate isomers in cartilage proteoglycans under associative conditions.

Associated proteoglycans were prepared with guanidine-HCl from bovine articular cartilage of various ages. They were purified and fractionated by equilibrium centrifugation in cesium chloride (CsCl) density gradients. The compositions of chondroitin sulfate (CS) isomers in associated proteoglycans of articular cartilages of three ages were compared based on the relative amounts of disaccharide units. The results indicated that the proportions of 4-sulfated disaccharide units comprised around 2/3, 1/3, and 1/6 of the total CS in the associated proteoglycans of calf, 18-month-old cow, and 8-year-old cow, respectively. In contrast, the proportions of 6-sulfated disaccharide units in the proteoglycans were in the reverse order; they comprised nearly 1/3, 1/2, and 2/3 of the total CS, respectively, at the three ages. Thus, with increasing age, the ratio of 4-sulfated disaccharide units to 6-sulfated disaccharide units decreased significantly. With the decrease of CsCl density in the gradients, the proportion of 4-sulfated disaccharide units to total CS as well as that of 4-sulfated disaccharide units to 6-sulfated disaccharide units increased in the associated proteoglycans of all ages. The increased ratios of 4-sulfated to 6-sulfated disaccharide units with decreasing CsCl density were significant among the individual proteoglycans: 1.84-2.36 in calf, 0.40-0.89 in 18-month-old cow, and 0.16-0.28 in 8-year-old cow.     

Immunocytochemical Localization of Chondroitin and Chondroitin 4- and 6-Sulfates in Developing Rat Cerebellum

Monoclonal antibodies specific for unsulfated, 4-sulfated, and 6-sulfated disaccharide "stubs" that remain attached to the core protein after chondroitinase ABC digestion of chondroitin/dermatan sulfate proteoglycans have been used to study the localization of chondroitin and the two isomeric chondroitin sulfates in developing rat cerebellum. At 1-2 weeks postnatal, unsulfated chondroitin is present in the granule cell layer, molecular layer, and prospective white matter, but there was no staining of the external granule cell layer other than light staining of Bergmann glia fibers. By 3 weeks postnatal, staining of the molecular layer has disappeared and has diminished in the white matter, whereas in adult cerebellum only the granule cell layer remains stained. The staining pattern of chondroitin 4-sulfate is similar to that for chondroitin at 1-2 weeks postnatal, but in contrast to chondroitin, chondroitin 4-sulfate increases in the molecular layer at 3 weeks, and this becomes the most densely stained region of adult cerebellum. Chondroitin 6-sulfate is present predominantly in the prospective white matter of 1-2 week postnatal cerebellum, although significant staining of the granule cell layer is also seen. By 3 weeks postnatal the granule cell staining of chondroitin 6-sulfate has decreased, and in adult cerebellum staining is seen only in the white matter and to a lesser extent in the granule cell layer. Electron microscopy confirmed the presence of chondroitin sulfate in the cytoplasm of neurons and glia of adult brain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human colon adenocarcinoma is associated with specific post-translational modifications of versican and decorin

In this study, the amounts and the fine structural characteristics of versican and decorin present in human colon adenocarcinomas (HCC) were investigated and compared with those in human normal colon (HNC). HCC is characterized by significant increase in the amounts of versican and decorin (13- and 8-fold in terms of protein, respectively). These two proteoglycans (PGs) were the predominant in HCC (86% of total uronic acid). In HNC, versican and decorin contained both chondroitin sulfate/dermatan sulfate chains (CS/DS), with DS to be the predominant one (90-93%). The molecular sizes (M(r)s) estimated for DS and CS chains were 25-28 and 21-28 kDa, respectively. In CS/DS chains isolated from both versican and decorin, 4-sulfated disaccharides accounted for 79-86% of total disaccharide units, respectively, whereas lower amounts of 6- and non-sulfated units were also recorded. In contrast, the tumor-associated versican and decorin were of smaller hydrodynamic size with lower glycosaminoglycan (GAG) content per PG molecule as compared with those found in HNC. In HCC, both PGs contained mainly CS chains (up to 86%) and the M(r)s of CS and DS chains were also found to be of smaller size (12 and 16 kDa, respectively). The sulfation patterns of CS/DS chains from both PGs were also significantly different. They were composed mainly of 6-sulfated disaccharides (63-70%), whereas 4-sulfated units accounted for 23-31%. A significant increase in the proportion of non-sulfated disaccharides was also recorded. These findings indicate that the colon adenocarcinoma is characterized by a remarkable increase in the concentration of versican and decorin. Furthermore, these PGs are significantly modified at the post-translational level, i.e. the type, length and the sulfation pattern of their GAG chains. These specific structural alterations of versican and decorin may influence the biology of cancer cells in HCC.     

Sulfation of chondroitin sulfate in human articular cartilage. The effect of age, topographical position, and zone of cartilage on tissue composition.

The chondroitin ABC lyase digestion products of normal human femoral condyle articular cartilage and of purified aggrecan were analyzed for their mono- and nonsulfated disaccharide composition. Changes in the total tissue chemistry were most pronounced during the period from birth to 20 years of age, when the -[GlcAbeta,3GalNAc6]- disaccharide content increased from approximately 50% to 85% of the total disaccharide content and there was a concomitant decrease in the content of the 4-sulfated disaccharide. In general, the disaccharide content of the deeper layers of immature cartilage were richer in the 4-sulfated residue than the upper regions of the tissue. As the tissue aged and decreased in thickness, the disaccharide composition became more evenly 6-sulfated. The newly synthesized chondroitin sulfate chains had a similar composition to the endogenous chains and also underwent the same age and zonal changes. The monoclonal antisera 3B3(+) and 2B6(+) were used to immunolocalize the unsaturated 6- and 4-sulfated residues generated at the reducing termini of the chondroitin sulfate chains by digestion with chondroitin ABC lyase, and these analyses indicated that the sulfation pattern at this position did not necessarily reflect the internal disaccharide composition of the chains. In summary, the sulfation pattern of chondroitin sulfate disaccharides from human normal articular cartilage varies with the age of the specimen, the position (topography) on the joint surface, and the zone of cartilage analyzed. Furthermore, these changes in composition are a consequence of both extracellular, post-translational processing of the core protein of aggrecan and changes in the sulfotransferase activity of the chondrocyte.     

Isolation of dermatan sulfate with high heparin cofactor II-mediated thrombin-inhibitory activity from porcine spleen

We prepared dermatan sulfate specimens from various porcine tissues, and compared their heparin cofactor II-mediated thrombin-inhibitory activities and chemical natures, including disaccharide composition. Electrophoresis of the specimens on cellulose acetate membrane indicated that spleen dermatan sulfate was the most acidic of the dermatan sulfates prepared from the various porcine tissues. Analysis of the disaccharide units of the dermatan sulfate specimens by high-performance liquid chromatography revealed that spleen dermatan sulfate was rich in 4,6-di-O-sulfated N-acetylgalactosamine residues as compared with those of the other tissues. Spleen dermatan sulfate exhibited the highest thrombin-inhibitory activity, which may be related to its high content of the disulfated N-acetylgalactosamine residue.     

Altered fine structures of corneal and skeletal keratan sulfate and chondroitin/dermatan sulfate in macular corneal dystrophy

The content and fine structure of keratan and chondroitin/dermatan sulfate in normal human corneas and corneas affected by macular corneal dystrophies (MCD) types I and II were examined by fluorophore-assisted carbohydrate electrophoresis. Normal tissues (n = 11) contained 15 microg of keratan sulfate and 8 microg of chondroitin/dermatan sulfate per mg dry weight. Keratan sulfates consisted of approximately 4% unsulfated, 42% monosulfated, and 54% disulfated disaccharides with number of average chain lengths of approximately 14 disaccharides. Chondroitin/dermatan sulfates were significantly longer, approximately 40 disaccharides per chain, and consisted of approximately 64% unsulfated, 28% 4-sulfated, and 8% 6-sulfated disaccharides. The fine structural parameters were altered in all diseased tissues. Keratan sulfate chain size was reduced to 3-4 disaccharides; chain sulfation was absent in MCD type I corneas and cartilages, and sulfation of both GlcNAc and Gal was significantly reduced in MCD type II. Chondroitin/dermatan sulfate chain sizes were also decreased in all diseased corneas to approximately 15 disaccharides, and the contents of 4- and 6-sulfated disaccharides were proportionally increased. Tissue concentrations (nanomole of chains per mg dry weight) of all glycosaminoglycan types were affected in the disease types. Keratan sulfate chain concentrations were reduced by approximately 24 and approximately 75% in type I corneas and cartilages, respectively, and by approximately 50% in type II corneas. Conversely, chondroitin/dermatan sulfate chain concentrations were increased by 60-70% in types I and II corneas. Such changes imply a modified tissue content of individual proteoglycans and/or an altered efficiency of chain substitution on the core proteins. Together with the finding that hyaluronan, not normally present in healthy adult corneas, was also detected in both disease subtypes, the data support the conclusion that a wide range of keratocyte-specific proteoglycan and glycosaminoglycan remodeling processes are activated during degeneration of the stromal matrix in the macular corneal dystrophies.     

Immunoelectron microscopic analysis of chondroitin sulfates during calcification in the rat growth plate cartilage

The proximal growth plate cartilage of rat tibia was fixed in the presence of ruthenium hexamine trichloride (RHT) in order to preserve proteoglycans in the tissue. Quantitative changes of chondroitin sulfates during endochondral calcification were investigated by immunoelectron microscopy using mouse monoclonal antibodies 1-B-5, 2-B-6, and 3-B-3, which recognize unsulfated, 4-sulfated, and 6-sulfated chondroitin sulfates, respectively. The content of chondroitin-4-sulfate in the cartilage matrix increased from the proliferative zone to the calcifying zone, while that of unsulfated chondroitin sulfate decreased. Chondroitin-6-sulfate remained constant from the proliferative zone to the upper hypertrophic zone, then decreased in the calcifying zone. The immunoreaction to each antibody increased conspicuously in the cartilagenous core of metaphysial bone trabeculae. The changes of sulfation in chondroitin sulfate chains of proteoglycans may play an important role in inducing and/or promoting calcification in growth plate cartilage.     

Purification and characterization of dermatan sulfate from the skin of the eel,Anguilla japonica

Glycosaminoglycans were isolated from the eel skin (Anguilla japonica) by actinase and endonuclease digestions, followed by a beta-elimination reaction and DEAE-Sephacel chromatography. Dermatan sulfate was the major glycosaminoglycan in the eel skin with 88% of the total uronic acid. The content of the IdoA2Salpha1-->4GalNAc4S sequence in eel skin, which shows anticoagulant activity through binding to heparin cofactor II, was two times higher than that of dermatan sulfate from porcine skin. The anti-IIa activity of eel skin dermatan sulfate was determined to be 2.4 units/mg, whereas dermatan sulfate from porcine skin shows 23.2 units/mg. The average molecular weight of dermatan sulfate was determined by gel chromatography on a TSKgel G3000SWXL column as 14 kDa. Based on 1H NMR spectroscopy, the presence of 3-sulfated and/or 2,3-sulfated IdoA residues was suggested. The reason why highly sulfated dermatan sulfate does not show anticoagulant activity is discussed. In addition to dermatan sulfate, the eel skin contained a small amount of keratan sulfate, which was identified by keratanase treatment.     

Characterization of a low-sulfated chondroitin sulfate from the body of Viviparus ater (mollusca gastropoda). Modification of its structure by lead pollution

A chondroitin sulfate was purified from the body of Viviparus ater (Mollusca gastropoda) and analyzed for molecular mass, constituent disaccharides, and structure by 1H NMR and 1H 2D NMR. A quite unique glycosaminoglycan species was isolated having a high molecular mass (greater than 45,000) and low charge density, about 0.60, due to the presence of 42% non-sulfated disaccharide, 5% 6-sulfated disaccharide, 48% 4-sulfated disaccharide, and 5% 4,6-disulfated disaccharide. Specimens of Mollusca were also submitted to lead exposure for different times, and the effect on chondroitin sulfate structure was studied. After 96 h treatment a strong decrease in chondroitin sulfate content was observed with a significant modification of its structure producing a more desulfated polymer, in particular in position 4 of the galactosamine unit. Simultaneously, the amount of unsaturated non-sulfated disaccharide increased with an overall decrease of the charge density.     

Dermatan sulfate formation in gastrulae of the sea urchin Clypeaster japonicus

Gastrullation of sea urchin embryos is arrested in sulfate-free sea water. This developmental arrest has been considered to be due to lack of sulfation of glycosaminoglycans in the extracellular matrix of the embryos. In the present study, we characterized a dermatan sulfate type component formed in gastrula-stage embryos of the sea urchin Clypeaster japonicus and examined the effects of sulfate deprivation on the formation. Glycosamino-glycans were prepared from gastrula-stage embryos incubated with [3H]acetate in normal and sulfate-free sea water. Enzymatic analyses indicated that embryos formed a glycosaminoglycan of the dermatan sulfate type which contained an N-acetylgalactosamine-6-sulfate-containing disaccharide as a major unit, plus a minor unidentified component. Under sulfate-free conditions, embryos formed an under-sulfated chondroitin/dermatan sulfate copolymer which mainly consisted of non-sulfate, glucuronic acid-containing (chondroitin) disaccharide units. These results suggest that sulfate deprivation diminishes not only the degree of sulfation but also the formation of L-iduronic acid-containing (dermatan) disaccharide units in dermatan sulfate in sea urchin embryos.     

Structural differences of heparan sulfates according to the tissue and species of origin

Some structural features of thirteen heparan sulfates isolated from different mammalian tissues and species are reported. Two N-acetylated disaccharides, one of then O-sulfated and two N-sulfated disaccharides, one of then 6-sulfated are formed from these compounds by the combined action of heparitinases I and II from Flavobacterium heparinum. The relative proportions of the four disaccharide units vary quite significantly among the thirteen heparan sulfates indicating that the structure of these polymers are tissue and species specific. Based on the frequency of appearance of each one of the disaccharides it was calculated that 10(36) types of heparan sulfates might theoretically be found. The possible role of these polyanions in cell-cell recognition is discussed in view of the present findings.     

Proteoglycan synthesis by rat lung cells cultured in vitro

Cells having a fibroblast-like morphology were cultured from explants of adult rat lung tissue. (35S)Sulfate was incorporated into sulfated proteoglycans in the medium at a linear rate for up to 96 h, while the rate of incorporation into the cell layer increased gradually until reaching a plateau at 40 h. The culture medium contained proteoglycans which migrated as a single peak with Kav = 0.10 on Bio-Gel A-15. Their glycosaminoglycan components (Kav = 0.70 on Bio-Gel A-15) contained predominantly chondroitin sulfate (33 to 44% of the total galactosaminoglycans) or dermatan sulfate chains. Based on the results of chondroitinase AC-II and periodate degradation, disaccharide repeating units of the dermatan sulfate were composed of 36% iduronic acid, 50% 2-sulfoiduronate, and 14% glucuronic acid. A similar composition was found for the dermatan sulfate in the cell fraction. Almost one-half of the sulfate label in the cell fraction was in a heparan sulfate proteoglycan which migrated on Bio-Gel A-15 with Kav = 0.30. The heparan sulfate chains (Kav = 0.81 on Bio-Gel A-15) had few, if any, sulfated N-acetylglucosamine residues and did not contain 2-sulfoiduronic acid in neighboring disaccharide repeat sequences. These results indicate that fibroblast-like lung cells synthesize several types of multichain sulfated proteoglycans which have properties in common with those found in lung tissues.     

Diversity in the degree of sulfation and chain length of the glycosaminoglycan moiety of urinary trypsin inhibitor isomers

Five isomers with different electric charge were fractionated from human urinary trypsin inhibitor (UTI) by anion exchange HPLC. Intact low-sulfated chondroitin 4-sulfate chains from the isomers were analyzed by HPLC and mass spectrometry. Unsaturated disaccharide composition analysis of the chondroitin sulfate chain revealed that the five isomers differ in the numbers of 4-sulfated disaccharide units. Intriguingly, we detected the presence of multiple novel isomers with different numbers of non-sulfated disaccharide units even in the same charge isomer fraction. Our results demonstrate that UTI can vary in terms of both the degree of sulfation and the length of the low-sulfated chondroitin 4-sulfate chain.     

Structure of a fucose-branched chondroitin sulfate from sea cucumber. Evidence for the presence of 3-O-sulfo-beta-D-glucuronosyl residues

The structure of a unique focose-branched chondroitin sulfate isolated from the body wall of a sea cucumber was examined in detail. This glycosaminoglycan contains side chain disaccharide units of sulfated fucopyranosyl units linked to approximately one-half of the glucuronic acid moieties through the O-3 position of the acid. The intact polysaccharide is totally resistant to chondroitinase degradation, whereas, after defucosylation, it is partially degraded by the enzyme. However, only after an additional step of desulfation, the chondroitin from sea cucumber is almost totally degraded by chondroitinase AC or ABC. This result, together with the methylation and NMR studies of the native and chemically modified polysaccharide, suggest that besides the fucose branches, the sea cucumber chondroitin sulfate contains sulfate esters at position O-3 of the beta-D-glucuronic acid units. Furthermore, the proteoglycan from the sea cucumber chondroitin sulfate is recognized by anti-Leu-7 monoclonal antibody, which specifically recognizes 3-sulfoglucuronic acid residues. In analogy with the fucose branched units, the 3-O-sulfo-beta-D-glucuronosyl residues are resistant to chondroitinase degradation. Regarding the position of the glycosidic linkage and site of sulfation in the fucose branches, our results suggest high heterogeneity. Tentatively, it is possible to suggest the preponderance of disaccharide units formed by 3,4-di-O-sulfo-alpha-L-fucopyranosyl units glycosidically linked through position 1----2 to 4-O-sulfo-alpha-L-fucopyranose. Finally, the presence of unusual 4/6-disulfated disaccharide units, together with the common 6-sulfated and non-sulfated units, was detected in the chondroitin sulfate core of this polysaccharide.     

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.