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Renal involvement in systemic lupus erythematosus (SLE) affects the disease outcome. In order to advance the diagnosis and the initiation of therapy, non-invasive diagnostic techniques are required. In this study, urinary glycosaminoglycans (GAG) and heparan sulphate (HS) were measured in 26 patients with biopsy-proven lupus nephritis and compared to 16 healthy controls. Uronic acid as a representative of GAGs in urine was determined spectrophotometrically with the meta-hydroxydiphenyl, following acid treatment. HS was determined as hexosamine by the method of Smith and Gilkerson. The median values of GAG (3.99 mg/g crea./day) and HS (2.41 mg/g crea./day) in patients were significantly ( P = 0.001) higher than in the control group (1.98 and 0.87, respectively). There was a positive correlation between GAG and HS values ( P = 0.000, r = 0.924) in SLE patients. There were no differences in HS excretion, microalbuminuria and SLE-DAI scores between different classes of lupus nephritis. However, GAG values in class 3 nephritis were significantly ( P = 0.033) higher than from both class 2 and class 4 lupus nephritis. There were no differences in all the measured parameters between normoalbuminuric, microalbuminuric and macroproteinuric patients. Furthermore, there were no correlations between GAG, HS excretions and SLE-DAI scores or microalbuminuria. These results suggest that urinary GAG and HS may serve as useful, independent and non-invasive markers of lupus nephritis.

Domain V of the major basement-membrane proteoglycan perlecan, a domain which consists of three laminin type G (LG) and four epidermal-growth-factor-like (EG) modules, was obtained in recombinant form by transfecting embryonic kidney cells with an episomal expression vector. A major 90-kDa fragment V was obtained together with fragments Va (74 kDa) and Vb (26 kDa) which were generated by endogenous proteolysis in front of the most C-terminal LG module. All three fragments bound to a heparin affinity column and could be displaced at a moderate (0.2 M) NaCl concentration. Rotary-shadowing electron microscopy demonstrated a three-globule structure for fragment V. Fragment V also showed a strong immunological cross-reaction with tissue-derived perlecan, indicating that it was folded into a native structure. A further, larger fragment, Vc, was apparently substituted with heparan sulphate and/or chondroitin sulphate chains and failed to bind to heparin. Fragment V but not fragment Vc promoted a distinct adhesion of several cell lines and this could be blocked by antibodies against the integrin beta1 chain. This domain may, however, represent only one of several cell-adhesive sites of perlecan. The recombinant perlecan fragment V bound in surface plasmon resonance assays to fibulin-2, laminin-nidogen complex, nidogen and two nidogen fragments. This indicated two different nidogen-binding epitopes on perlecan domain V with about a 10-fold difference in their affinities (Kd = 0.05-0.2 microM and about 2 microM). Perlecan domain V therefore seems to participate in the supramolecular assembly and cell connections of basement membranes.

The qualitative and quantitative pattern of GAGs was examined by electrophoresis in aging chicken brain in four different stages starting from day 1 to 30 months. GAG content referred to defatted dry tissue exhibits constant decrease. Four main GAGs have been identified with a mobility corresponding to hyaluronate, condroitin sulfate, heparan sulfate and dermatan sulfate controls. Hyaluronate appears the main GAG represented while dermatan sulfate the minor one. Our data show that in chicken brain GAG percentage undergoes age-related changes.

The proteoglycans (PGs) and glycosaminoglycans (GAGs) of buffalo ovarian follicular fluid (FF) have been studied in small (2-4.9 mm), medium (5-9.9 mm) and large (> or = 10 mm) follicles. GAGs in different categories of follicles were isolated, assayed and analysed. On the basis of hexosamine analysis, glucosamine accounted for all the free GAGs in FF of small and medium follicles. No free GAG was found in large follicles. The concentration of GAGs in the form of PGs decreased significantly with follicular maturation. Qualitative analysis of GAGs from PGs showed higher galactosamine than glucosamine. The ratios of GalNH2:GluNH2 and neutral sugars were highest in small follicles followed by medium and large follicles. On the other hand, the percentage of sialic acid in GAGs was highest in large follicles followed by medium and small follicles. The fractionation of PGs by gel filtration indicated the presence of two types of PGs in buffalo ovarian FF. Difference in distribution of two types of PGs in small and large follicles was also noted.

The effect of reactive oxygen species (ROS) on the chemical structure of glycosaminoglycans (GAG) was studied in order to consider their role in connective tissue damage during an inflammatory disease state such as periodontal disease. GAG were exposed to a radical generating system for 1 h and analysed by gel filtration for fragmentation and chemically with respect to uronic acid, hexosamine and sulfate content. Non-sulfated GAG, hyaluronan and chondroitin, were most susceptible to depolymerisation and chemical modification of uronic acid and hexosamine residues by ROS. Depolymerisation and chemical modification of sulfated GAG, chondroitin 4-sulfate, dermatan sulfate and heparan sulfate was significantly less than for non-sulfated GAG. The highly sulfated GAG heparin showed minimal depolymerisation by ROS, but uronic acid residues were readily modified. Analysis of the ROS-exposed residues suggests that uronic acid is capable of degrading to a 3-carbon aldehyde, malondialdehyde. Chondroitin sulfate exposed to ROS resulted in marginal desulfation. The results suggest that the presence of sulfate on the GAG chain may protect the molecule against ROS attack. However, chemical modification of GAG may affect proteoglycan function and be of importance in considering connective tissue destruction in a variety of pathological situations, including periodontal disease.

Glycosaminoglycan (GAG) hexosamines were measured after deacetylation (2 h acid hydrolysis), deaminative cleavage by nitrous acid, and coupling of the 2,5-anhydro sugars thus produced with 3,5-diaminobenzoic acid (DABA) to give a fluorescent product. Analyses were performed after the addition of an aliquot of potassium acetate solution to the acid hydrolysates, to adjust the pH. Results on a series of GAGs were compared with an Elson-Morgan (E-M) procedure. Our method is faster, more convenient, 10-20 times more sensitive, and always gave higher figures for hexosamine content. In particular it avoids long, destructive hydrolysis in concentrated strong acid and the complications of the Moggridge-Neuberger effect. Results agreed with calculations and titrimetric data. Using internal standards, our method is tolerant of cetylpyridinium chloride, peptides, and salts. A simple fluorometer easily handled less than 1 microgram GAG per sample in 1.0 ml volume. Results are available in 4-5 h. The method is suitable for automation. A strongly polycationic environment, as in chitosan, markedly slowed nitrous acid deamination, possibly because of Donnan-type exclusion from the domain of the polycation of the cationic nitrosonium species. Whereas all other alpha- and beta-hexosaminides gave yield-hydrolysis time profiles that peaked in approximately 1 h in the DABA method, the E-M profiles, particularly for 2-sulfamato-alpha-hexosaminides, took longer and/or achieved lower optimal yields. All the usually encountered forms of the 2-amino-2-deoxy group (free, acetylated, sulfamato) are readily assayed using the same reagents, with appropriate prefluorometric stages.

Glycosaminoglycan fractions isolated from either comb or wattle tissue were examined using ELISA for their antigenicities to AH12, a monoclonal antibody recognizing keratan sulfate. The results showed a positive antibody binding to a sulfated glycosaminoglycan fraction from either tissue. The treatment of the same fraction with keratan sulfate degrading enzyme, keratanase, or endo-beta-galactosidase resulted in a decrease in its antigenicity. These findings indicated the presence of AH12 epitope (keratan sulfate disaccharide) in both tissues.

Few details are available on the heterogeneity of glycosaminoglycans (GAGs) in healing fetal wound tissue. We used a sensitive assay for hexosamines to examine changes occurring in the development of normal sheep skin and of wound healing tissue in PVA sponges inserted subcutaneously at different stages of gestation. It was assumed that glucosamine was derived mainly from hyaluronan and galactosamine mainly from dermatan sulphate and chondroitin sulphate. Hexosamine-containing tissue infiltrating the sponges was deposited more rapidly in the first week than in the second week. Three days after wounding, approximately 70% of the total GAGs in wound tissue was hyaluronan. The proportion of hyaluronan then fell progressively and by the 14th day contributed 57% to the total GAGs. In uninjured skin the contribution of hyaluronan to the total GAGs fell progressively with increasing fetal maturity, the level being 70% at 75 days gestation, but only 35-40% in newborn or adult skin. At no stage of development was there a sudden change in GAG composition suggestive of a transition from regeneration to scar formation. It is concluded that hyaluronan may play an important role in the biochemical sequence leading to collagen fibrillogenesis and mature scar formation.

Glycosaminoglycans (GAGs) are one of the important components of articular cartilage. The present study had determined the contents of uronic acid, galactomine, glucosamine, and hexosamine of temporomandibular joint discs in 10 dogs by Bitter's carbozole reaction method and derivative spectrophotometry. The data indicated that the contents of these components in different bands of the disc are not significantly different and that the discs of dogs contained averagely 0.6%, 0.987%, 0.4% and 1.4% of uronic acid, galactomine, glucosamine, and hexosamine respectively. The data also suggested that the discs contained keratan sulfate and chondroitin sulfate in some degree. This study is significant for analysing TMJ disc functions and its biomechanical properties in the future.

Measurements of total collagen, of the ratio of collagen types III/(I+III) and of sulphated glycosaminoglycans (GAGs) were compared with mechanical strength for individual ulcerated and non-ulcerated human aortic plaque caps and with intima adjacent to the plaques. The distributions of the collagen type ratio were similar for both ulcerated and non-ulcerated plaque caps but different from that of the adjacent intima. The proportions of different collagen types were not related to fracture stress and are thus unlikely to affect the potential to ulcerate. The distributions of the sulphated GAGs showed lower amounts for the plaque caps compared with the nearby intima, with the centres of ulcerated plaque caps having the lowest values. Total collagen had higher values in the peripheries of plaque caps compared with the nearby intima, but was distinctly lower in the centres of ulcerated plaque caps. Plaque caps appeared to require a higher collagen content than adjacent intima to support a given level of mechanical strength, suggesting that while collagen production had occurred in the plaque caps it was not as efficiently organized to resist fracture as a similar amount of collagen in the adjacent intima. Ulcerated plaque caps are notable for much larger transverse (centre vs. periphery) gradients of connective tissue constituents than for non-ulcerated plaque caps. The development of these transverse gradients may be a critical aspect in determining the propensity of a plaque to ulcerate.

Glycosaminoglycan (GAG) fractions were isolated from papain digests of comb and wattle tissues of 52-wk-old Single Comb White Leghorn roosters and the chemical composition of each fraction was examined. Total GAG concentrations were greater in the comb than in the wattle tissue with the ratio of hyaluronic acid to sulfated GAG being similar between the two tissues. L-iduronosyl-N-acetylgalactosamine-4-sulfate was the predominant disaccharide unit in the sulfated GAG fraction.

Mucopolysaccharides (glucosaminoglycans, GAGs) were extracted from otosclerotic and non-otosclerotic human stapes footplate, stapes superstructure and guinea pig stapes footplate. Cellulose acetate electrophoresis revealed an unknown highly anionic band (Rf = 1.05) beside the conventional hyaluronic acid, chondroitin sulfate and heparan sulfate fractions. Chemical analysis resulted in a high sulfate/hexosamine molar ratio in the otosclerotic GAG extract (1.7 and 1.9) and a very low ratio in non-otosclerotic human stapes and normal guinea pig stapes footplate GAG extract (0.20 and 0.21, respectively). Hexuronic acid content was highest in guinea pig stapes GAG preparation. Our results serve as indirect evidence of the presence of an oversulfated GAG fraction in the otosclerotic bone, which can potentially initiate otosclerotic bone resorption.

1. Follow-up studies of two siblings with mental retardation, progressive paraplegia and dementia were reported. 2. The brain and visceral organs of a patient (elder brother) who died recently were investigated histopathologically, electronmicroscopically and neurochemically. a. Moderate, diffuse demyelination occurred throughout the white matter of the central nervous system. b. Two abnormal materials were deposited in the white matter: one showed metachromasia containing sulfatide and another had staining characteristics of acid mucopolysaccharide histochemically. Electronmicroscopically, the former was a conglomerate of electron-dense materials of various degrees and the latter had a membrane-limited granular structure. The myocardium contained the same mucopolysaccharide material as that in the brain. c. Slight increase of sulfatide was found in the cerebral white matter. Arylsulfatase A activities were preserved in the brain as well as in the liver. Contents of hexosamine and uronic acid in the white matter were about three or five times as much as that of the controls. Electrophoresis on cellulose acetate membrane showed that acid glucopeptide was the main component of the mucopolysaccharide extracted from the brain.

Periodontal ligament and gingivae of bovine and porcine periodontium were analyzed for relative amounts of carbohydrates, collagen, and acid mucopolysaccharides. The sugar content was 3.6% and 3.4% of dry weight in bovine and porcine periodontal ligament, respectively. The values were lower in the gingivae being 2.34% and 2.30%, respectively. Approximately 50% of hexosamine in gingivae was present in acid mucopolysacchrides as compared to 36% in periodontal ligament. Relative to collagen there was a considerable amount of non-collagenous glycoproteins present in the periodontium as judged by carbohydrate content of the tissue.

A new procedure for the fractionation of glycosaminoglycans by electrophoresis on Pevikon has been described. Mixtures of glycosaminoglycans were fractionated by preparative electrophoresis on Pevikon in pyridine formate or glycine-HCl buffers. By this procedure, 200 mg of a mixture of hyaluronic acid, heparan sulphate, dermatan sulphate and chondroitin sulphate isolated from arterial wall could be successfully separated without loss of material. The purified fractions were analysed by enzymatic and chemical procedures. The molar ratios of uronic acid to hexosamine and of sulphate to hexosamine and the amino acid content of each glycosaminoglycan have been determined. The peptidic content (less than 1%) is represented by five amino acids, viz. serine, glycine, alanine, aspartic acid, and glutamic acid.

The two glycosaminoglycans found in FF, DS and HS were isolated from fluid of atretic and non-atretic bovine follicles and simultaneously separated by anion exchange high performance liquid chromatography. Heparan sulfate eluted with the apex of the peak at .071 M NaCl. Dermatan sulfate eluted with the apex of the peak at .353 M NaCl. The concentrations of DS and HS were greater in atretic follicles compared to non-atretic follicles and decreased as follicles size increased, as previously reported. The proportion of DS was approximately 90% of the total glycosaminoglycan concentration of all FF samples. The ratio of galactosamine to glucosamine in DS increased as follicle size increased, being 3.44 +/- .35, 7.65 +/- 2.4, and 15.9 +/- 3.8 (P less than .05) for small, medium and large follicles, respectively.

Male albino rabbits were exposed to intermittent nitrogen breathing every 30 sec for 5 sec, 15 min daily over a period of 3 weeks, and every 30 sec for 5 sec over a period of 10 hr. A third group of animals was exposed continuously to 8% oxygen breathing for 2 weeks. Neither intermittent not continuous hypoxia induced gross or microscopic alteration in the aorta. The effects of hypoxia upg which hypoxia was distributed than upon the total period or the degree of hypoxia. Exposure to hypoxia over a short period stimulated the synthesis of glycosaminoglycans, whereas distribution of the hypoxia over a longer period resulted in a reduction in the amount of glycosaminoglycans, probably secondary to an inhibition of the synthesis. Similarly, continuous exposure to 8% oxygen for a longer period decreased the aortic content of collagen. The alterations in the glycosaminoglycans and collagen induced by hypoxia may cause changes in the passage of macromolecules through the aortic wall. The changes may also influence the mechanical properties of the aorta and lead to impaired healing of vascular injury.

The tight-skin (TSK) mouse has cutaneous changes similar to those found in the skin of patients with progressive systemic sclerosis (PSS). Previous studies have shown that both have common abnormalities in skin thickness, dry weight, and hydroxyproline content. In this study, glycosaminoglycans (GAGs), major components of the ground substance, were quantitated in skins from TSK mice and compared with age-matched normal mice. Biochemical studies included determinations of hexosamines, uronic acids, and total GAGs by cetylpyridinium chloride precipitation. Dry weights and water-fat content of skin biopsy specimens from TSK mice were also compared with those of normal mice. Hexosamine, uronic acid, total GAGs, and dry weight were increased in TSK mouse skin when compared with normal mouse skin. The water-fat content did not differ significantly. These findings were similar to those known to occur in PSS skin, further suggesting that the TSK mouse might serve as an animal model for the skin changes found in PSS patients.

The composition of acidic glycosaminoglycans in pooled blood vessels, mostly of fetal origin, from a normal human term placenta was investigated by a series of procedures consisting of chromatography on a cetyl pyridinium chloride-cellulose column, cellulose acetate electrophoresis, susceptibility to testicular hyaluronidase and thin layer chromatography of the products of digestion with bacterial chondroitinases. The results indicate the presence of hyaluronic acid (36%), chondroitin-6-sulfate (27%), dermatan sulfate (21%), heparan sulfate (8%) and chondroitin-4-sulfate (8%). Several of the fractions obtained from the cetyl pyridinium chloride-cellulose column exhibited anticoagulant activity, which might be physiological importance for the maintainance of the fluidity of the intensively circulating placental blood.

Glycosaminoglycans (GAG) were extracted by digestion with papain followed by ultrafiltration and separated by cellulose-acetate electrophoresis and by chromatography of their cetyl-pyridinium complexes on cellulose microcolumns. The uronic-acid content of the tissues ranged from 0.8 to 2.4 mg/g of dry defatted tissue. Hyaluronic acid and dermatan sulphate were found in all tissues with chondroitin-4-sulphate also in skin, palatal mucosa and gingiva. There was 3-fold more hyaluronic acid in palatal mucosa than in any other tissue; it was concentrated in the antemolar rugae. A substance of presumptive salivary origin staining with alcian blue was found in cheek and floor of mouth mucosa. It migrated differently from reference GAG by electrophoresis and was not degraded by testicular hyaluronidase.

Glycosaminoglycans (GG) were isolated from commercial Ateroid and compared with those from bovine duodenal mucosa and pancreas. The major GG in Ateroid is heparin. Heparan sulfate (HS) and dermatan sulfate were also found. HS, chondroitin sulfates, and heparin were isolated from duodenal mucosa after papain digestion, but a residue, non-digestible, was mostly heparin. Pancreas contains very little GG, and the GG composition is similar to that of mucosa. The heparin isolated from Ateroid and mucosa have similar lipoprotein lipase-releasing activity, but the former has considerably less anticoagulant activity. Interestingly, papain digestion of mucosa and pancreas did not release all heparin from the tissue, suggesting that the protein to which heparin is linked is not readily accessible to the enzyme.

The daily urinary excretions of total polymeric glycosaminoglycans and of polymeric heparan sulfate have been measured in the urine of juvenile-onset and adult-onset diabetics of both sexes and in those of normal controls. The results indicate that diabetic patients excrete more polymeric heparan sulfate than their controls, either in an absolute amount or as a percentage of the total glycosaminoglycans excreted. These results suggest that in the course of diabetes there is an increased degradation of heparan sulfate to large oligosaccharide fragments. These are excreted before being completely degraded to monosaccharides and inorganic sulfate.

The variation with age and spinal level of the glycosaminoglycan distribution in the greyhound (a non-chondrodystrophoid breed) intervertebral disc has been assessed by means of histochemical and chemical methods. The principal glycosaminoglycans of the greyhound disc are the chondroitin sulphates. With ageing, there is evidence of a decline in these components and the replacement by keratosulphate. The discs of this breed contain high proportions of sialic acid and hexosamines well into old age which are thought to be associated with non-collagenous proteins (glycoproteins). The maintenance of glycosaminoglycans and glycoprotein levels in the disc nucleus pulposus into old age is possibly associated with the relatively low incidence of disc disorders in the non-chondrodystrophoid breeds.

A proteoglycan was isolated from ascites fluid produced by a rat yolk sac tumor. The glycosaminoglycan chains of the proteoglycan are all sensitive to digestion with chondroitinase ABC and about 90% are sensitive to chondroitinase AC. The proteoglycan contains 5% protein. Amino acid analysis revealed a high content of serine and glycine which together constitute 37% of the amino acids. Glutamic acid (glutamine) and aspartic acid (asparagine) are also abundant. Galactosamine accounts for 97% of the hexosamine and the remainder is glucosamine. These characteristics indicate that the glycosaminoglycan side chains of this proteoglycan are predominantly chondroitin sulfate with a smaller amount of dermatan sulfate. Antibodies to the proteoglycan were prepared by immunization of a rabbit with purified alkali-treated proteoglycan. Affinity-purified antibodies from the antiserum immunoprecipitated (35S)sulfate-labeled radioactivity from culture media of the yolk sac tumor cells known to contain chondroitin sulfate proteoglycan. This binding was inhibited by the intact purified proteoglycan but not by proteoglycan treated with papain, suggesting dependence of the reactivity of the antibodies on integrity of the protein part of the proteoglycan. Immunofluorescence of the cultured yolk sac tumor cells revealed localization of immune reactive proteoglycans at the cell surface.

The analyses of the products formed from heparitin sulfates by the action of two heparitinases and a heparinase from Flavorbacterium heparinum is reported. Heparitin sulfates A and B are degraded by heparitinase I yielding two disaccharides, one of them composed of N-acetylucosamine and an unsaturated uronic, joined by alpha(1 lead to 4) linkage, and the other, with the same composition but with an O-sulfate at the hexosamine moiety. A third disaccharide is also formed from heparitin sulfate B, by the action of the same enzyme, composed of glucosamine N-sulfate and an unsaturated uronic acid joined probably by alpha(1 lead to 4) linkage. Besides these three disaccharides, heparitin sulfate B yields, by the action of heparitinase I, an oligosaccharide (with an average molecular weight of 6000) which is completely degraded by the heparitinase II yielding a disaccharide composed of glucosamine 2,6-disulfate and unsaturated uronic acid. All the disaccharides are further degraded by alpha-glycuronidase from Flavobacterium heparinum yielding the respective monosaccharides. Based on these and other analyses the possible structures of the heparitin sulfates are proposed.

The study examined the subcellular distribution of [3H]glucosamine-labeled glycoconjugates undergoing axonal transport in 100,000 x g soluble and two membranous subfractions of the garfish olfactory nerve. Analysis was made of intact glycoconjugates and of glycopeptides and glycosaminoglycans derived from these molecules by limit protease digestion. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed labeling of a variety of high-molecular-weight molecules with a lower molecular weight distribution in the soluble fraction than in the membranous fractions. Following protease digestion, nearly two-thirds of transported radioactivity in glycopeptides was recovered in the plasma membrane-enriched subfraction, with the remainder equally divided between soluble and higher density membrane fraction. Comparison of the distribution of glycopeptide radioactivity and chemically assayed hexosamine revealed transport labeling of a large variety of different-sized neutral and acidic glycopeptides in all subfractions. Transport labeling of most glycoprotein carbohydrate chains was in proportion of their hexosamine content. Transported glycosaminoglycan label was most heavily concentrated in the plasma membrane fraction, whereas hexosamine was most concentrated in the higher density membrane fraction. The labeling pattern suggested both transported and nontransported pools of these molecules. The specific glycosaminoglycans chondroitin sulfate and heparan sulfate were recovered in all subfractions, whereas hyaluronic acid was confined to the soluble fraction.

A method is described for the quantitative determination of urine and plasma glycosaminoglycans (GAGs) by gas chromatography of the acetylated amino sugars. GAGs were first recovered by precipitation from urine with alkyltrimethylammonium bromide and from plasma by mini-column chromatography after papain digestion. Urine samples (24) analysed for total hexosamines by gas chromatography and for uronic acid by colorimetry had a correlation coefficient of 0.85. The within-run coefficient of variation (C.V.) for nineteen samples from a pooled urine was 5.2% for total hexosamines and that for the ratio of galactosamine to total hexosamines was 3.7%. The corresponding C.V. values for twelve plasma samples from a common pool were 6.5 and 3.7%. The mean ratio of galactosamine to total hexosamine in ten pre-breakfast spot urines was 51.5%. The corresponding ratio in the plasma from twenty adolescent blood donors was 76.3% and the mean total hexosamine content of the GAGs was 47.36 mumol/l.

The glycosaminoglycans of bovine interphotoreceptor matrix were analyzed biochemically and compared with those of neural retina and retinal pigment epithelium. Chondroitin sulfates extracted from these three tissues were electrophoretically low-sulfated and composed of various kinds of chondroitin sulfate isomers. Interphotoreceptor matrix and retinal pigment epithelium contained hyaluronic acid as another glycosaminoglycan. With regard to neural retina, chemical characteristics of another component identified on the membrane are still unknown.

A reliable and reproducible method that allows the combined, simultaneous gas chromatographic (GC) determination of neutral sugars, hexosamines, alditols, identification and quantitation of the reducing aldose end-group in oligo and polysaccharides and glycosaminoglycans has been described. It involves the following steps: release of the reducing end-group from its protein linkage in glycosaminoglycans and reduction of this reducing end-group into alditol, release of the components of the reduced polymer by resin-catalysed hydrolysis, nitrous acid deamination of the resin-bound hexosamines in this hydrolysate into anhydroaldoses and a combined derivatization and GC determination of the neutral sugars as aldononitrile acetates, anhydroaldoses as peracetylated oximes and alditols as alditol acetates. Application of the method to determination of degree of polymerization of oligo-and polysaccharides and chain weights of proteoglycans has been described. This method has several advantages over the previous methods.

The concentration, composition and sulfate labeling of glycosaminoglycans and glycoproteins have been studied in purified nuclei isolated in bulk from rat brain. The concentration of total glycosaminoglycans is 0.142 mumol hexosamine/100 mg protein, comprising 57% chondroitin 4-sulfate, 7% chondroitin 6-sulfate, 29% hyaluronic acid and 7% heparan sulfate. Control experiments demonstrated that less than 5% of the sulfated glycosaminoglycans associated with nuclei could be accounted for by the nonspecific adsorption of soluble acidic proteoglycans to basic nuclear proteins. Glycoprotein carbohydrate is present at a level of 206 mug/100 mg protein, and has an average composition of 30% N-acetylglucosamine, 29% mannose, 19% N-acetylneuraminic acid, 15% galactose, 4% N-acetylgalactosamine, and 3% fucose. Labeling studies also indicated the presence of ester sulfate residues on the glycoprotein oligosaccharides.