Search

A method was developed for the determination of urinary chondroitin sulphate (CS), including dermatan sulphate and chondroitin 4 and 6-sulphates, using an enzymatic degradation with chondroitinase-ABC followed by precipitation with Alcian blue, whereby CS was determined as the difference between undigested and chondroitinase digested material. The method was linear in the range 0-100 mg l-1 with a detection limit of 1 mg l-1 and allowed determinations on small urine volumes without pretreatment of the urine. It could be demonstrated that males excreted more CS than females, and growing children had the highest urinary content of CS. Renal calcium stone formers did not differ from healthy controls in urinary CS. Patients with acromegaly had a higher excretion of CS compared with controls. There was also, in these patients, a positive correlation between the serum growth hormone levels and the urinary CS, indicating that CS-excretion may be an estimate of the activity of the pituitary disorder.

Vitamin A decreased the urinary excretion of total mucopolysaccharides in a patient with Hurler-Scheie compound (type IH-S mucopolysaccharidosis). Vitamin A was administered orally in daily doses of 1,000 to 2,000 IU/kg body weight for 10 years. Adverse clinical responses such as irritability, bone pain, dizziness, vomiting and diarrhea appeared in the patient and were controlled by reduction of the dose administered. No clinical improvement was observed, although it is possible that the clinical course of the disease may have been retarded.

Abnormally elevated hyaluronic acid and dermatan sulfate were isolated from lesional skin of a patient with severe pseudoxanthoma elasticum. These glycosaminoglycans (estimated as uronic acid) surpassed the normal controls by 33.6 and 4.8 magnitudes, respectively. Urine chondroitin 6-sulfate of the same patient moved faster by electrophoresis on cellulose acetate than its counterpart isolated from urine of another four patients or from the normal controls. Hyaluronic acid and chondroitin 6-sulfate exceeded their counterparts in normal urine by 2- to 10- and 4- to 18-folds, respectively. These increments correlated with the pseudoxanthoma elasticum severity in three of the five patients studied. The data showed: the first conclusive evidence that dermatan sulfate increased in lesional skin of a patient, with severe pseudoxanthoma elasticum, who also had considerably augmented HA, alteration of the same patient's urine chondroitin 6-sulfate, and diversified urine glycosaminoglycans in pseudoxanthoma elasticum.

The investigation included 15 psoriatic patients and 14 healthy controls. By using a simple method based on susceptibility to chondroitinases the excretion of dermatan sulphate, chondroitin-4/6-sulphate and heparan sulphate in the psoriatics was found to be increased with 104, 62 and 47% from uronic acid mean excretions of 1.97, 6.37 and 5.10 mumol/24 h, respectively. The excretion of hydroxyproline was not increased. In both groups the excretion of hyaluronic acid was insignificant. The absolute increase in the excretion of a major skin component like dermatan sulphate was exceeded by the excretion of chondroitin-4/6-sulphate and heparan sulphate which are both small components of skin. This indicates a comparatively high turnover of those two fractions in psoriatic lesions. The fact, that only the excretion of dermatan sulphate correlated with the fraction of skin surface involved in the psoriatic disease, indicated an important origin of this fraction, as well as the possibility of dermatan sulphate as a means of following dermal metabolism.

Some hitherto undetected differences in chemical and macromolecular structure between both dermatan sulphates and heparan sulphates excreted in the Hurler and Hunter syndromes are demonstrated. 1. Of Hunter dermatan sulphate, 37-43% is resistant to periodate oxidation, as opposed to 25% of the corresponding Hurler material. It is likely that the resistance is conferred by the presence of sulphate groups on carbon atoms 2 or 3 of the iduronate residues, correlating with the recently established deficiency of a sulphoiduronate sulphatase in Hunter fibroblasts. 2. Two distinct electrophoretic species of dermatan sulphate are found in Hunter urine, but only one in Hurler preparations. 3. Ion-exchange chromatography and gel filtration reveal that Hurler dermatan sulphate is more heterogeneous with respect to molecular weight distribution than the other. The dermatan sulphates were degraded by hyaluronidase to a limited extent. 4. Hurler heparan sulphate contains a higher proportion of sulphoamino-glucose than material from Hunter urine. Similar high levels in Sanfilippo patients, representing 65-78% of the total glucosamine suggest a direct correlation with mental deficiency.

Urinary dermatan sulfate (DS) and heparan sulfate (HS) were purified from mucopolysaccharidosis patients. DS shows average mol. wt 8600-12,000 (approx. one half of tissue DS), iduronic acid content 99.1-99.6% (81.8% in tissue DS), core peptide mostly di- or tri-peptide (--Ser--Gly--or--Ser--Gly--Glu--). Molecular weight of HS ranged from 2500 to 20,000, averaging about 5000. Highly sulfated HS was found in the low molecular weight fraction, and no bound core peptide. By contrast, HS in the high molecular weight fraction bound one sulfate per repeating unit, which include core peptide.

Crude glycosaminoglycan (GAG) fraction was directly precipitated with cetylpyridinium chloride without prior dialysis of urine of orthopedic patients. The crude GAG fraction was then fractionated with trichloroacetic acid (TCA). The TCA-insoluble peptide-bound GAG fraction thus obtained was treated with alkali to eliminate the peptide moiety for enzymatic analysis. The GAG compositions of this fraction and the TCA-soluble fraction were determined by digestion with mucopolysaccharidases (chondroitinase AC, chondroitinase B, chondroitinase C, heparitinase and Streptomyces hyaluronidase). When the amount of the crude GAG fraction was small, no significant amount of the TCA-insoluble peptide-bound GAG fraction was obtained. The GAG composition of this case was also determined by the same procedures after direct alkali-treatment of the crude GAG fraction. The data indicated that the proportion of the TCA-insoluble peptide-bound GAG fraction was very small. The alkali-treated TCA-insoluble peptide-bound GAG fraction contained a larger proportion of heparan sulfate than the TCA-soluble GAG fraction. It was clearly demonstrated that the patients with Werner's syndrome and mucopolysaccharidosis I-S (Scheie) excreted large amounts of hyaluronic acid and dermatan sulfate respectively, into urines. It was indicated in most cases that major urinary GAG were chondroitin 4-sulfate, chondroitin 6-sulfate plus chondroitin and heparan sulfate, while minor ones were dermatan sulfate and hyaluronic acid. In addition, the data suggested a wide range of the degree of desulfation or urinary GAG, and the presence of significant amounts of keratan sulfate plus acidic glycopeptides in the urinary GAG fractions. The present data provided more precise information on urinary GAG from orthopedic patients than those reported previously.

The chemical structure of dermatan sulfate (DS) in the urine of a patient the Hunter syndrome was studied through the analysis of disaccharide units which were derived from the urinary DS by digestion with chondroitinase ABC and separated on a Dowex 1 column. The DS was basically composed of repeating disaccharide units of iduronyl N-acetylgalactosamine 4-sulfate. About 90% of the excess sulfate were linked to the iduronate residues as an additional sulfate group in the unit. N-Acetylgalactosamine 6-sulfate and N-acetylgalactosamine 4,6-disulfate residues were minor components. No non-sulfated disaccharide unit was detected in the digestion products. Only sulfoiduronate residue was found as the non-reducing terminal sugar of the DS molecule, consistent with the lack of iduronosulfate sulfatase in this disease.

The present investigation confirmed a former report of a diurnal variation with an increased urinary excretion rate of total glycosaminoglycans in the daytime [4]. Fractionation showed that this increase was solely due to three electrophoretic fractions which resisted degradation with chondroitinase ABC. Fasting in the daytime had no influence on the increased excretion of one of these fractions, whereas it prevented the increased excretion of the other two (probably both heparan sulphates). The excretion rates of chondroitin 4/6 sulphate, dermatan sulphate and a fourth chondroitinase ABC resistant glycosaminoglycan (heparan sulphate) were uninfluenced by time of day and fasting. This was contrary to the excretion of hydroxyproline which decreased during the fasting period.

The study group consisted of 82 children aged 9 to 14 years with the following forms of myopia: progressive (37 cases), stable (22 cases), congenital stable (11 cases) and incipient (12 cases). During the period of progression in myopia, a significant increase was found in the 24-hour-excretion of acid mucopolysaccharides. In the patients with stable and congenital stable forms the excretion of acid mucopolysaccharides did not differ significantly from that in the control group of emmetropic and hypermetropic children. The results of electrophoresis on cellulose acetate and of the carbazol and orcinol reactions showed predominant excretion of A and C chondroitinsulphates in patients with myopia.

To elucidate precise nature of urinary glycosaminoglycans (GAG) GAG in the acidic subfractions obtained in a previous paper (Endo et al. 1980) were quantitated by digestion with mucopolysaccharidases, nitrous acid treatment and by quantitative cellulose acetate membrane (Separax) electrophoresis. Hyaluronic acid, chondroitin sulfates and chondroitin, dermatan sulfate and dermatan, heparan sulfate, keratan sulfate, and acidic glycopeptide were found in wide distribution in these subfractions. The results suggested the presence of various degree of the depolymerization products of each GAG and of wide range of the desulfation products of each sulfated GAG. Distribution map of each GAG in the subfractions showed more detailed polydispersity of GAG in normal human urine than that reported previously.

Glycosaminoglycans were isolated from the urines of 46 patients with mucopolysaccharidosis; 11 with Type I (Hurler), 8 with Type II (Hunter), 16 with Type III (Sanfilippo A and B), 9 with Type V (Scheie), one with Type VI (Marateaux-Lamy), and one unclassified. All 46 patients excreted in their urine excessive amounts of dermatan sulfate, heparan sulfate or both. In addition, patients of certain types excreted excessive amounts of chondroitin sulfates A and/or C. There is a trend in each type of the disease towards the same carbazole/orcinol ratio, glucosamine/galactosamine ratio and glycosaminoglycan composition. Molecular weight distribution of the urinary glycosaminoglycans by gel filtration from Sephadex G-200 is characteristic for each different type of mucopolysaccharidosis and is distinguished from normal controls and patients without mucopolysaccharidosis. Preparation of elution diagrams from Sephadex G-200 allows an estimation of the composition of the glycosamino-glycans. Practically all heparan sulfate and a sizable part of dermatan sulfate from the urinary glycosaminoglycans of all these patients have been highly degraded. In all the patients in which the specific enzyme defect was demonstrated, the assignment of the type of mucopolysaccharidosis, on the basis of the elution diagrams, was correct. Patients with mucopolysaccharidosis Type V displayed two conspicuously different types of elution patterns, suggesting heterogeneity. Indeed, only a portion of these patients showed alpha-L- iduronidase deficiency. Carriers had normal urinary glycosaminoglycan output and composition and exhibited normal elution diagrams.

Urinary glycosaminoglycan excretion was examined in 25 individuals with bladder cancer in comparison to glycosaminoglycan excretion by eight normal individuals. Urinary glycosaminoglycan was isolated by gel filtration and quantified as macromolecular uronate concentration. Electrophoresis in calcium acetate and densitometry of Alcian blue-stained electrophoretograms were used to separate and quantify the relative amounts of individual glycosaminoglycans. Elevated excretion of macromolecular uronate was noted in 53% of the cancer cases. The highest levels were found among individuals with metastatic disease. Three electrophoretic bands were always detected in the control and cancer groups: chondroitin sulfate, heparan sulfate (both confirmed by chemical and enzymatic degradation), and a third band (Band 1) of unknown composition. A fourth band, corresponding to dermatan sulfate, was seen in some high-grade metastatic tumors. Band 1 excretion was elevated in a significant fraction of all patients. Seven of 12 metastatic cases but only two of 13 localized cases showed increased heparan sulfate excretion. Diagnostic limits were drawn from the observed distributions of normals, and with these limits 92% of the cancer cases, including 12 of 12 metastatic cases, could be identified. The results strongly suggest noninvasive urinary glycosaminoglycan analysis may well provide a new biochemical approach for detecting and monitoring the pathogeneses of bladder cancer.

Oligosaccharides of testicular hyaluronidase-degraded dermatan sulfate were separated from undegraded dermatan sulfate by chromatography on Sephadex G-75, but not by chromatography on Sephadex G-25. All but the smallest of these oligosaccharides were recovered in excellent yield following dialysis and precipitation with cetyl pyridinium chloride (CPC). G-75 chromatography of dialyzed, concentrated Hunter urine mucopolysaccharides precipitated with CPC resolved most of the large dermatan sulfate into a void volume related peak which was free of heparan sulfate. Decreasing amounts of dermatan sulfate oligosaccharides were eluted with sephadex-retarded polysaccharides, including small amounts which appeared with otherwise pure heparan sulfate.

Using methods which have been described earlier in detail [23] the sulphate/uronic acid molar ratio of glycosaminoglycans was determined in corresponding specimens of skin and urine obtained from 14 healthy adults. The method of dermis was provided with an electrophoretic separation that made it possible to determine the ratio of the sulphate subfraction without increasing the weight of the biopsies. The sulphate/uronic acid ratios were uninfluenced by age and were exactly equal in males and females whether measured in total glycosaminoglycans of dermis (0 X 51 +/- 0 X 11, means +/- SD), in the sulphated subfraction of dermal glycosaminoglycans (1 X 17 +/- 0 X 25) or in urinary glycosaminoglycans (0 X 96 +/- 0 X 15). The latter ratio was surprisingly high and the significance of this was discussed. Furthermore an inverse relationship between the urinary ratio and the ratio of sulphated glycosaminoglycans of dermis was found. However the exact nature of this relationship remains to be explained. The results demonstrate the applicability of a method that can be used on small biopsies without ethical obstacles and make it possible to discover pathological conditions.

Abnormal urinary excretion of acid mucopolysaccharides in two children affected with Albers-Schönberg disease is described. The urine specimens were first checked for their acid glycosaminoglycan (AGAG) content by a screening test. The AGAGs were then evaluated by uronic acid determination using the carbazole method, and the ratio between high molecular mass and low molecular mass AGAGs was determined. Qualitative characterization, before and after enzymatic digestion with chondroitinases AC (ChAC) and ABC (ChABC), of the total AGAGs contained in the specimen and of each AGAG separated with Dowex 1X2 was also performed by electrophoresis. The pattern of the urinary mucopolysaccharides in the two patients showed an abnormal quantity of heparan sulphate (HS) and a smaller quantity of dermatan sulphate (DS). Such abnormal urinary AGAGs excretion should confirm the assumption of some authors that malignant infantile osteopetrosis may be caused by a deficiency in the lysosomal enzymatic activity, or by some anomaly in the cell membrane function.

Glycosaminoglycan isolated from the urine of a patient with the Hunter syndrome was composed of heparan sulfate (59.9%), dermatan sulfate (30.6%) and chondroitin sulfate (9.5%), and was heterogeneous in molecular weight (1,500-10,000) and in sulfate content (0.35-2.05 moles/mole of hexosamine). About 60% of dermatan sulfate and 10% of heparan sulfate had molecular weight of 7,000 to 10,000, while about 10% of the former and 60% of the latter had those of 1,500 to 3,500. Sulfate contents of dermatan sulfate and heparan sulfate were inversely related to their molecular weights. Higher total- and N-sulfate contents were measured in smaller molecular-weight heparan sulfate, and higher acetyl content was in larger molecular-weight heparan sulfate. On the basis of the chemical properties of dermatan sulfate and heparan sulfate isolated in this experiment, their catabolic processes in the Hunter syndrome were discussed.

Urinary excretion of acid mucopolysaccharides and glycopeptides in the urine of an untreated patient with cretinism were measured before and after thyroid hormone replacement. Urinary uronic acid and hexose excretion in the CPC-precipitated fraction increased four to ten times after thyroid hormone administration. The maximum excretion was observed after 2 months of thyroid replacement. The excreted acid mucopolysaccharides consisted of chondroitin sulphate A/C and minor quantities of heparan sulphate. Urinary excretion of glycopeptides, particularly small molecular glycopeptides rose also four to five-fold with thyroid hormone administration. These data suggest that thyroid hormone markedly influences the metabolism of acid mucopolysaccharides and glycoproteins. Possibly, the lack of thyroid hormone caused a decreased activity of various lysosomal glycosidases and sulphatases.

60 patients with different forms of neuromuscular disorders were examined. Morphological studies of skeletal muscles and of diurnal excretion with urine of acetic GAG were carried out. It was established that the changes in the stromal connective tissue in the progressive muscular dystrophia appear in the early stages of the disease and affect both the essential substance and the fibrillar structures. The excretion with the urine of acetic GAG was increased. In denervative amyotrophy the changes became apparent against the background of marked clinical symptoms. The data obtained may be important for a differential diagnosis of progressive muscular dystrophy and denervative amyotrophy, and for the development of differential drug therapy.

A fluorogenic substrate for alpha-L-iduronidase, 4-methylumbelliferyl alpha-L-iduronide, has been newly synthesized and the enzyme activity has been measured in urine samples obtained from normal persons and patients suffering from mucopolysaccharidosis. Urine samples derived from a patient with Scheie syndrome showed greatly reduced activity compared with a normal adult at a similar age. This patient exhibited a high level of urinary excretion of dermatan sulfate and heparan sulfate, which could be interpreted in terms of her low alpha-L-iduronidase activity. The use of the fluorogenic substrate has some advantages over existing methods because of the high sensitivity and the relative ease of handling, and it should be useful not only for diagnosis but also for following the purification process of the enzyme.