Determination of urinary hexosamines for diagnosis of bladder pain syndrome.

INTRODUCTION AND HYPOTHESIS: Bladder pain syndrome (BPS) is a chronic disease characterized by urgency, bladder pain, and frequency, and urinary glycosaminoglycans are thought to reflect bladder epithelial deficiency in BPS. Sensitive and specific evaluation of total urinary glycosaminoglycans may be useful for the clinical diagnosis of BPS and its treatment. METHODS: A procedure for the simultaneous determination of glucosamine and galactosamine produced from urinary glycosaminoglycans has been performed in BPS patients and healthy subjects. RESULTS: The total content of urinary hexosamines in BPS patients significantly increased by ~130% with the increase in glucosamine greater than galactosamine. CONCLUSIONS: A significant increase in total hexosamines content and in particular in glucosamine belonging to urinary heparan sulfate was determined in BPS patients compared with controls. We propose HS and in particular its low-molecular mass fragments and glucosamine assay as useful markers for a biochemical diagnosis of BPS and for monitoring this syndrome.

High-throughput determination of urinary hexosamines for diagnosis of mucopolysaccharidoses by capillary electrophoresis and high-performance liquid chromatography.

Mucopolysaccharidoses (MPS) diagnosis is often delayed and irreversible organ damage can occur, making possible therapies less effective. This highlights the importance of early and accurate diagnosis. A high-throughput procedure for the simultaneous determination of glucosamine and galactosamine produced from urinary galactosaminoglycans and glucosaminoglycans by capillary electrophoresis (CE) and HPLC has been performed and validated in subjects affected by various MPS including their mild and severe forms, Hurler and Hurler-Scheie, Hunter, Sanfilippo, Morquio, and Maroteaux-Lamy. Contrary to other analytical approaches, the present single analytical procedure, which is able to measure total abnormal amounts of urinary GAGs, high molecular mass, and related fragments, as well as specific hexosamines belonging to a group of GAGs, would be useful for possible application in their early diagnosis. After a rapid urine pretreatment, free hexosamines are generated by acidic hydrolysis, derivatized with 2-aminobenzoic acid and separated by CE/UV in ∼10min and reverse-phase (RP)-HPLC in fluorescence in ∼21min. The total content of hexosamines was found to be indicative of abnormal urinary excretion of GAGs in patients compared to the controls, and the galactosamine/glucosamine ratio was observed to be related to specific MPS syndromes in regard to both their mild and severe forms. As a consequence, important correlations between analytical response and clinical diagnosis and the severity of the disorders were observed. Furthermore, we can assume that the severity of the syndrome may be ascribed to the quantity of total GAGs, as high-molecular-mass polymers and fragments, accumulated in cells and directly excreted in the urine. Finally, due to the high-throughput nature of this approach and to the equipment commonly available in laboratories, this method is suitable for newborn screening in preventive public health programs for early detection of MPS disorders, diagnosis, and their treatment.

Effect of lupeol and lupeol linoleate on lysosomal enzymes and collagen in adjuvant-induced arthritis in rats.

Lysosomal enzymes play important roles in the inflammatory process. The pentacyclic triterpenes, lupeol and lupeol linoleate were administered orally (50 mg/kg) for 8 days to arthritic rats, after 11th day of adjuvant injection. The lysosomal enzymes were significantly increased in arthritic condition, which are involved in the destruction of structural macromolecules in connective tissue and cartilage in rheumatoid arthritis. Hence the level of collagen was significantly decreased and the excretion of urinary hydroxyproline, hexosamine, hexuronic acid and glycosaminoglycans were increased in arthritic rats. Treatment of arthritic rats with triterpenes reversed the above changes, which may be due to stabilization of the lysosomal membrane. Out of the two triterpenes tested, lupeol linoleate showed better ameliorating action than lupeol.

Significance of variation in turnover of glycated albumin on indices of diabetic control.

We measured the half-time of disappearance of 125I-labelled glycated albumins in a rat model of diabetes with continuous infusion of physiological saline and insulin. Our results indicate that (i) in non-diabetic rats, continuous infusion of saline per se did not affect the concentrations of glucose or of fructosamine, and the half-time of disappearance of albumin was unaffected by degree of glycation; (ii) hyperglycaemia (mean plasma glucose concentration of 18-27 mmol/l) caused a small but significant increase in half-time of labelled glycated albumin disappearance from a mean of 42 h to a mean of 47 h; (iii) this effect of hyperglycaemia outweighed any effect of increase in albumin excretion detected in poorly controlled diabetic rats without infusion. We conclude that the effect of hyperglycaemia in slowing turnover of glycated albumin is likely to be insignificant in relation to its effect in promoting glycation, and may be species-dependent. However, in nondiabetics, variation of turnover of glycated albumin may well be significant in explaining the wide interindividual variation in concentration of glycated protein.

[Assessment of criteria for forming follow-up groups of patients at high risk for ulcerogenesis in ambulatory care]. / Otsenka kriteriev formirovaniia grupp nabliudeniia sredi lits s faktorami vysokogo riska ul'tserogeneza v usloviiakh polikliniki.

A method of clinico-laboratory examinations to reveal persons at high risk for the development of duodenal ulcer, suitable for wide use during prophylactic medical screenings was devised. The rise of the levels of serum pepsinogen-I, pepsin and hexosamines in the urine, being of prognostic importance as applicable to ulcerogenesis, was the most significant indicator in screening risk group patients. During 3 to 5 years of the screened group follow-up and carrying out health measures, peptic ulcer was ascertained in 8.4% of the patients with chronic gastroduodenitis. Of these, 66.6% had initially suffered from pylorobulbitis. It is shown that there is a real opportunity of preventing ulcer formation in patients with chronic primary gastroduodenitis under outpatient conditions.

A screening method for mucopolysaccharidoses with increased urinary excretion of sulfated N-acetylhexosamines.

Patients with mucopolysaccharidosis have been reported to excrete elevated amounts of sulfated N-acetylhexosamines in their urine. Based on this finding, a new and simple colorimetric screening test for these disorders is presented. Chromatography of whole urine on Dowex AG 1-X8, from each of 23 normal controls, 5 patients with mucopolysaccharidosis and one patient with multiple sulfatase deficiency, was used to separate the sulfated hexosamines. The fractions eluted with 2M NaCl were analyzed according to the method of Reissig. Patients with Sanfilippo syndrome, type A, Sanfilippo syndrome, type D, Maroteaux-Lamy syndrome, Morquio syndrome, type A, and multiple sulfatase deficiency were clearly distinguished from normal controls. The procedure appeared most sensitive for Sanfilippo syndrome, type D, and multiple sulfatase deficiency, each of which involves deficient activity of N-acetylglucosamine 6-sulfate sulfatase.

Studies on carbohydrate moieties of glycoproteins in established adjuvant induced arthritis.

The investigation presents the metabolic changes in the carbohydrate components of glycoproteins in several tissues of adjuvant arthritic rats. The experimental arthritis induces a significant modification of total carbohydrate moieties of glycoproteins in arthritic tissues. In both acute and chronic phases of the disease, the adjuvant arthritis caused a significant increase in the levels of carbohydrate moieties of tissue glycoproteins viz. total hexose, hexosamine, fucose, sialic acid, total neutral sugar content and neutral sugar monosaccharides. In addition, the urinary excretions of hexosamine and uronic acid in arthritic rats were found to be elevated significantly. The data from the investigation clearly indicate that the experimental arthritis induces an increased glycoprotein synthesis in most of the tissues examined.

Urinary excretion of connective tissue metabolites under the influence of a new non-steroidal anti-inflammatory agent in adjuvant induced arthritis.

The therapeutic effect of boswellic acids and salai guggal in adjuvant induced arthritic rats in relation to urinary excretion of connective tissue metabolites viz. hydroxyproline, hexosamine and uronic acid was thoroughly investigated. Compared to controls, the arthritic animals showed an increase in the excretion of these metabolites in urine. The elevated levels of urinary hydroxyproline (free, total, nondialysable and dialysable), hexosamine and uronic acid in the arthritic animals were found to be slightly decreased in the acute phase and significantly decreased in the chronic phase of the disease following the administration of boswellic acids or salai guggal. The results of the investigation indicated that both these anti-inflammatory drugs could offer a partial protective action against changes induced by adjuvant induced arthritis.

Quantitative gas chromatographic measurement of glycosaminoglycan hexosamines in urine and plasma.

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.

Terminal monosaccharides of carbohydrate chains of glycosaminoglycans in normal human urine.

To elucidate the mode of the exertion of glycosidase activities in the catabolism of the tissue glycosaminoglycans (GAG), the terminal monosaccharides of the carbohydrate chains of urinary GAG in the most prominent subfraction (40% Fr-1.25 M Fr) among the subfractions obtained in a previous paper (Endo et al. 1980a) were investigated. The results of determination of the reducing hexuronic acid and N-acetylhexosamine before and after digestion of the subfraction with beta-glucuronidase and beta-N-acetylhexosaminidase, together with previous data indicated that 0.36 and 0.37 mol of glucuronic acid and N-acetylgalactosamine, respectively, per mol of the subfraction were located at the non-reducing terminals of the carbohydrate chains. The remaining portion (0.27 mol per mol) of the non-reducing ends might be mostly occupied by the sulfated N-acetylgalactosamine residues. On the other hand, 0.25, 0.16 and 0.34 mol of glucuronic acid, N-acetylgalactosamine and xylose, respectively, per mol of the subfraction were indicated to the present at the reducing terminals of the carbohydrate chains. The remaining portion (0.25 mol per mol) of the reducing ends might be mostly occupied by the galactose residues and/or the N-acetylgalactosamine 4-sulfate residues. The present observations provided with evidence for the action of endo-beta-glucuronidase and endo-beta-N-acetylhexosaminidase on the tissue GAG, specifically on chondroitin sulfates.

Effect of bioflavonoids on the urinary excretion of hydroxyproline, hydroxylysyl glycosides and hexosamine in adjuvant arthritis.

The effects of (+)--Catechin (AC) and 0--(beta hydroxyethyl) rutosides (HR) on the urinary collagen metabolites were studied up to 49 days in rats with adjuvant-induced arthritis. The elevated levels of urinary total, non-dialysable and dialysable hydroxyproline, hydroxylysyl glycosides and total hexosamine in the arthritic animals were found to be slightly decreased in the acute phase and significantly decreased in the chronic phase of the disease due to the administration of bioflavonoids. Of the two bioflavonoids tests, CA was found to afford more protective action than HR.

Increased urinary excretion of free N-acetylneuraminic acid in thirteen patients with Salla disease.

Thirteen severely retarded patients with Salla disease, a new type of lysosomal storage disorder, have been studied biochemically. All patients excreted approximately ten times more free sialic acid than normal individuals. The isolated sialic acid was characterized by paper chromatography, thin-layer chromatography, optical rotation, 13C and 1H nuclear magnetic resonance spectroscopy, and mass spectrometry of its permethylated derivative. The results clearly indicated that the excreted sialic acid was identical to N-acetylneuraminic acid. The main sialylated trisaccharide present in the urine of the patients was identified as 3'-sialyllactose by sugar and methylation analysis. The excreted amounts were found to be within normal range.

Convenient method for the gas chromatographic analysis of hexosamines in the presence of neutral monosaccharides and uronic acids.

A convenient gas chromatographic method has been devised for the analysis of hexosamines in the presence of neutral and acidic sugars, which involves sequential derivatization reactions of nitrous acid deamination, mercaptalation, and trimethylsilylation. This method allows rapid, simultaneous determination of 0.1-1 micromole samples of hexosamines with coefficients of variation of less than 3%.

2-Acetamidoglucal, a new metabolite isolated from the urine of a patient with sialuria.

A new metabolite, namely 2-acetamidoglucal, has been found in the urine of a patient with sialuria in addition to the metabolites N-acetylneuraminic acid, N-acetylmannosamine, N-acetylglucosamine and 2-deoxy-2,3-dehydro-N-acetylneuraminic acid reported earlier. the structure has been identified by mass spectrometry and 360 MHz proton nuclear magnetic resonance spectroscopy and verified by synthesis. All accumulated compounds fit into the metabolic pathway for the biosynthesis of CMP-N-acetylneuraminic acid. Sialuria is discussed in terms of a failure of regulation of UDP-N-acetylglucosamine 2-epimerase.

Urinary excretion of glycosaminoglycans in disseminated neoplasm.

Urinary glycosaminoglycan excretion was studied in 24 cases of disseminated neoplasm, 12 of which had unequivocal evidence of skeletal involvement. Urinary hydroxyproline, cetylpyridinium chloride (CPC)-precipitable uronic acid, and CPC-precipitable hexosamine were expressed as a ratio to urinary creatinine. Glycosaminoglycans contained in urine concentrated x 1000 by vacuum-dialysis were separated by electrophoresis on cellulose acetate and stained with alcian blue. Of the 12 cases with clear evidence of skeletal involvement, eight (66%) showed elevation of serum alkaline phosphatase, five (42%) showed elevation of urinary hydroxyproline, and three (25%) showed elevation of urinary uronic acid. It is concluded that urinary uronic acid is not a sensitive index of skeletal involvement in disseminated neoplasm. The most striking feature of the study was the identification of a well-defined fraction indist inguishable from hyaluronic acid in seven (58%) of the cases with evidence of skeletal involvement. Hyaluronic acid is not normally identifiable in adult human urine. The hyaluronic acid excretors showed more consistent biochemical evidence of bone disease (elevation of serum alkaline phosphatase and urinary hydroxyproline) than the non-excretors. The possibility that the urinary hyaluronic acid is derived from degradation of skeletal hyaluronic acid is discussed. An alternative explanation is that the hyaluronic acid is derived from neoplastic cells as part of a reversion of glycosaminoglycan synthesis to a more ;fetal' state, a glycosaminoglycan counterpart of the production of oncofetal antigens by neoplastic cells.