Glycomics expression analysis of sulfated glycosaminoglycans of human colorectal cancer tissues and non-neoplastic mucosa by electrospray ionization mass spectrometry / Análise da expressão de glicosaminoglicanos sulfatados no tecido humano neoplásico colorretal e na mucosa não neoplásica por espectrometria de massa por ionização por electrospray

ABSTRACT Objective To determine the presence of glycosaminoglycans in the extracellular matrix of connective tissue from neoplastic and non-neoplastic colorectal tissues, since it has a central role in tumor development and progression. Methods Tissue samples from neoplastic and non-neoplastic colorectal tissues were obtained from 64 operated patients who had colorectal carcinoma with no distant metastases. Expressions of heparan sulphate, chondroitin sulphate, dermatan sulphate and their fragments were analyzed by electrospray ionization mass spectrometry, with the technique for extraction and quantification of glycosaminoglycans after proteolysis and electrophoresis. The statistical analysis included mean, standard deviation, and Student’st test. Results The glycosaminoglycans extracted from colorectal tissue showed three electrophoretic bands in agarose gel. Electrospray ionization mass spectrometry showed characteristic disaccharide fragments from glycosaminoglycans, indicating their structural characterization in the tissues analyzed. Some peaks in the electrospray ionization mass spectrometry were not characterized as fragments of sugars, indicating the presence of fragments of the protein structure of proteoglycans generated during the glycosaminoglycan purification. The average amount of chondroitin and dermatan increased in the neoplastic tissue compared to normal tissue (p=0.01). On the other hand, the average amount of heparan decreased in the neoplastic tissue compared to normal tissue (p= 0.03). Conclusion The method allowed the determination of the glycosaminoglycans structural profile in colorectal tissue from neoplastic and non-neoplastic colorectal tissue. Neoplastic tissues showed greater amounts of chondroitin sulphate and dermatan sulphate compared to non-neoplastic tissues, while heparan sulphate was decreased in neoplastic tissues.

RESUMO Objetivo Determinar a presença de glicosaminoglicanos na matriz extracelular do tecido conjuntivo colorretal neoplásico e não neoplásico, tendo em vista seu papel central no desenvolvimento e na progressão dos tumores. Métodos Amostras de tecidos colorretais neoplásicos e não neoplásicos foram obtidas de 64 pacientes operados com carcinoma colorretal sem metástases a distância. As expressões de heparan sulfato, sulfato de condroitina e sulfato de dermatan e seus fragmentos foram analisadas por espectrometria de massa por ionização por electrospray, com técnica de extração e quantificação de glicosaminoglicanos após proteólise e eletroforese. Para análise estatística, utilizaram-se média, desvio padrão e teste t de Student. Resultados Em gel de agarose, os glicosaminoglicanos extraídos de tecido colorretal mostraram três bandas eletroforéticas. A espectrometria de massa por ionização por electrospray mostrou fragmentos de dissacarídeos característicos de glicosaminoglicanos e indicou sua característica estrutural. Alguns picos na espectrometria de massa por ionização por electrospray não foram caracterizados como fragmentos de açúcares, sugerindo a presença de fragmentos de proteínas estruturais dos proteoglicanos, formadas durante a purificação dos glicosaminoglicanos. A quantidade média de condroitina e dermatan aumentou no tecido neoplástico em relação ao tecido normal (p=0,01). Por outro lado, a quantidade média de heparan foi menor no tecido neoplásico em relação ao tecido normal (p=0,03). Conclusão O método empregado permitiu determinar o perfil estrutural dos glicosaminoglicanos nas amostras. Tecidos neoplásicos apresentaram maiores quantidades de sulfato de condroitina e sulfato de dermatan em comparação com os não neoplásicos, enquanto o sulfato de heparan foi encontrado em menores quantidades nos tecidos neoplásicos.

Mucopolysaccharidosis VI: Evaluation After 2 Years of Treatment

Abstract Introduction: Mucopolysaccharidosis VI (MPS VI) is the result of the absence of arylsulfatase B leading to the abnormal lysosomal accumulation of glycosaminoglycans. Two different phenotypes have been described to date, namely, rapidly progressive and slowly progressive. Aim: To present the evolution of a slowly progressive phenotype of MPS VI in a patient after 2 years of enzyme replacement therapy. Case report: A 26-year-old man diagnosed with MPS VI at 9 years of age started enzyme replacement therapy with galsulfase due to cardiac, pulmonary, neurologic, and joint involvement. After 10 months of treatment, improvement in quality-of-life scales and walk test was evident. Because of persistent symptomatology associated with narrow cervical spinal canal, decompressive surgery was performed. After 2 years of treatment, there was a clear improvement in the respiratory, motor, and cardiac functions as well as in the spinal symptoms. Discussion: The evolution of our patient leads to the conclusion that the combined treatment of galasulfase and decompressive surgery should be indicated at an early stage in order to achieve best outcome for the patient.

Dermatan sulfate reduces monocyte chemoattractant protein 1 and TGF-β production, as well as macrophage recruitment and myofibroblast accumulation in mice with unilateral ureteral obstruction

Selectins play an essential role in most inflammatory reactions, mediating the initial leukocyte-rolling event on activated endothelium. Heparin and dermatan sulfate (DS) bind and block P- and L-selectin function in vitro. Recently, we reported that subcutaneous administration of DS inhibits colon inflammation in rats by reducing macrophage and T-cell recruitment and macrophage activation. In the present study, we examined the effect of porcine intestinal mucosa DS on renal inflammation and fibrosis in mice after unilateral ureteral obstruction (UUO). Twenty-four adult male Swiss mice weighing 20-25 g were divided into 4 groups: group C (N = 6) was not subjected to any surgical manipulation; group SH (N = 6) was subjected to surgical manipulation but without ureter ligation; group UUO (N = 6) was subjected to unilateral ureteral obstruction and received no treatment; group UUO plus DS (N = 6) was subjected to UUO and received DS (4 mg/kg) subcutaneously daily for 14 days. An immunoblot study was also performed for TGF-β. Collagen (stained area ~3700 µm²), MCP-1 (stained area ~1700 µm²), TGF-β (stained area ~13 percent of total area), macrophage (number of cells ~40), and myofibroblast (stained area ~1900 µm²) levels were significantly (P < 0.05) higher in the UUO group compared to control. DS treatment significantly (P < 0.05) reduced the content of collagen (stained area ~700 µm²), MCP-1 (stained area ~160 µm²) and TGF-β (stained area ~5 percent of total area), in addition to myofibroblast (stained area ~190 µm²) and macrophage (number of cells ~32) accumulation in the obstructed kidney. Overall, these results indicate that DS attenuates kidney inflammation by reducing macrophage recruitment, myofibroblast population and fibrosis in mice submitted to UUO.

Estudo bioquímico do glicosaminoglicano dermatam sulfato em homens adultos portadores de hérnia inguinal tipo II de Nyhus / Biochemical study of dermatan sulfate glycosaminoglycan in adult male patients with Nyhus type II inguinal hernia

OBJETIVO: Comparar a quantidade do glicosaminoglicano dermatam sulfato entre pacientes homens, portadores de hérnia inguinal tipo II de Nyhus e, indivíduos sem hérnia inguinal, com idade entre 20 e 40 anos. MÉTODOS: Foram constituídos dois grupos. Um de 15 pacientes do sexo masculino com hérnia inguinal tipo II de Nyhus e idade entre 20 e 40 anos, com risco ASA I e II, e um grupo controle com dez indivíduos, também do sexo masculino entre 20 e 40 anos, que morreram em período de até 24 h. Foram excluídos os pacientes do sexo feminino, diabéticos, portadores de doença do tecido conjuntivo, tabagistas e com risco cirúrgico ASA III e IV. Foi retirada uma amostra de 1cm² da fáscia transversal na parte intermediária do trígono inguinal, e 1cm² na bainha anterior do músculo reto abdominal na região inguinal correspondente e quantificados os glicosaminoglicanos dermatam sulfato por densitometria, após eletroforese em gel de agarose. RESULTADOS: A quantidade de dermatam sulfato não apresentou diferença estatisticamente significante entre os pacientes com hérnia inguinal e os indivíduos sem hérnia inguinal, tanto na fáscia transversal (p=0,108) quanto na bainha anterior do músculo reto abdominal (p=0,292). CONCLUSÃO: Não se encontrou diferença na quantidade do glicosaminoglicano dermatam sulfato entre os pacientes portadores de hérnia inguinal tipo II de Nyhus e indivíduos sem hérnia inguinal em homens adultos.

OBJECTIVE: To compare the amount of the dermatan sulfate glycosaminoglycan between male patients with Nyhus type II inguinal hernias and subjects without inguinal hernia, aged between 20 and 40 years. METHODS: Two groups were formed: One with 15 male patients with Nyhus type II inguinal hernia and aged between 20 and 40 years with ASA risk I and II, and a control group of ten individuals, also males between 20 and 40, who had died up to 24 h before. We excluded female patients, diabetic patients with connective tissue disease, smokers and surgical risk ASA III and IV. We resected a sample of 1 cm² of the transversalis fascia in the middle of the inguinal trigone, and 1 cm² of the anterior sheath of the rectus abdominis muscle in the groin for the quantification of dermatan sulfate glycosaminoglycans by densitometry after agarose gel electrophoresis. RESULTS: The amount of dermatan sulfate showed no statistically significant difference between patients with inguinal hernia and individuals without inguinal hernia in both the transverse fascia (p = 0.108) and anterior sheath of the rectus abdominis muscle (p = 0.292). CONCLUSION: There was no difference in the amount of the dermatan sulfate glycosaminoglycan among patients with Nyhus type II inguinal hernias and subjects without inguinal hernia in adult males.

Glycosaminoglycan distribution in the rat uterine cervix during the estrous cycle

OBJECTIVE: To analyze the amount of glycosaminoglycans in the uterine cervix during each phase of the rat estrous cycle. DESIGN: Based on vaginal smears, forty female, regularly cycling rats were divided into four groups (n = 10 for each group): GI - proestrous, GII - estrous, GIII - metaestrous and GIV - diestrous. Animals were sacrificed at each phase of the cycle, and the cervix was immediately removed and submitted to biochemical extraction and determination of sulfated glycosaminoglycans and hyaluronic acid. The results were analyzed by ANOVA followed by the Bonferroni post-hoc test. RESULTS: The uterine cervix had the highest amount of total sulfated glycosaminoglycans and dermatan sulfate during the estrous phase (8.90 ± 0.55 mg/g of cetonic extract, p<0.001; and 8.86 ± 0.57 mg/g of cetonic extract, p<0.001). In addition, there was more heparan sulfate at the cervix during the proestrous phase (0.185 ± 0.03 mg/g of cetonic extract) than during any other phase (p<0.001). There were no significant changes in the concentration of hyaluronic acid in the uterine cervix during the estrous cycle. CONCLUSION: Our data suggest that the amount of total sulfated glycosaminoglycans may be influenced by hormonal fluctuations related to the estrous cycle, with dermatan sulfate and heparan sulfate being the glycosaminoglycans most sensitive to hormonal change.

Separation of sulfated urinary glycosaminoglycans by highresolution electrophoresis for isotyping of mucopolysaccharidoses in Malaysia

Mucopolysaccharidoses (MPS) are a group of inherited disorders caused by the defi ciency of specifi c lysosomal enzymes involved in glycosaminoglycans (GAGs) degradation. Currently, there are 11 enzyme defi ciencies resulting in seven distinct MPS clinical syndromes and their subtypes. Different MPS syndromes cannot be clearly distinguished clinically due to overlapping signs and symptoms. Measurement of GAGs content in urine and separation of GAGs using high-resolution electrophoresis (HRE) are very useful initial screening tests for isotyping of MPS before specifi c enzyme diagnostics. In this study, we measured total urinary GAGs by a method using dimethylmethylene blue (DMB), and followed by isolation and separation of GAGs using high resolution electrophoresis (HRE) technique. Of 760 urine samples analyzed, 40 have abnormal GAGs HRE patterns. Thirty-fi ve of these 40 cases have elevated urinary GAGs levels as well. These abnormal HRE patterns could be classifi ed into 4 patterns: Pattern A (elevated DS and HS; suggestive of MPS I, II or VII; 16 cases), Pattern B (elevated HS and CS; suggestive of MPS III; 17 cases), and Pattern C (elevated KS and CS; suggestive of MPS IV, 5 cases), and Pattern D (elevated DS; suggestive of MPS VI; 2 cases). Based on the GAGs HRE pattern and a few discriminating clinical signs, we performed selective enzymatic investigation in 16 cases. In all except one case with MPS VII, the enzymatic diagnosis correlated well with the provisional MPS type as suggested by the abnormal HRE pattern. Our results showed that GAGs HRE is a useful, inexpensive and practical fi rst-line screening test when MPS is suspected clinically, and it provides an important guide to further enzymatic studies on a selective basis.

Glicosaminoglicanos en hemostasia: Acción del dermatán sulfato sobre el sistema fibrinolítico / Glycosaminoglycans on hemostasis: Dermatan sulfate action on the fibrinolytic system

El dermatán sulfato (DS) es un glicosaminoglicano endógeno, ampliamente conocido por su acción anticoagulante mediante su interacción con el cofactor II de la heparina para potenciar la inhibición de trombina. En los últimos años se ha sugerido que además el DS aumentaría la actividad fibrinolítica, aunque el mecanismo aún no ha sido completamente dilucidado. En este trabajo se presenta una revisión detallada de los resultados propios y una discusión de la bibliografía disponible respecto de la evaluación del efecto del DS sobre el sistema fibrinolítico. En estudios de activación fibrinolítica, por métodos amidolíticos y coagulométricos, el DS mostró tener efecto pro-fibrinolítico mediante potenciación de la activación de plasminógeno por t-PA y uPA, efecto independiente de su conocida acción anticoagulante. En estudios de caracterización de fibrina, las redes obtenidas en presencia de DS presentaron fibras más largas y delgadas que las redes control, mayor grado de compactación y de lisabilidad; efecto pro-fibrinolítico asociado a su acción anticoagulante. Los resultados presentados contribuyen al esclarecimiento del mecanismo de acción del DS sobre el sistema plasminógeno-plasmina y permiten plantear hipótesis sobre el rol fisiológico de este glicosaminoglicano.

Dermatan sulfate (DS) is well-known for its anticoagulant activity by binding to heparin cofactor II in order to enhance the antithrombin action. It has also been suggested that DS has a profibrinolytic effect, although the exact molecular mechanism is unknown. This review exposes the results obtained and discusses on the available literature on DS effect on the fibrynolytic system. DS exhibited a stimulating effect on the activation of plasminogen by plasminogen activators (t-PA and u-PA), by in vitro amidolytic and coagulometric methods, showing a pro-fibrinolytic effect independent of its known anticoagulant action. Studies of fibrin networks obtained in the presence of DS showed longer and thinner fibers than controls, increased degree of compaction and lisability. Thus, DS displayed a pro-fibrinolytic effect associated to its anticoagulant action. These results contribute to clarify the mechanism of DS action on the plasminogen-plasmin system and to the understanding of the physiologic role of this glycosaminoglycan.

Determination of the Concentration of New Antithrombotic Medicine Dermatan Sulfate in Blood or Urine by Spectrophotometry / 中国药房

OBJECTIVE:To determine the concentration of Dermatan Sulfate,a new antithrombotic medicine in blood or urine by spectrophotometry.METHODS:In Britton Robinson buffer solution(pH=5.8),color fading reaction occurred when Dermatan sulfate combined with neutral red dye to form ionic associate.The reduction of absorbance of the system was positively correlated to dermatan sulfate concentration,with the maximum absorption wavelength at 526 nm.RESULTS:The linear range of dermatan sulfate was 0.18～4.0?g?mL~(-1)(r=0.999 1) with a detection limit of 0.054?g?m~(-1).The average recovery was 100.3%(RSD=1.2%),with RSD of dermatan sulfate at low,medium,and high concentrations at 2.9%, 1.5%,and 1.1%,respectively.The coexisting substances did no interference on the determination results.CONCLUSION: The method is simple,accurate,and sensitive with good methodological selectivity and it achieved satisfactory results in the determination of dermatan sulfate in blood or urine.