Quantitation of Proteoglycans as Glycosaminoglycans in Biological Fluids Using an Alcian Blue Dot Blot Analysis

Björnsson, Sven

doi:10.1006/abio.1997.2494

Cited by 89 publications

(58 citation statements)

References 30 publications

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“…After digestion, the samples are loaded into the LC-MS/MS for quantitation, and the results are compared with control samples (Figure 2). The LC-MS/MS method for analysis of disaccharides not only shows sensitivity and specificity for detecting all subtypes of MPS but also can monitor therapeutic efficacy in MPS patients and animal models [27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45]. This method has an advantage of being both GAG-specific and quantitative.…”

Section: History Of Gag Assay By Tandem Mass Spectrometry (Ms/ms)mentioning

confidence: 99%

“…Total urinary GAG, a potential biomarker for MPS, is measured spectrometrically by using DMB [17,18,33,34] or Alcian blue [23,35]; however, these methods cannot be applied to blood without protease treatment, since protein in the specimen hinders the binding of the dye to the GAG. Moreover, the dye itself tends to decompose, leading to a high background and false positive results.…”

Section: Introductionmentioning

confidence: 99%

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Establishment of glycosaminoglycan assays for mucopolysaccharidoses

Tomatsu

Shimada

Mason

et al. 2015

Molecular Genetics and Metabolism

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Mucopolysaccharidoses (MPS) are a group of lysosomal storage disorders caused by deficiency of the lysosomal enzymes essential for catabolism of glycosaminoglycans (GAGs). Accumulation of undegraded GAGs results in dysfunction of multiple organs, resulting in distinct clinical manifestations. A range of methods have been developed to measure specific GAGs in various human samples to investigate diagnosis, prognosis, pathogenesis, GAG interaction with other molecules, and monitoring therapeutic efficacy. We OPEN ACCESSMetabolites 2014, 4 656 established ELISA, liquid chromatography tandem mass spectrometry (LC-MS/MS), and an automated high-throughput mass spectrometry (HT-MS/MS) system (RapidFire) to identify epitopes (ELISA) or disaccharides (MS/MS) derived from different GAGs (dermatan sulfate, heparan sulfate, keratan sulfate, and/or chondroitin sulfate). These methods have a high sensitivity and specificity in GAG analysis, applicable to the analysis of blood, urine, tissues, and cells. ELISA is feasible, sensitive, and reproducible with the standard equipment. HT-MS/MS yields higher throughput than conventional LC-MS/MS-based methods while the HT-MS/MS system does not have a chromatographic step and cannot distinguish GAGs with identical molecular weights, leading to a limitation of measurements for some specific GAGs. Here we review the advantages and disadvantages of these methods for measuring GAG levels in biological specimens. We also describe an unexpected secondary elevation of keratan sulfate in patients with MPS that is an indirect consequence of disruption of catabolism of other GAGs.

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Section: History Of Gag Assay By Tandem Mass Spectrometry (Ms/ms)mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Establishment of glycosaminoglycan assays for mucopolysaccharidoses

Tomatsu

Shimada

Mason

et al. 2015

Molecular Genetics and Metabolism

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“…3) and the dot blot/reflection Alcian Blue assay [30] is even more sensitive. However, there are multiple issues associated with the Alcian Blue assay.…”

Section: Discussionmentioning

confidence: 99%

The Quantification of Glycosaminoglycans: A Comparison of HPLC, Carbazole, and Alcian Blue Methods

Frazier

Roodhouse²,

Hourcade³

et al. 2008

TOGLYJ

111

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Glycosaminoglycans (GAGs) are linear polysaccharides that are found in the extracellular matrix and biological fluids of animals where they interact with hundreds of proteins and perform a variety of critical roles. There are five classes of animal GAGs: heparan sulfate (HS), chondroitin sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS), and hyaluronan (HA). Many biological functions can be monitored directly by their impact on GAG quantity. Thus, simple, sensitive, and robust GAG quantification methods are needed for the development of biomarkers. We have systematically compared three available GAG quantification assays including an HPLC-based assay, a simplified Alcian Blue assay, and a miniaturized carbazole assay. The carbazole and Alcian Blue assays were reproducible and simple to perform in general lab settings, but had important limitations: The carbazole assay could not detect KS and it overestimated GAGs that were contaminated with salts or dissolved in PBS. The Alcian Blue assay detected only those GAGs that were sulfated. In contrast, while the HPLC method was time-consuming, it was a robust and sensitive assay that not only detected all GAGs but also quantified glucosamine-GAGs and galactosamine-GAGs simultaneously. The HPLC assay was not affected by salt or level of GAG sulfation and it yielded reproducible values for all types of GAGs tested. These results suggest that an automated HPLC assay would be generally useful for the routine measurement of a panel of GAG-based biomarkers while the carbazole assay and the Alcian Blue assays could prove valuable for more specific purposes.

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“…The urinary mucus concentration was measured using a modified version of the method of Björnsson (1993Björnsson ( , 1998. The dye solution was prepared by dissolving 900 mg Alcian Blue GX a in 100 ml of a solution that consisted of 30 ml sulfuric acid 0.5 % and 5ml of 8 M urea solution.…”

Section: Renal Functionmentioning

confidence: 99%

Effects of using Flunixin Meglumine, Metamizole, and Phenylbutazone on equine kidney functions, urinary mucus, and secretory Immunoglobulin A (IgA) concentrations

Adam¹,

Koller²,

Arnold³

et al. 2017

PHK

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Summary: Nonsteroidal anti-inflammatory drugs (NSAIDs) are routinely used in equine practices to manage inflammation, endotoxemia, pain or fever in horses. This study was carried out to investigate the effects of the most commonly used nonsteroidal anti-inflammatory drugs on urinary parameters, mucus and immunoglobulin A (IgA) concentrations in horses. Thirty healthy horses (control group) and 20 horses with indications of using either flunixin meglumine (FM), metamizole or phenylbutazone (PHZ) have been used and assigned to group 1, 2 or 3, respectively. Serum creatinine, blood urea nitrogen and electrolytes, in addition to urinary creatinine (U-Cr), urine urea nitrogen and urinary electrolytes, were measured using an automatic analyser. Fractional excretions (FE) of sodium, chloride, potassium, calcium, magnesium and inorganic phosphate, in addition to urinary protein (U-Pro):U-Cr and urinary gamma glutamyl transferase (U-GGT):U-Cr ratios were calculated. Urinary mucus and IgA concentrations and their ratios to the urinary creatinine were measured. The FEMg was significantly higher in group 3 (P < 0.033) than the control group. The U-GGT:U-Cr ratio was also significantly increased in group 3 (P < 0.001) compared with the control group. The U-Pro:U-Cr ratio was significantly higher in groups 1 and 2 (P < 0.007 and P < 0.001, respectively) than in the control group. Phenylbutazone (PHZ) had a significantly increased mucus:U-Cr ratio (P < 0.005). Significant increases were observed regarding the IgA:U-Cr ratio in groups 1 (P < 0.007) and 2 (P < 0.014). In conclusion, long-term use of PHZ has an influence on the renal ascending limb of the loop of Henle, and all these drugs could have effects on the proximal tubules. Phenylbutazone causes an increase in urinary mucus secretion, probably as a protective mechanism against the necrotic effect of PHZ. Parameters such as U-Pro:U-Cr and U-GGT:U-Cr ratios and FEMg are helpful in detecting these renal abnormalities.

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Quantitation of Proteoglycans as Glycosaminoglycans in Biological Fluids Using an Alcian Blue Dot Blot Analysis

Cited by 89 publications

References 30 publications

Establishment of glycosaminoglycan assays for mucopolysaccharidoses

Establishment of glycosaminoglycan assays for mucopolysaccharidoses

The Quantification of Glycosaminoglycans: A Comparison of HPLC, Carbazole, and Alcian Blue Methods

Effects of using Flunixin Meglumine, Metamizole, and Phenylbutazone on equine kidney functions, urinary mucus, and secretory Immunoglobulin A (IgA) concentrations

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