High Performance Liquid Chromatographie Assay of Disaccharides and Oligosaccharides Produced by the Digestion of Glycosaminoglycans with Chondroitin Sulphate Lyases

Gurr, Eberhard; G, Pallasch; Tunn, Sabine; Tamm, Ch.; Delbrück, A.

doi:10.1515/cclm.1985.23.2.77

Cited by 11 publications

(11 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Regarding the sequence of application of the GAG lyases, recovery rates using the order employed in the present study proved to be superior to other methods that used hyaluronate lyase digestion as the first degradation step [23], since CA and DS can also be digested by hyaluronate lyase [24]. Comparison of the specific-enzyme digestion and HPLC analysis method with the commonly used dye reaction showed the higher recovery rate, sensitivity and reproducibility of the HPLC analysis.…”

Section: Discussionmentioning

confidence: 69%

“…Enzymic digestion and HPLC analysis of GAG compounds has been successfully applied in human articular cartilage [23] and in serum and plasma samples [24] using different chromatographic columns, such as strong anion-exchange and reversedphase columns. In this study we employed an aminopropyl column which is designed for disaccharide separation based on polar and hydrophobic interactions dependent upon the mobile phase used.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

HPLC Glycosaminoglycan Analysis in Patients with Graves' Disease

et al. 1997

View full text Add to dashboard Cite

1. Orbital accumulation of hydrophilic, interstitial glycosaminoglycans (GAGs) and subsequent expansion of retrobulbar tissue lead to the clinical manifestation of exophthalmos in patients with Graves' eye disease. 2. A highly specific method to determine the concentration and biochemical composition of different GAGs was developed in order to obtain a sensitive test system for the activity of the disease. By means of this method, GAG excretion in 24 h urine collections of 56 patients and 21 controls was analysed by precipitation with cetylpyridinium chloride and potassium acetate in ethanol, followed by sequential enzymic hydrolysis with chondroitin AC lyase, chondroitin ABC lyase and hyaluronate lyase, with HPLC analysis of the resulting alpha, beta-unsaturated disaccharides by anion-exchange chromatography. 3. Concentrations of GAG, chondroitin sulphate A (CA), dermatan sulphate (DS) and hyaluronic acid (HA) were determined in patients and controls, with high recovery rates [72.2 +/- 5.3%, mean +/- SEM; detection limit, 4.2 micrograms/l (0.01 mumol/l)], revealing marked differences in urinary concentrations of total GAG and HA, as well as an elevation of CA in patients compared with controls. 4. Method sensitivity was 0.86 for patients with active Graves' eye disease, and 0.87 for patients with untreated ophthalmopathy, whereas specificity was 1.0 for patients with inactive disease. Patients with increased GAG concentration responded well to steroids and/or orbital irradiation (before therapy: GAG, 111.49 +/- 40.32; CA, 59.58 +/- 21.34; DS, 25.05 +/- 8.12; HA, 26.88 +/- 11.63 mg/24 h; during therapy: GAG, 54.22 +/- 10.94; CA, 20.52 +/- 4.58; DS, 17.65 +/- 3.46; HA, 16.05 +/- 3.69 mg/24 h), whereas GAG excretion increased markedly 2-3 months after stopping prednisone therapy in patients with still active eye disease (GAG, 109.9 +/- 10.51; CA, 63.8 +/- 7.34; DS, 24.1 +/- 5.07; HA, 22.0 +/- 6.28 mg/24 h). 5. This sensitive method determines the nature of renally excreted GAGs, reflecting the aberrant synthesis pattern of fibroblasts in patients with Graves' disease.

show abstract

Section: Discussionmentioning

confidence: 69%

Section: Discussionmentioning

confidence: 99%

HPLC Glycosaminoglycan Analysis in Patients with Graves' Disease

et al. 1997

View full text Add to dashboard Cite

show abstract

“…. -, n< e "i«i^t-/i ^mnr^ntc ratory Medicine, university Hospital "BergmannsheiT Bo-P hated > C4-sulphated and C6-sulphated components chum, FRG (11 …”

mentioning

confidence: 99%

The Distribution of Unsulphated and Sulphated Glycosaminoglycans in Palmar Fascia from Patients with Dupuytren’s Disease and Healthy Subjects

Tunn¹,

Gurr²,

Delbrück³

et al. 1988

Clinical Chemistry and Laboratory Medicine

Self Cite

View full text Add to dashboard Cite

Eighty specimens from 20 patients with Dupuytren's disease and 7 biopsies of healthy palmar fascia were analysed for their glycosaminoglycan isomer patterns with a combined enzymatic/HPLC method. The diseased portions of palmar fascia tissue were characterized by elevated total glycosaminoglycans together with a relative increase in the sulphated fractions. The macroscopic stages of nodules, bands and unaffected tissue could be classified very well by multivariate statistical analysis on the basis of their glycosaminoglycan patterns. The biochemical analysis provided evidence of the pathological process even in those specimens that did not yet show any clinical symptoms of the disease.Because of the role played by sulphated glycosamiThe mechanisms involved in the pathogenesis of Du-noglycans in the interaction with collagen (12), the puytren's disease are still unknown (1). Recent studies question arises of whether there are changes in the on the morphological changes in diseased tissue have proportions of glycosaminoglycans and their unsuldemonstfated occlusioii of capillaries by cells that phated and sulphated isomers in affected palmar fasmigrate into the perivascular space. These cells are cia that may have an influence on the collagen pattern thought to be the origin of dedifferentiated cells that and the macromolecular structure. The present study proliferate and metabolize in such Way that they cause was conducted to determine the types and amounts contracture of the aponeurosis (2, 3). As to the pa-of the various sulphated and unsulphated glycosathobiochemistry, an increase in collagen type III pro-minoglycans and to reassess the data published so far tein has been reported (4-7), as has an increase in on the glycosaminoglycan content of both healthy the proportion of chondroitin sulphate (4, 8 -11). human palmar fascia and tissue from Dupuytren'ŝ contracture, by employing a method of determination ') With support of the Deutsche Forschungsgemeinschaft (SFB more specific than any used previously. The new 54). method also enabled differentiation of the unsul-2 ) Present address: Institute for Clinical Chemistry and Labo-, , ~A . . -, n< e "i«i^t-/i ^mnr^ntc ratory Medicine, university Hospital "BergmannsheiT Bo-P hated > C4-sulphated and C6-sulphated components chum, FRG (11).

show abstract

“…The HPLC analysis of the disaccharide was carried out according to a modified method of E. Gurr et al (4).…”

Section: Analysis Of Chondroitin Sulphates By High Performance Liquidmentioning

confidence: 99%

“…Several methods for the determi-nation of chondroitin sulphate have been described. These techniques use enzymatic digestion, precipitation and Chromatographie methods, especially high performance liquid chromatography (3,4).…”

Section: Introductionmentioning

confidence: 99%

Determination of Chondroitin-6-Sulphate by a Competitive Enzyme Immunoassay Using a Biotinylated Antigen

Kähnert

Brinkmann²,

Gässler³

et al. 1994

CCLM

View full text Add to dashboard Cite

Summary: A competitive enzyme immunoassay was developed to determine chondroitin-6-sulphate in body fluids and cell cultures. The assay uses a monoclonal anti-chondroitin-6-sulphate antibody, immobilised to microtitre plates, and it involves a competitive binding reaction between chondroitih-6-sulphate in the samples and the biotinylated antigen.This assay enables the quantification of chondroitin-6-sulphate in the low concentration ränge of 16-120 g/l. The intra-assay and inter-assay coefficients of Variation are below 6.5% and 9.0%, respectively. More than 90% of chondroitin-6-sulphate was recovered when added to 0.1 mol/1 phosphate-buffered saline, an albumin solution (40 g/l in phosphäte-buffered saune) and cell culture medium (containing 100 ml/l foetal calf serum).Chondroitin-6-sulphate was also determined in sera of healthy male (n = 90) and female (n = 90) blood donors. The normal ränge was 55-169 g/l. In men the mean value was estimated at 102.2 ±37.1 g/l and in women at 98.7 i 26.4 g/l. Np age or sex dependence was observed.The urine excretion of chondroitin-6-sulphate in men (n = 16) was 44.5 ±21.1 mg/kg creatinine (mean ± Standard deviation) and in fernales (n = 10) 53.5 ± 21.3 mg/kg creatinine. The clearance rate in men was 0.41 ± 0.22 ml X min" 1 and in women €.38 ± 0.15 ml X min" 1 . No sex dependence was found.Furthermore, the enzyme immunoassay was modifted to measure the specific incorporation of a radioactively labelled precursor ([ 14 C]galactosamine) into chondroitin-6-sulphate. This modification rapidly gives Information on the cellular glycosaminoglycan synthesis in cell culture. Usmg this method our experiments with cultivated human chondrocytes showed that the synthesis of chpndroitin^o-sulphate decreased in the presence of interleukin-la (60.0% less), turnour necrosis factor a (64.4%), -interferön (21.6%) and lipopolysaccharide (53.4%).

show abstract

High Performance Liquid Chromatographie Assay of Disaccharides and Oligosaccharides Produced by the Digestion of Glycosaminoglycans with Chondroitin Sulphate Lyases

Cited by 11 publications

References 19 publications

HPLC Glycosaminoglycan Analysis in Patients with Graves' Disease

HPLC Glycosaminoglycan Analysis in Patients with Graves' Disease

The Distribution of Unsulphated and Sulphated Glycosaminoglycans in Palmar Fascia from Patients with Dupuytren’s Disease and Healthy Subjects

Determination of Chondroitin-6-Sulphate by a Competitive Enzyme Immunoassay Using a Biotinylated Antigen

Contact Info

Product

Resources

About