heparitin-sulfate and 1-9-dimethylmethylene-blue

The assessment of the clinical significance of chondroitin sulfate in patients with type 2 diabetes mellitus (T2DM) and diabetic nephropathy (DN) for the detection of the relationship between chondroitin sulfate (CS) structure and disease.. Healthy control (n=15), type 2 diabetic patients with normalbuminuria (n=12), and patients with microalbuminuria (n=13) were enrolled in the study. Total sulfated glycosaminoglycans (GAGs) concentration in the first morning urine was evaluated by 1,9-dimethylmethylene blue method and the composition was determined by agarose gel electrophoresis. Urinary chondroitin sulfate was quantified by a combination of treatment with specific lyase digestions and separation of products by SAX-HPLC.. GAGs concentration significantly increased in diabetic patients with microalbuminuria compared to diabetic patients with normalbuminuria. Qualitative analysis of urinary GAGs revealed the presence of chondroitin sulfate, heparan sulfate, and low-sulphated chondroitin sulphate-protein complex (LSC-PG). There was a decrease in CS and an increase in LSC-PG in the urine of patients with diabetes compared to healthy controls. Moreover, in diabetic patients, chondroitin sulfate contains more 6-sulfated disaccharide and less 4-sulfated disaccharide. There was a statistically significant difference in ratio of 6-sulfated disaccharide to 4-sulfated disaccharide among the three groups.. GAGs were significantly increased in diabetic patients with microalbuminuria. The levels of urinary GAGs, ratio of LSC-PG/CS, as well as ratio of 6-sulfated to 4-sulfated disaccharides could be useful markers for diagnosis of patients with diabetic nephropathy.

Topics: Chondroitin Sulfates; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Electrophoresis, Agar Gel; Female; Heparitin Sulfate; Humans; Male; Methylene Blue; Middle Aged

Topics: Chondroitin Sulfates; Coloring Agents; Glycosaminoglycans; Heparitin Sulfate; Humans; Methylene Blue; Reference Values; Sensitivity and Specificity; Spectrophotometry

A simple and reliable spectrophotometric method for the determination of heparan sulfate is described. The method is based on the 1,9-dimethylmethylene blue assay for sulfated glycosaminoglycans. Addition of bovine serum albumin, together with a specific NaCl concentration and pH, results in a specific decrease of heparan sulfate-based absorbance. The amount of heparan sulfate can be calculated by subtracting the values obtained in the presence of albumin from those obtained in its absence. The sensitivity is 0.5 microgram heparan sulfate. Two applications are given: the quantification of heparan sulfate in urine, including urine from patients with mucopolysaccharidosis, and the evaluation of fractions from gel filtration and ion exchange column chromatography for isolation of heparan sulfate proteoglycans.

Topics: Glycosaminoglycans; Heparitin Sulfate; Humans; Hydrogen-Ion Concentration; Methylene Blue; Mucopolysaccharidoses; Proteoglycans; Sensitivity and Specificity; Serum Albumin, Bovine; Sodium Chloride; Spectrophotometry

We measured the concentrations of urine glycosaminoglycans (GAG) by the modified dimethylmethylene blue method and the concentration of urine heparan sulfate (HS) by enzyme-linked immunosorbent assay (ELISA) in patients with various glomerular diseases. The GAG/creatinine(Crea) ratios in patients with IgA nephropathy (mean +/- SD, 0.31 +/- 0.056) and membranous nephropathy (0.41 +/- 0.115) were significantly greater than in healthy controls (0.18 +/- 0.045). Urine GAG/Crea ratios in minimal change nephrotic patients increased during remission (0.38 +/- 0.102) and decreased to normal values during the nephrotic stage (0.25 +/- 0.088). In contrast, urine HS/Crea ratios in patients with minimal change nephrotic syndrome decreased during remission (0.0069 +/- 0.0029) and increased markedly during the nephrotic period (0.047 +/- 0.0007 versus controls 0.0158 +/- 0.0046). Serial measurement in three minimal change nephrotic patients showed the similar change for the HS/Crea ratio and urine albumin excretion in the course of steroid therapy. The loss of HS from the glomerular basement membrane (GBM) may therefore be related to the pathogenesis of increased albumin excretion and measurement of urine HS excretion may be helpful for studying metabolism in renal disease, especially in patients with minimal change lesions.

Topics: Adult; Enzyme-Linked Immunosorbent Assay; Female; Glomerulonephritis, IGA; Glomerulonephritis, Membranous; Glycosaminoglycans; Heparitin Sulfate; Humans; Male; Methylene Blue; Nephrosis, Lipoid; Spectrophotometry; Urinalysis