chondroitin-sulfates 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

The fact that mucopolysaccharidoses (MPSes) are now treatable, and that the earlier treatment is initiated the better, is an indication for neonatal screening. The most efficient approach seems likely to be a multi-tier procedure in which screening for urinary glycosaminoglycan (GAG) is followed by enzyme determinations in heelprick blood of newborns screening positive. Hitherto the method of choice for the determination of GAG has been the measurement of absorbance by a complex of GAG and 1,9-dimethylmethylene blue (DMB).. We evaluated a DMB method in which absorbance by DMB is measured following its addition to the eluate obtained from paper-borne newborn urine samples and is normalized relative to urinary creatinine. Calibration is performed with chondroitin-6-sulfate (Ch-6-S).. The limits of detection and quantification of GAG were 1.98 and 5.94 mg/dl, respectively. The within-run coefficients of variation (CVs) of the GAG/creatinine ratio for 25, 31, and 70 mg/dl solutions of Ch-6-S in urine were 21.8, 16.4, and 10.5%, respectively, and the corresponding between-run CVs were 25.0, 13.5, and 10.1%. Recovery from the urine spiked with 31 mg Ch-6-S/dl was 94.8%. Accuracy was also acceptable for all other GAGs except hyaluronic acid. For neonatal screening, the diagnostic threshold was tentatively established as 800 mg GAG/g creatinine, the 95th centile of samples from 903 infants aged 3-28 days, but the value of the GAG/creatinine ratio was negatively correlated with age. Application of the new method to samples from older individuals with and without MPS achieved 100% sensitivity and specificity when used with an age-dependent threshold taken from the literature on the original DMB method.. If used in the first tier of a multi-tier screening protocol, the proposed method would allow the detection of abnormal levels of all GAGs except hyaluronic acid.

Topics: Aging; Calibration; Chondroitin Sulfates; Creatinine; Dermatan Sulfate; Glycosaminoglycans; Heparin; Heparinoids; Humans; Hyaluronic Acid; Infant, Newborn; Methylene Blue; Mucopolysaccharidoses; Neonatal Screening; Paper; Reproducibility of Results; Sensitivity and Specificity

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

The dye 1,9-dimethylmethylene blue may be used for the specific and quantitative precipitation of sulphated glycosaminoglycans as well as proteoglycans from the extracts of articular cartilage. The consumption of the dye DMMB enables to determine the amount of the proteoglycans present in the extract, by measuring the decrease of the absorption at 595 nm. The precipitated complex of DMMB and proteoglycans is used for qualitative investigations into the electrophoretic mobility of the different proteoglycans in agarose slab gel and their immunological characterization after blotting. Because of its higher sensitivity and greater simplicity the method described in this report is a promising alternative to the procedure based on alcian blue.

Topics: Animals; Cartilage, Articular; Chondroitin Sulfates; Coloring Agents; Dogs; Humerus; Methylene Blue; Proteoglycans; Sensitivity and Specificity; Staining and Labeling; Tissue Extracts

Topics: Alginates; Animals; Bacteriological Techniques; Cartilage; Cattle; Chondroitin Sulfates; Glucuronic Acid; Glycosaminoglycans; Hexuronic Acids; Hydrogen-Ion Concentration; Methylene Blue

Chondroitin sulfate was administered orally to six healthy volunteers, six patients with rheumatoid arthritis and six patients with osteoarthritis. Blood was collected at intervals before and after treatment and the glycosaminoglycan concentration was analyzed in serum using a sensitive assay based on the metachromatic reaction with 1,9-dimethylmethylene blue. The glycosaminoglycan concentration in serum before and after ingestion of chondroitin sulfate was statistically unchanged in all of the subjects studied. We suggest that chondroprotection by orally administered chondroitin sulfate is a biologically and pharmacologically unfounded theory. Any possible benefit to osteoarthritic patients after ingestion of chondroitin sulfate should be sought at the gastrointestinal rather than at the plasmatic or articular cartilage level.

Topics: Administration, Oral; Arthritis, Rheumatoid; Artifacts; Chondroitin Sulfates; Digestive System; Glycosaminoglycans; Humans; Methylene Blue; Osteoarthritis

A rapid spectrophotometric assay for sulfated glycosaminoglycans, based on reaction of these compounds with the dye 1,9-dimethylmethylene blue (DMMB), is gaining widespread acceptance. The limitations of the assay have not been explored, and we have investigated the absorption spectra of the dye in the presence of a variety of compounds in order to understand the mechanism of colour formation. The dye exhibits classical metachromasia in the presence of sulfated glycosaminoglycans, but appears to do so by a much simpler mechanism than other thiazine dyes such as Azure A. Evidence is offered to support the conclusion that dye dimer reacts with the polyanion to produce a single metachromatic species by ionic perturbation of the chromophore. As predicted, factors which disrupt dimerization, such as pyridine and pyridine nucleotides, preclude use of the assay in their presence, and high salt concentrations can give false positive readings by inducing metachromasia in the absence of polyanion. When these factors are understood, the assay can be used with convenience. In particular, it allows direct measurement of the glycosaminoglycan content of a few mg of alkaline-solubilized basement membrane.

Topics: Animals; Anions; Basement Membrane; Borohydrides; Cations; Cattle; Chondroitin Sulfates; Dermatan Sulfate; Glycosaminoglycans; Hyaluronic Acid; Kidney Glomerulus; Macromolecular Substances; Methylene Blue; Solubility; Spectrophotometry; Sulfuric Acids; Uronic Acids

The absorption spectrum of the dye 1,9-dimethylmethylene blue shifts if complexed with sulfated glycosaminoglycans. The present method uses the decrease in A633 rather than the increase in A535, described in a recent method, to measure the sulfated glycosaminoglycan content of biological samples. A conventional spectrophotometer was used to estimate the levels of sulfated glycosaminoglycan in papain extracts from intestinal wall tissue, by measuring both the A535 and the A633 and comparing them with a chondroitin sulfate standard: a highly significant correlation (r = 0.974, n = 17) was obtained. Also, interference by substances like RNA, DNA, and hyaluronic acid was similar for both methods. These results allowed us to employ a laser densitometer with a helium/neon laser emitting at 633 nm to improve the sensitivity and the capacity of the assay. The combination of a small reaction volume and a high-intensity light source allows the detection of less than 0.1 microgram chondroitin sulfate, a 40-fold improvement in sensitivity as compared with the original method. A very significant correlation (r = 0.885, n = 17) existed between results obtained with the macroassay, using a spectrophotometer, and those found by employing the microassay, using the laser densitometer. The use of microtiter plates and the screening potential of the densitometer yields an assay which is fast, very sensitive, and suitable for processing large numbers of samples.

Topics: Animals; Chondroitin Sulfates; Densitometry; Glycosaminoglycans; Humans; Intestines; Lasers; Methylene Blue; Rabbits; Reference Standards; Spectrophotometry

The release of proteoglycans from explant cultures of articular cartilage from immature and mature rabbits has been studied with the following results. At both ages the tissue proteoglycan was released in two phases: an initial extensive release (day 0 to 3) and a period of slow release (day 4 to 15). The percentage released in the initial phase was, however, significantly greater for mature (55%) than immature (38%) explants. At both ages the newly synthesized proteoglycans (in vivo labeled) were also released in two kinetic pools. Thus, graphical analysis of release data readily resolved the disappearance curves into two linear components with in vitro half-lives of 1 day and 22 days. Again, the percentage in the short half-life pool was much greater for mature (70%) than immature (40%) explants. At both ages the initial release was largely chondrocyte-mediated since freeze-thawing the tissue before culture markedly reduced proteoglycan release. At both ages the released proteoglycans were smaller than equivalent preparations of extracted proteoglycans and they were much less capable of forming aggregates with hyaluronate. The results show that there are age-dependent changes in rabbit articular cartilage that increase the proportion of proteoglycans, both total and newly synthesized, that are susceptible to rapid chondrocyte-mediated catabolism in explant cultures.

Topics: Age Factors; Animals; Cartilage, Articular; Cell Aggregation; Chondroitin Sulfates; Culture Techniques; Glycosaminoglycans; Half-Life; Hyaluronic Acid; Keratan Sulfate; Kinetics; Knee Joint; Methylene Blue; Proteoglycans; Rabbits; Time Factors