Navegando por Palavras-chave "glycosaminoglycan"

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    Changes in cat urinary glycosaminoglycans with age and in feline urologic syndrome
    (Elsevier B.V., 2004-04-07) Pereira, D. A.; Aguiar, JAK; Hagiwara, M. K.; Michelacci, Y. M.; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP)
    The aim of the present study was to characterize the urinary excretion of glycosaminoglycaris in kittens and adult healthy cats, as well as in cats with a low urinary tract disease, the feline urologic syndrome (FUS). the main urinary glycosamitioglycan in cats was found to be chondroitin sulfate, with smaller amounts of dermatan sulfate and heparan sulfate. There was no difference in the urinary glycosaminoglycan concentration with sex, but a marked decrease occurred with age, due to chondroitin sulfate. Trace amounts of keratan sulfate were also detected in the urine of kittens, but not of healthy adult cats. Dermatan sulfate and heparan sulfate were the only glycosaminoglycans found in the urinary tract and kidney, and chondroitin sulfate was the only glycosaminoglycan found in the plasma. These data suggest that the main urinary glycosaminoglycan chondroitin sulfate is of systemic origin and filtered in the kidney, while the minor components dermatan sulfate and heparan sulfate may come from the urinary tract. the urinary glycosaminoglycan concentration was greatly decreased in animals with FUS, as compared to normal adults. We hypothesize that these low glycosaminoglycan levels reflect a damage to the bladder surface, resulting in absorption and/or degradation of the endogenous urinary glycosaminoglycans. (C) 2004 Elsevier B.V. All rights reserved.
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    Characterization of glycosaminoglycans in tubular epithelial cells: Calcium oxalate and oxalate ions effects
    (Blackwell Publishing, 2005-10-01) Borges, Fernanda Teixeira [UNIFESP]; Michelacci, Yara Maria [UNIFESP]; Aguiar, Jair Adriano Kopke [UNIFESP]; Dalboni, Maria Aparecida [UNIFESP]; Garofalo, Andrezza S.; Schor, Nestor [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Background. the interaction between tubular epithelial cells and calcium oxalate crystals or oxalate ions is a very precarious event in the lithogenesis. Urine contains ions, glycoproteins and glycosaminoglycans that inhibit the crystallization process and may protect the kidney against lithogenesis. We examined the effect of oxalate ions and calcium oxalate crystals upon the synthesis of glycosaminoglycans in distal [Madin-Darby canine kidney (MDCK)] and proximal (LLC-PK1) tubular cell lines.Methods. Glycosaminoglycan synthesis was analyzed by metabolic labeling with S-35-sulfate and enzymatic digestion with specific mucopolysaccharidases. Cell death was assessed by fluorescent dyes and crystal endocytosis was analised by flow cytometry.Results. the main glycosaminoglycans synthesized by both cells were chondroitin sulfate and heparan sulfate most of them secreted to the culture medium or present at cellular surface. Exposition of MDCK cells to oxalate ions increased apoptosis rate and the incorporation of S-35-sulfate in chondroitin sulfate and heparan sulfate, while calcium oxalate crystals were endocyted by LLC-PK1, induced necrotic cell death, and increased S-35-sulfate incorporation in glycosaminoglycans. These effects seem to be specific and due to increased biosynthesis, since hydroxyapatite and other carboxylic acid did not induced cellular death or glycosaminoglycan synthesis and no changes in sulfation degree or molecular weight of glycosaminoglycans could be detected. Thapsigargin inhibited the glycosaminoglycan synthesis induced by calcium oxalate in LLC-PK1, suggesting that this effect was sensitive to the increase in cytosolic calcium.Conclusion. Tubular cells may increase the synthesis of glycosaminoglycans to protect from the toxic insult of calcium oxalate crystals and oxalate ions, what could partially limit the lithogenesis.
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    Effect of epithelial debridement on glycosaminoglycan synthesis by human corneal explants
    (Elsevier B.V., 2000-05-01) Soriano, E. S.; Campos, MSQ; Michelacci, Y. M.; Universidade Federal de São Paulo (UNIFESP)
    The purpose of the present work was to investigate the effects of mechanical epithelial debridement upon glycosaminoglycan synthesis by human corneal explants Corneal explants were maintained under tissue culture conditions for 2-72 days and the glycosaminoglycans synthesized in 24 h were metabolically labeled by addition of S-35-sulfate to the culture medium. These compounds were isolated from the tissue explants and analyzed by a combination of agarose gel electrophoresis and enzymatic degradation with specific mucopolysaccharidases. the glycosaminoglycans synthesized by isolated epithelial cells and by corneas previously submitted to epithelial cell debridement were compared to controls. Keratan sulfate (26 kDa) and dermatan sulfate (43 kDa) were the main corneal glycosaminoglycans,each one corresponding to about 50% of the total. Nevertheless, the main S-35-labeled glycosaminoglycan was S-35-dermatan sulfate (73%), with smaller amounts of S-35-keratan sulfate (15%) and S-35-heparan sulfate (12%), suggesting a lower synthesis rate for keratan sulfate. the main glycosaminoglycan synthesized by isolated epithelial cells was heparan sulfate. the removal of epithelial layer caused a decrease in heparan sulfate labeling and induced the synthesis of dermatan sulfate by stromal cells. This increased synthesis of dermatan sulfate suggest a relationship between epithelium and stroma and could be related to the corneal opacity that may appear after epithelial cell debridement. (C) 2000 Elsevier Science B.V. All rights reserved.
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    Effect of epithelial debridement on human cornea proteoglycans
    (Associação Brasileira de Divulgação Científica, 2001-03-01) Soriano, Eduardo Sone [UNIFESP]; Campos, Mauro Silveira de Queiroz [UNIFESP]; Aguiar, Jair Adriano Kopke [UNIFESP]; Michelacci, Yara Maria [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Corneal transparency is attributed to the regular spacing and diameter of collagen fibrils, and proteoglycans may play a role in fibrillogenesis and matrix assembly. Corneal scar tissue is opaque and this opacity is explained by decreased ultrastructural order that may be related to proteoglycan composition. Thus, the objectives of the present study were to characterize the proteoglycans synthesized by human corneal explants and to investigate the effect of mechanical epithelial debridement. Human corneas unsuitable for transplants were immersed in F-12 culture medium and maintained under tissue culture conditions. The proteoglycans synthesized in 24 h were labeled metabolically by the addition of 35S-sulfate to the medium. These compounds were extracted by 4 M GuHCl and identified by a combination of agarose gel electrophoresis, enzymatic degradation with protease and mucopolysaccharidases, and immunoblotting. Decorin was identified as the main dermatan sulfate proteoglycan and keratan sulfate proteoglycans were also prominent components. When the glycosaminoglycan side chains were analyzed, only keratan sulfate and dermatan sulfate were detected (~50% each). Nevertheless, when these compounds were 35S-labeled metabolically, the label in dermatan sulfate was greater than in keratan sulfate, suggesting a lower synthesis rate for keratan sulfate. 35S-Heparan sulfate also appeared. The removal of the epithelial layer caused a decrease in heparan sulfate labeling and induced the synthesis of dermatan sulfate by the stroma. The increased deposit of dermatan sulfate proteoglycans in the stroma suggests a functional relationship between epithelium and stroma that could be related to the corneal opacity that may appear after epithelial cell debridement.
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    Effects of Training and Overtraining on Intervertebral Disc Proteoglycans
    (Lippincott Williams & Wilkins, 2018) Ueta, Renato H. S. [UNIFESP]; Tarini, Victor A. F. [UNIFESP]; Franciozi, Carlos E. S. [UNIFESP]; Tamaoki, Marcel J. S. [UNIFESP]; Medeiros, Valquiria P.; Nader, Helena B. [UNIFESP]; Faloppa, Flavio [UNIFESP]
    Study Design. Animal experimental study. Objective. Evaluate the effect of physical activity and overtraining condition on glycosaminoglycan concentration on the intervertebral disc (IVD) using a rat running model. Summary of Background Data. Some guidelines recommend the implementation of a physical exercise program as treatment for low back pain however, cyclic loading impact on the health of the IVD and whether there is a dose-response relationship is still incompletely understood. Methods. Thirty-two rats ages 8 weeks were divided into four groups with eight animals each. The first 8 weeks were the adaptive phase, the overtraining phase was from the ninth to the eleventh week, which consisted of increasing the number of daily training sessions from 1 to 4 and the recovery phase was represented by the 12th and 13th weeks without training. Control group 1 (CG1) did not undergo any kind of training. Control group 2 (CG2) completed just the adaptive phase. Overtraining group 1 (OT1) completed the overtraining phase. Overtraining group 2 (OT2) completed the recovery phase. Running performance tests were used to assess the "overtraining'' status of the animals. IVD glycosaminoglycans were extracted and quantified, and identified by electrophoresis. Results. Glycosaminoglycans showed a distribution between chondroitin sulfate and dermatan sulfate. Glycosaminoglycans quantification showed decreasing concentration at the following order: OT1 > CG2 > OT2 > CG1. Increased expression of dermatan sulfate was verified at the groups submitted to any training. Conclusion. Overtraining condition, as assessed by muscle and cardiovascular endurance did not lessen glycosaminoglycan concentration in the IVD. In fact, physical exercise increased glycosaminoglycan concentration in the IVD in proportion to the training load, even at overtraining condition, returning to normal levels after the recovery phase and glycosaminoglycan production is a reversible acute positive response for mechanical stimulation of the IVD.
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    Enzyme and integrin expression by high and low metastatic melanoma cell lines
    (Lippincott Williams & Wilkins, 2003-02-01) Staquicini, F. I.; Moreira, C. R.; Nascimento, F. D.; Tersariol, ILS; Nader, H. B.; Dietrich, C. P.; Lopes, J. D.; Universidade Federal de São Paulo (UNIFESP); Univ Mogi Cruzes
    Dissemination of a malignant tumour is the result of a cascade of events beginning with detachment of cells from primary tumour followed by extravasation and growth at secondary sites. the differences in metastatic ability could be attributed to properties intrinsic to the various tumour types. Thus the clonal selection of tumour cells from successive metastases apparently results in cells better equipped for survival and formation of colonies in secondary sites, indicating that survival is not a random phenomenon. Many studies of malignant cells have correlated the overexpression of adhesion receptors such as integrins and the production of cysteine proteases and glycosidases with the progression of malignancy. the interaction of cysteine proteases with basement membrane components has been implicated in tumour invasion, activation of hormones and growth factors. On the other hand, the expression of the heparanase gene and its protein has been associated with the metastatic potential of several human and mouse tumour cell lines. This study aimed to investigate the correlations between the metastatic properties of clones with high and low metastatic potential and their ability to adhere to the extracellular matrix and to degrade proteins and sulphated glycosaminoglycans present there. Clonal selection of the B16F10 cell line was performed, and the clones were examined for the expression of an integrin-type laminin receptor. A significantly higher level was detected in a high metastatic clone. Enzymatic assays showed higher activity for alpha-D-N-acetylglucosaminidase, beta-D-N-acetylgalactosaminidase and beta-D-glucuronidase in conditioned medium from low metastatic clones compared with that from high metastatic clones. However, higher endopeptidase activity was observed in conditioned medium from high metastatic clones. in summary, these results showed a positive correlation between high metastatic potential and endopeptidase secretion. Similarly, a positive correlation was observed between low metastatic cells and the secretion of glycosaminoglycan-degrading glycosidases.
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    Galactosaminoglycans from normal myometrium and leiomyoma
    (Associação Brasileira de Divulgação Científica, 2001-05-01) Berto, A.g.a. [UNIFESP]; Oba-Shinjo, Sueli Mieko [UNIFESP]; Michelacci, Yara Maria [UNIFESP]; Sampaio, L.o. [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    In many tumors, the amount of chondroitin sulfate in the extracellular matrix has been shown to be elevated when compared to the corresponding normal tissue. Nevertheless, the degree of chondroitin sulfate increase varies widely. In order to investigate a possible correlation between the amount of chondroitin sulfate and tumor size, several individual specimens of human leiomyoma, a benign uterine tumor, were analyzed. The glycosaminoglycans from eight tumors were extracted and compared with those from the respective adjacent normal myometrium. The main glycosaminoglycan found in normal myometrium was dermatan sulfate, with small amounts of chondroitin sulfate and heparan sulfate. In leiomyoma, both dermatan sulfate and chondroitin sulfate were detected and the total amounts of the two galactosaminoglycans was increased in all tumors when compared to normal tissue. In contrast, the heparan sulfate concentration decreased in the tumor. To assess the disaccharide composition of galactosaminoglycans, these compounds were incubated with bacterial chondroitinases AC and ABC. The amounts of L-iduronic acid-containing disaccharides remained constant, whereas the concentration of D-glucuronic acid-containing disaccharides increased from 2 to 10 times in the tumor, indicating that D-glucuronic acid-containing disaccharides are responsible for the elevation in galactosaminoglycan concentration. This increase is positively correlated with tumor size.
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    Heparan sulfate and cell division
    (Associação Brasileira de Divulgação Científica, 1999-05-01) Porcionatto, Marimélia Aparecida [UNIFESP]; Nader, Helena Bonciani [UNIFESP]; Dietrich, Carl Peter [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Heparan sulfate is a component of vertebrate and invertebrate tissues which appears during the cytodifferentiation stage of embryonic development. Its structure varies according to the tissue and species of origin and is modified during neoplastic transformation. Several lines of experimental evidence suggest that heparan sulfate plays a role in cellular recognition, cellular adhesion and growth control. Heparan sulfate can participate in the process of cell division in two distinct ways, either as a positive or negative modulator of cellular proliferation, or as a response to a mitogenic stimulus.
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    An improved methodology to produce Flavobacterium heparinum chondroitinases, important instruments for diagnosis of diseases
    (Wiley-Blackwell, 2003-04-01) Aguiar, JAK; Lima, C. R.; Berto, AGA; Michelacci, Y. M.; Universidade Federal de São Paulo (UNIFESP)
    Chondroitinases are very important tools for the identification and structural analysis of proteoglycans. Enzymic analysis with Flavobacterium heparinum chondroitinases has shown that chondroitin sulphate and dermatan sulphate structures are modified in many human diseases, suggesting a diagnostic value for these enzymes. Furthermore, it was recently shown that F. heparinum chondroitinases AC and B inhibit tumoural cell growth, invasion and angiogenesis. Due to the increasing importance of F. heparinum chondroitinases, we investigated optimized conditions for preparation and assay of chondroitinases AC, B and C. the Dimethylmethylene Blue assay was modified and fully developed to measure the chondroitinase activities of crude extracts of F. heparinum. This method estimates chondroitin sulphate or dermatan sulphate depolymerization upon the digestion of chondroitinase, and was compared with A(232), which measures the unsaturated products formed. Trypticase was the best culture medium, both for bacterial growth and enzyme induction. the chondroitinases were solubilized by ultrasound under conditions that do not completely disrupt the cells, suggesting that they are located at the periplasmic space. Maximum chondroitinase induction occurred in the presence of 0.2-1.0 g/l chondroitin sulphate. Chondroitin sulphate-degraclation products were also inducers, but heparin and heparan sulphate were not. Chondroitinases AC, B and C were separated from each other by hydrophobic-interaction chromatography on Phenyl-Sepharose HP. When contaminant proteins were first removed from crude extract by Q-Sepharose, the chondroitinases could be purified to homogeneity in this phenyl-Sepharose chromatographic step.
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    Interaction of heparin with internally quenched fluorogenic peptides derived from heparin-binding consensus sequences, kallistatin and anti-thrombin III
    (Portland Press, 2002-09-01) Pimenta, Daniel C.; Nantes, Iseli L.; Souza, Eduardo S. de; Le Bonniec, Bernard; Ito, Amando S.; Tersariol, Ivarne Luis dos Santos [UNIFESP]; Oliveira, Vitor; Juliano, Maria Aparecida [UNIFESP]; Juliano, Luiz [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); CEPID; UMC; Universidade de São Paulo (USP); Univ Paris 05
    Internally quenched fluorogenic (IQF) peptides bearing the fluorescence donor/acceptor pair o-aminobenzoic acid (Abz)/N-(2,4-dinitrophenyl)ethylenediamine (EDDnp) at N- and C-terminal ends were synthesized containing heparin-binding sites from the human serpins kallistatin and antithrombin, as well as consensus heparin-binding sequences (Cardin clusters). the dissociation constant (K-d), as well as the stoichiometry for the heparin-peptide complexes, was determined directly by measuring the decrease in fluorescence of the peptide solution. Experimental procedures were as sensitive as those used to follow the fluorescence change of tryptophan in heparin-binding proteins. the conformation of the peptides and the heparin-peptide complexes were obtained from measurements of time-resolved fluorescence decay and CD spectra. Kallistatin (Arg(300)-Pro(319))-derived peptide (HC2) and one derived from antithrombin III helix D [(AT3D), corresponding to Ser(112)-Lys(139)], which are the heparin-binding sites in these serpins, showed significant affinity for 4500 Da heparin, for which K-d values were 17 nM and 100 nM respectively. the CD spectra of the heparin-HC2 peptide complex did not show any significant alpha-helix content, different from the situation with peptide AT3D, for which complex-formation with heparin resulted in 24% alpha-helix content. the end-to-end distance distribution and the time-resolved fluorescence-decay measurements agree with the CD spectra and K-d values. the synthetic alpha-methyl glycoside pentasaccharide AGA*IA(M) (where A represents N,6-O-sulphated alpha-D-glucosamine; G, beta-D-glucuronic acid; A*, N,3, 6-O-sulphated alpha-D-glucosamine; 1, 2-O-sulphated alpha-L-iduronic acid; and A(M), alpha-methyl glycoside of A) also binds to AT3D and other consensus heparin-binding sequences, although with lower affinity. the interaction of IQF peptides with 4500 Da heparin was displaced by protamine. in conclusion, IQF peptides containing Abz/EDDnp as the donor/acceptor fluorescence pair are very promising tools for structure-activity relationship studies on heparin-peptide complexes, as well as for the development of new peptides as heparin reversal-effect compounds.
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    Proteoglycans and glycosaminoglycans synthesized in vitro by mesangial cells from normal and diabetic rats
    (Elsevier B.V., 1996-05-21) Hadad, S. J.; Michelacci, Y. M.; Schor, N.; Universidade Federal de São Paulo (UNIFESP)
    In the renal glomerulus, two extracellular matrices have been identified, the glomerular basement membrane and the mesangial matrix. Accumulation of glomerular extracellular matrix is a conspicuous feature of most forms of progressive glomerular disease, including diabetic nephropathy. Since proteoglycans are prominent components of the extracellular matrix, we examined the glycosaminoglycans and proteoglycans synthesized in vitro by mesangial cells from normal and diabetic rats. A mixture of dermatan sulfate and heparan sulfate was recovered. Dermatan sulfate was the predominant glycosaminoglycan synthesized and most of it was released to the culture medium, in contrast to heparan sulfate which was found to be cell associated to a higher degree. the dermatan sulfate chains are composed by D-glucuronic and L-iduronic acid-containing disaccharides and are highly sulfated. Mesangial cells from diabetic rats produce much more glycosaminoglycans than mesangial cells from normal rats, especially dermatan sulfate and this increase was proportional to the duration of diabetes. in contrast, exposure of mesangial cell from normal rats to elevated glucose did not lead to any changes in glycosaminoglycan synthesis, indicating that this short-term culture conditions may not adequately simulate diabetes mellitus. Other factors related to diabetes environment may be responsible for the observed alterations. the dermatan sulfate was secreted to the medium as proteoglycan, Two dermatan sulfate proteoglycans were identified, with molecular weights of 120 and 85 kDa respectively. the proteoglycan core protein M(r) was 45 kDa and the dermatan sulfate chains were 35 kDa. It is possible that the two proteoglycans represent two populations, one with two dermatan sulfate side chains (120 kDa) and the other with only one side chain (85 kDa), presumably fitting in the decorin/biglycan family of small proteoglycans.
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    Putative role of heparan sulfate proteoglycan expression and shedding on the proliferation and survival of cells after photodynamic therapy
    (Elsevier B.V., 2007-01-01) Pazos, Marcelo de Castro; Ricci, Ritchelli; Simioni, Andreza R.; Lopes, Carla C.; Tedesco, Antonio C.; Nader, Helena B.; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP)
    Introduction: Photodynamic therapy is based on the selective retention of a photosensitizer by highly proliferating cells and its activation with light at the appropriate wavelength. This combination generates reactive oxygen species that ultimately kill the cells. Some cells, however, may survive photodynamic therapy and the interaction of these cells with the extracellular matrix has profound effect in tumor biology. the knowledge of photodynamic therapy action on the extracellular matrix has not been fully explored. It has been focused mainly on integrins, matrix metalloproteinases and on growth factors and immunological mediators. Other important molecules involved in the regulation of many cell processes are the glycosaminoglycans, polymers of disaccharide units, present on the cell surface and in the extracellular matrix. in most cases, the glycosaminoglycans occur as proteoglycans.Aims: the purpose of the present investigation is to evaluate heparan sulfate proteoglycan expression and shedding, and its relation to the survival of the remaining cells, after a liposomal-AlClPc based photodynamic treatment.Materials: A wild-type endothelial cell derived from rabbit aorta and its counterpart transfected with EJ-ras oncogene were used.Results: Both cell lines presented augmented heparan sulfate proteoglycan syndecan-4 mRNA expression, augmented synthesis of heparan sulfate chains and increased shedding. Also, the formation of stress fibers on the border of the cells and the arrest in G, phase of the cell cycle was observed.Conclusions: These results show that surviving cells after photodynamic therapy exhibit changes in their morphology and cell processes that differ from that of non-treated cells, and these changes are probably hindering the cells from resuming normal proliferation. (C)2007 Elsevier B.V. All rights reserved.
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    Reduced urinary excretion of sulfated polysaccharides in diabetic rats
    (Elsevier B.V., 2005-06-30) Lima, Cilene Rebouças de [UNIFESP]; Aguiar, Jair Adriano Kopke [UNIFESP]; Michelacci, Yara Maria [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    The aim of the present study was to further understand the changes in renal filtration that occur in the early stages of diabetes mellitus. Diabetes was induced in male Wistar rats by a single injection of streptozotocin. Glycemia, body weight, 24-h urine volume and urinary excretion of creatinine, protein and glycosaminoglycans were measured 10 and 30 days after diabetes induction. All the diabetic animals used in the present study were hyperglycemic, did not gain weight, and presented proteinuria and creatinine hyperfiltration. in contrast, the glycosaminoglycan excretion decreased. Dextrain sulfates of different molecular weights (6.0 to 11.5 kDa) were administered to the diabetic rats, and to age-matched, sham-treated controls. Most of the dextran sulfate was excreted during the first 24 h, and the amounts excreted in the urine were inversely proportional to the dextran sulfate molecular weight for all groups. Nevertheless, diabetic rats excreted less and accumulated more dextran sulfate in kidney and liver, as compared to controls. These differences, which were observed only for the dextran sulfates of higher molecular weights (> 7 kDa), increased with the duration of diabetes. Our findings suggest differential renal processing mechanisms for proteins and sulfated polysaccharides, with the possible involvement of kidney cells. (c) 2004 Elsevier B.V. All rights reserved.
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    Reliability of 1,9-dimethylmethylene blue tests in comparison to agarose gel electrophoresis for quantification of urinary glycosaminoglycans
    (Elsevier B.V., 2007-03-01) Lima, Cilene R. de; Baccarin, Raquel Yvonne Arantes; Michelacci, Yara M.; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP)
    Background: the relevance of glycosaminoglycan determination in biological fluids is gradually gaining importance in the literature. Nevertheless, the results obtained by different methods vary widely. We evaluated 1,9-dimethylmethylene blue (DMB) dye-binding assays for quantification of urinary glycosaminoglycans, in comparison to densitometry after agarose gel electrophoresis.Methods: Urinary glycosaminoglycans from different mammalian species were quantified by 3 different DMB dye-binding assays. the results were compared to those obtained by densitometry after agarose gel electrophoresis of glycosaminoglycans isolated from urine samples by ion exchange chromatography.Results: Densitometry after agarose gel electrophoresis showed glycosaminoglycan urinary concentrations of 1-20 mg/l, and glycosaminoglycan/creatinine ratios of 2-25 x 10(-3), for all the mammalian species here studied. A decrease with age was observed for humans, cats and horses. in comparison, DMB assays gave much higher results - up to 200 mg/l and 500 x 10(-3) glycosaminoglycan/creatinine ratios. These values were greatly reduced after 4-h dialysis, suggesting that low molecular weight compounds do interfere. Furthermore, urinary anions such as sulfate, phosphate and citrate, react with metachromatic dyes, such as Toluidine Blue and DMB.Conclusion: DMB assays, although rapid and simple, are not appropriate to quantify urinary glycosaminoglycans in normal mammalians, since other urinary components interfere with the reactions. (c) 2006 Elsevier B.V. All rights reserved.
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    Synovial fluid chondroitin sulphate indicates abnormal joint metabolism in asymptomatic osteochondritic horses
    (Wiley-Blackwell, 2012-07-01) Machado, T. S. L.; Correia da Silva, L. C. L.; Baccarin, Raquel Yvonne Arantes [UNIFESP]; Michelacci, Y. M. [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP)
    Reasons for performing study: Alternative methods to evaluate the joint condition in asymptomatic osteochondrosis dissecans (OCD) and other joint diseases may be useful. Objectives: To investigate possible changes in synovial fluid composition that may lead to joint conditions in asymptomatic OCD, in mature horses. Methods: Animals aged >2 years, of different breeds, with OCD in the intermediate ridge of distal tibia, symptomatic or not, were studied. Synovial fluid samples (10 healthy; 11 asymptomatic OCD; 25 symptomatic OCD) were collected by arthroscopy from 29 horses. Glycosaminoglycans (GAGs) were analysed by a combination of agarose gel electrophoresis and enzymatic degradation with specific GAG lyases. the viscosity, white blood cell (WBC) count, protein concentration and hyaluronic acid (HA) molecular weight were also determined. Results: the method used here to analyse synovial fluid GAGs is reliable, reproducible and specific. the main synovial fluid GAGs are HA and chondroitin sulphate (CS), 93% and 7% respectively in normal horses. in symptomatic OCD, the concentrations of both increased (expressed as GAG/urea ratios), but CS increased more. the CS increased also in asymptomatic OCD. An inflammatory reaction was suggested by the increased WBC counts in OCD. the molecular weight of the synovial fluid HA was reduced in OCD, explaining the lower viscosity observed. Conclusions: the increased CS in synovial fluid of OCD joints in mature horses suggests that the synovial fluid CS and the WBC count are good markers of the joint conditions, allowing the identification of pathological phase in joint diseases. Potential relevance: the analysis of synovial fluid GAGs shows that cartilage damage occurs even in asymptomatic OCD, implying that arthroscopic removal of osteochondral fragments should be performed even in asymptomatic OCD.
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    Urinary excretion of glycosaminoglycans and albumin in experimental diabetes mellitus
    (Oxford Univ Press, 2000-02-01) Cadaval, Augusto de Miranda [UNIFESP]; Kohlman, Oswaldo [UNIFESP]; Michelacci, Yara Maria [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Diabetes mellitus was induced in one group of rats by a single injection of streptozotocin. the glycemia, the body weight, and the blood systolic pressure were measured every week, and the 24 h urine volume and urinary excretions of creatinine, albumin and glycosaminoglycans were measured every 2 weeks, At the end of the experiment (12 weeks) the weight and the glycosaminoglycan composition of the kidneys were determined. All the diabetic animals were hyperglycemic, hypertense, and did not gain weight during all the experimental period. Albuminuria appeared from the second week on. Rat urine was shown to contain heparan sulfate, chondroitin sulfate, and dermatan sulfate, and the glycosaminoglycan excretion decreased in all diabetic animals. the onset of the change in glycosaminoglycan excretion rate was a very early event, appearing in the second week after diabetes induction. the main glycosaminoglycan found in normal rat kidney was heparan sulfate and, in contrast to the urine, the total kidney glycosaminoglycans increased in diabetic kidney, due to chondroitin sulfate and dermatan sulfate accumulation. the heparan sulfate concentration (per tissue dry weight) did not change. Our results suggest that quantification of urinary glycosaminoglycans may be a useful tool for the early diagnosis of diabetic nephropathy.