HPLC Glycosaminoglycan Analysis in Patients with Graves' Disease

1997 ◽  
Vol 92 (5) ◽  
pp. 511-517 ◽  
Author(s):  
CH Hansen ◽  
B. Fraiture ◽  
R. Rouhi ◽  
E. Otto ◽  
G. Förster ◽  
...  
Keyword(s):  
Chondroitin Sulphate ◽  
Cetylpyridinium Chloride ◽  
Potassium Acetate ◽  
Anion Exchange Chromatography ◽  
Test System ◽  
Exchange Chromatography ◽  
Inactive Disease ◽  
Eye Disease ◽  
Graves Disease ◽  
Specific Method

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 α,β-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 μg/l (0.01 μmol/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.

scholarly journals The glycosaminoglycans of human tracheobronchial cartilage

1970 ◽  
Vol 120 (4) ◽  
pp. 777-785 ◽  
Author(s):  
R. M. Mason ◽  
F. S. Wusteman
Keyword(s):  
Molecular Weight ◽  
Potassium Hydroxide ◽  
Chondroitin Sulphate ◽  
Cetylpyridinium Chloride ◽  
Exchange Chromatography ◽  
Chemical Heterogeneity ◽  
Keratan Sulphate ◽  
Sulphate Content ◽  
Age Dependent ◽  
Dowex 1

1. The glycosaminoglycans of human tracheobronchial cartilages from subjects of various ages were liberated by proteolysis of the tissue and purified by ion-exchange chromatography. Purified glycosaminoglycans were fractionated on Dowex 1 resin and cetylpyridinium chloride was used to separate chondroitin sulphates and keratan sulphates occurring in the same fraction. 2. The total chondroitin sulphate content of the cartilages decreased linearly with increasing age. Age-dependent changes in the chemical heterogeneity of chondroitin sulphate were observed, a low-sulphated compound making up 25% of the total glycosaminoglycan at birth but rapidly diminishing in content during the first 6 months of life. Of the total chondroitin sulphate the 6-isomer became rather more prominent than the 4-isomer with increasing age. 3. The total keratan sulphate content of the cartilages increased from trace amounts only at birth to a plateau value by the beginning of the fifth decade. Of the total keratan sulphate approx. 70% was due to a high-molecular-weight compound with a sulphate/hexosamine ratio of 1.5–1.8: 1.0. The degree of sulphation varied between compounds isolated from different individuals. The remaining 30% of the keratan sulphate appeared to be intimately associated with chondroitin 6-sulphate and could only be separated from it after treatment with 0.45m-potassium hydroxide. The hybrid glycosaminoglycans were of lower molecular weight and had a lower sulphate/hexosamine ratio than the major keratan sulphate compound.

Increased incidence of unsulphated and 4-sulphated residues in the chondroitin sulphate linkage region observed by high-pH anion-exchange chromatography

2000 ◽  
Vol 347 (2) ◽  
pp. 339-348 ◽  
Author(s):  
Robert M. LAUDER ◽  
Thomas N. HUCKERBY ◽  
Ian A. NIEDUSZYNSKI
Keyword(s):  
Articular Cartilage ◽  
Anion Exchange ◽  
Chondroitin Sulphate ◽  
Basic Structure ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
High Ph ◽  
Tracheal Cartilage ◽  
Bovine Articular Cartilage ◽  
Linkage Region

We report the isolation, characterization and quantification of five octasaccharides, four hexasaccharides and two tetrasaccharides, derived from the chondroitin sulphate (CS) linkage region of 6-8-year-old bovine articular cartilage aggrecan, following digestion with chondroitin ABC endolyase. Using a novel high-pH anion-exchange chromatography (HPAEC) method, in conjunction with one- and two-dimensional 1H-NMR spectroscopy, we have identified the following basic structure for the CS linkage region of aggrecan: ∆UA(β1-3)GalNAc[0S/4S/6S](β1-4)GlcA(β1-3)GalNAc[0S/4S/6S](β1-4)GlcA(β1-3)Gal[0S/6S](β1-3)Gal(β1-4)Xyl, where ∆UA represents 4,5-unsaturated hexuronic acid, and 4S and 6S represent an O-ester sulphate group on C-4 and C-6 respectively. The octa-, hexa- and tetra-saccharide linkage region fragments were used to develop a HPAEC fingerprinting method, with detection at A232 nm, and a linear response to approx. 0.1 nmol of substance. The sulphation patterns of CS linkage regions, of up to octasaccharide in size, from articular and tracheal cartilage aggrecan were examined. The results show that in articular cartilage, for the majority (53%) of octasaccharides the 2-deoxy-2-N-acetyl amino-D-galactose (GalNAc) residues closest to the linkage region are both 6-sulphated; however, in a significant portion (34%), one or more of these GalNAc residues are unsulphated, and in 8% both are unsulphated. Approximately 10-18% of the chains have a 4-sulphated GalNAc in the first disaccharide, and 12% have a sulphated linkage region Gal residue. No evidence was found for uronic acid sulphation. These data show that there is a significant increase in the incidence of unsulphated and 4-sulphated GalNAc residues adjacent to the linkage region compared with the rest of the chain. Bovine tracheal cartilage linkage regions displayed very similar sulphation profiles to those from articular cartilage, despite the presence of a higher level of GalNAc 4-sulphation within the repeat region of the main CS chain.

Age-related changes in the sulphation of the chondroitin sulphate linkage region from human articular cartilage aggrecan

2001 ◽  
Vol 358 (2) ◽  
pp. 523-528 ◽  
Author(s):  
Robert M. LAUDER ◽  
Thomas N. HUCKERBY ◽  
Gavin M. BROWN ◽  
Michael T. BAYLISS ◽  
Ian A. NIEDUSZYNSKI
Keyword(s):  
Articular Cartilage ◽  
Chondroitin Sulphate ◽  
Basic Structure ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Human Articular Cartilage ◽  
Linkage Region ◽  
Age Related ◽  
Age Related Changes

The chondroitin sulphate (CS) linkage regions have been isolated from human articular cartilage aggrecan (from 10- to 72-year-olds) by chondroitin ABC endolyase digestion and size-exclusion chromatography. Linkage region hexasaccharides have been characterized and their abundance estimated by high-pH anion-exchange chromatography. The basic structure for the CS linkage region oligosaccharides identified from human aggrecan is as follows: ΔUA(β1–3)GalNAc[0S/4S/6S](β1–4)GlcA(β1–3)Gal[0S/6S](β1–3)Gal(β1–4)Xyl, where ΔUA represents 4,5-unsaturated hexuronic acid, 4S and 6S represent an O-ester sulphate group on C-4 and C-6 respectively, and 0S represents zero sulphation. There are significant age-related changes in the abundance of the various N-acetylgalactosamine (GalNAc) sulphation forms identified, occurring up to approx. 20 years old. During the period from 10 to 20 years old the level of GalNAc 6-sulphation at the linkage region increases from approx. 43% to approx. 75%, while there is a corresponding reduction in unsulphated (approx. 30% to approx. 20%) and 4-sulphated (approx. 25% to approx. 6%) GalNAc residues. There is also an increase in the incidence of linkage region galactose 6-sulphation (approx. 2% to approx. 10%) which was only observed in linkage regions with GalNAc 6-sulphation. Beyond 20 years old there are few changes in the relative abundance of these GalNAc sulphation variants; however, there is a slight increase in the abundance of 6-sulphation between approx. 20 years old and approx. 40 years old and a slight decrease in its abundance beyond approx. 40 years old. Our data show that in the majority of chains from tissues of all ages the GalNAc residue closest to the linkage region is 6-sulphated, but the level of GalNAc 6-sulphation within the linkage region is lower than the average level observed within the repeat region.

scholarly journals Levels of Urinary Trypsin Inhibitor and Structure of Its Chondroitin Sulphate Moiety in Type 1 and Type 2 Diabetes

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Antonio Junior Lepedda ◽  
Gabriele Nieddu ◽  
Silvia Rocchiccioli ◽  
Nadia Ucciferri ◽  
Michela Idini ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Trypsin Inhibitor ◽  
Chondroitin Sulphate ◽  
Albumin Excretion Rate ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Urinary Trypsin Inhibitor ◽  
Potential Marker

Background. Diabetes mellitus is a global health problem representing the fifth leading cause of mortality and a major risk factor for cardiovascular diseases. In the last years, we reported an association among urinary trypsin inhibitor (UTI), a small proteoglycan that plays pleiotropic roles in many inflammatory processes, and both type 1 and 2 diabetes and developed a method for its direct quantitation and structural characterization. Methods. Urine from 39 patients affected by type 1 diabetes, 32 patients with type 2 diabetes, and 52 controls were analysed. UTI was separated from the main glycosaminoglycans physiologically present in urine by anion exchange chromatography, treated for chondroitin sulphate (CS) chain complete depolymerisation, and analysed for both UTI content and CS structure. UTI identification was performed by nano-LC-MS/MS analysis. Results. We evidenced increased UTI levels, as well as reduced sulphation of its CS moiety in association with diabetes, regardless of both age and medium-term glycaemic control. Furthermore, no association between UTI and albumin excretion rate was found. Conclusions. Evidences suggest that UTI levels are not directly correlated with renal function or, otherwise, that they may increase before the onset of renal impairment in diabetes, representing a potential marker for the underlying inflammatory condition.

Extracellular polysaccharides from suspension cultures of Nicotiana plumbaginifolia

2020 ◽  
Author(s):  
Ian Sims ◽  
A Bacic
Keyword(s):  
Uronic Acid ◽  
Cell Suspension Cultures ◽  
Anion Exchange Chromatography ◽  
Nicotiana Plumbaginifolia ◽  
Suspension Cultures ◽  
Exchange Chromatography ◽  
Arabinogalactan Protein ◽  
Extracellular Polysaccharides ◽  
Selective Precipitation ◽  
The Individual

The soluble polymers secreted by cell-suspension cultures of Nicotiana plumbaginifolia contained 78% carbohydrate, 6% protein and 4% inorganic material. The extracellular polysaccharides were separated into three fractions by anion-exchange chromatography using a gradient of imidazole-HCl at pH 7 and the individual polysaccharides in each fraction were then isolated by selective precipitation and enzymic treatment. Monosaccharide and linkage compositions were determined for each polysaccharide after reduction of uronic acid residues and the degree of esterification of the various uronic acid residues in each polysaccharide was determined concurrently with the linkage types. Six components were identified: an arabinoxyloglucan (comprising 34% of the total polysaccharide) and a galactoglucomannan (15%) in the unbound neutral fraction, a type II arabinogalactan (an arabinogalactan-protein, 11%) and an acidic xylan (3%) in the first bound fraction, and an arabinoglucuronomannan (11%) and a galacturonan (26%) in the second bound fraction. © 1995.

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