Analytical and preparative separation of acidic glycosaminoglycans by electrophoresis in barium acetate

doi:10.1016/0003-2697(68)90205-4

Analytical Biochemistry

Volume 26, Issue 3, December 1968, Pages 439-444

https://doi.org/10.1016/0003-2697(68)90205-4 Get rights and content

Abstract

Acidic glycosaminoglycans have been separated by analytical and preparative electrophoresis in barium acetate. This method was found to fractionate the polysaccharides into three groups of increasing mobility: heparan sulfate-heparin, dermatan sulfate-hyaluronic acid, and the chondroitin sulfates. The electrophoretic mobility in barium acetate thus depends primarily on the structure of the polysaccharide backbone, whereas the sulfate content is of less importance.

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Cited by (181)

Molecular genetics and metabolism, special edition: Diagnosis, diagnosis and prognosis of Mucopolysaccharidosis IVA
2018, Molecular Genetics and Metabolism
Citation Excerpt :
The qualitative analysis of total urinary GAGs is usually performed by spectrophotometric analysis using dimethylmethylene blue (DMMB) [28]. For qualitative urine-based testing methods, the GAGs are first isolated from the urine and then separated by thin layer chromatography or electrophoresis [29–32]. KS is mostly synthesized and accumulated in the cartilage and cornea in MPS IVA patients, and excessive accumulation of KS in lysosome leads to disruption of chondrocytes and subsequent increase of KS levels in blood and urine [33].
Mucopolysaccharidosis IVA (MPS IVA, Morquio A syndrome) is an autosomal recessive disorder caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase. Deficiency of this enzyme leads to the accumulation of specific glycosaminoglycans (GAGs), chondroitin-6-sulfate (C6S) and keratan sulfate (KS), which are mainly synthesized in the cartilage. Therefore, the substrates are stored primarily in the cartilage and its extracellular matrix (ECM), leading to a direct impact on bone development and successive systemic skeletal spondylepiphyseal dysplasia. The skeletal-related symptoms for MPS IVA include short stature with short neck and trunk, odontoid hypoplasia, spinal cord compression, tracheal obstruction, obstructive airway, pectus carinatum, restrictive lung, kyphoscoliosis, platyspondyly, coxa valga, genu valgum, waddling gait, and laxity of joints. The degree of imbalance of growth in bone and other organs and tissues largely contributes to unique skeletal dysplasia and clinical severity. Diagnosis of MPS IVA needs clinical, radiographic, and laboratory testing to make a complete conclusion. To diagnose MPS IVA, total urinary GAG analysis which has been used is problematic since the values overlap with those in age-matched controls. Currently, urinary and blood KS and C6S, the enzyme activity of GALNS, and GALNS molecular analysis are used for diagnosis and prognosis of clinical phenotype in MPS IVA. MPS IVA can be diagnosed with unique characters although this disorder relates closely to other disorders in some characteristics.
In this review article, we comprehensively describe clinical, radiographic, biochemical, and molecular diagnosis and clinical assessment tests for MPS IVA. We also compare MPS IVA to other closely related disorders to differentiate MPS IVA. Overall, imbalance of growth in MPS IVA patients underlies unique skeletal manifestations leading to a critical indicator for diagnosis.
Isolation and characterization of hyaluronic acid from marine organisms
2014, Advances in Food and Nutrition Research
Citation Excerpt :
Most of these involve the digestion of polymeric HA with the endohydrolase, testicular hyaluronidase, followed by purification by size exclusion chromatography (Lesley, Hascall, Tammi, & Hyman, 2000; Tammi et al., 1998), ion exchange chromatography (Almond, Brass, & Sheehan, 1998; Holmbeck & Lerner, 1993; Toffanin et al., 1993), and reversed-phase ion pair high-performance liquid chromatography (Cramer & Bailey, 1991). The electromigration procedures developed to analyze and characterize complex polysaccharides including electrophoresis on cellulose acetate (Wessler, 1968) and nitrocellulose membrane (Volpi, 1996), agarose gel electrophoresis (Dietrich & Dietrich, 1976; Dietrich, McDuffie, & Sampaio, 1977), electrophoresis on polyacrylamide gel (Edens et al., 1992; Hakim & Linhardt, 1990; Min & Cowman, 1986; Rice, Rottink, & Linhardt, 1987), high-performance capillary electrophoresis (Maccari & Volpi, 2003; Volpi, 2004a, 2004b), and fluorophore-assisted carbohydrate electrophoresis (Calabro et al., 2001; Volpi & Maccari, 2005) analyses were critically important in HA evaluation. These methods allow on direct detection or prederivatization for both qualitative and quantitative analysis with a high level of sensitivity (Volpi & Maccari, 2006).
Hyaluronic acid (HA) being a viscous slippery substance is a multifunctional glue with immense therapeutic applications such as ophthalmic surgery, orthopedic surgery and rheumatology, drug delivery systems, pulmonary pathology, joint pathologies, and tissue engineering. Although HA has been isolated from terrestrial origin (human umbilical cord, rooster comb, bacterial sources, etc.) so far, the increasing interest on this polysaccharide significantly aroused the alternative search from marine sources since it is at the preliminary level. Enthrallingly, marine environments are considered more biologically diverse than terrestrial environments. Although numerous methods have been described for the extraction and purification of HA, the hitch on the isolation methods which greatly influences the yield as well as the molecular weight of the polymer still exists. Adaptation of suitable method is essential in this venture. Stimulated by the developed technology, to sketch the steps involved in isolation and analytical techniques for characterization of this polymer, a brief report on the concerned approach has been reviewed.
Review of clinical presentation and diagnosis of mucopolysaccharidosis IVA
2013, Molecular Genetics and Metabolism
Citation Excerpt :
Qualitative urine-based testing methods separate the various GAG species and therefore, provide a starting path for differential diagnosis. For these procedures, the GAGs are first isolated from the urine and then separated by thin layer chromatography or electrophoresis [72–75]. A GAG specific stain such as dimethylmethylene blue is then used to visualize the GAGs and their relative position on the gel or plate is used for identification [76].
Mucopolysaccharidosis type IVA (MPS IVA) was described in 1929 by Luis Morquio from Uruguay and James Brailsford from England, and was later found as an autosomal recessive lysosomal storage disease. MPS IVA is caused by mutations in the gene encoding the enzyme, N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Reduced GALNS activity results in impaired catabolism of two glycosaminoglycans (GAGs), chondroitin-6-sulfate (C6S) and keratan sulfate (KS). Clinical presentations of MPS IVA reflect a spectrum of progression from a severe “classical” phenotype to a mild “attenuated” phenotype. More than 180 different mutations have been identified in the GALNS gene, which likely explains the phenotypic heterogeneity of the disorder.
Accumulation of C6S and KS manifests predominantly as short stature and skeletal dysplasia (dysostosis multiplex), including atlantoaxial instability and cervical cord compression. However, abnormalities in the visual, auditory, cardiovascular, and respiratory systems can also affect individuals with MPS IVA. Diagnosis is typically based on clinical examination, skeletal radiographs, urinary GAG, and enzymatic activity of GALNS in blood cells or fibroblasts. Deficiency of GALNS activity is a common assessment for the laboratory diagnosis of MPS IVA; however, with recently increased availability, gene sequencing for MPS IVA is often used to confirm enzyme results. As multiple clinical presentations are observed, diagnosis of MPS IVA may require multi-system considerations.
This review provides a history of defining MPS IVA and how the understanding of the disease manifestations has changed over time. A summary of the accumulated knowledge is presented, including information from the International Morquio Registry. The classical phenotype is contrasted with attenuated cases, which are now being recognized and diagnosed more frequently. Laboratory based diagnoses of MPS IVA are also discussed.
Newborn screening and diagnosis of mucopolysaccharidoses
2013, Molecular Genetics and Metabolism
Citation Excerpt :
Overall, establishment of the method to measure the disaccharides rather than hetergenous oligosaccharides make it feasible to interpret individual GAG values, leading to rapid and accurate diagnosis, prognosis, and monitoring therapies for MPS. At present, the following LC–MS/MS instruments have been confirmed to provide good resolution for disaccharide analysis by multiple laboratories; Alliance 2795XE HPLC system/Quattro micro tandem quadrupole (Waters Corp, Milford, MA, USA); Ultra performance liquid chromatography (UPLC) Acquity system/Xevo TQ-S (Waters Corp, Milford, MA, USA); HP1100 system (Agilent Technologies, Palo Alto, CA, USA)/API-4000 or API-5000 (AB Sciex, Foster City, CA, USA); Acquity HPLC system/Quattro Premier XE (Waters Corp, Milford, MA, USA); and 1260 infinity LC/6460 Triple Quad (Agilent Technologies, Palo Alto, CA, USA) [56–58,95–98,9–101,103–106, personal communication from Dr. K. Kida]. ΔDiHS-6S (HS); ΔDiHS-NS (HS); ΔDiHS-0S (HS); ΔDi-4S (DS); mono-sulfated KS — Galβ1-4GlcNAc(6S); di-sulfated KS — Gal(6S)β1-4GlcNAc(6S); and KS I (bovine cornea) digested with keratanase II to yield Galβ1-4GlcNAc(6S), Gal(6S)β1-4GlcNAc(6S), have all been used to produce standard curves for each specific GAG (Fig. 3).
Mucopolysaccharidoses (MPS) are caused by deficiency of lysosomal enzyme activities needed to degrade glycosaminoglycans (GAGs), which are long unbranched polysaccharides consisting of repeating disaccharides. GAGs include: chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), keratan sulfate (KS), and hyaluronan. Their catabolism may be blocked singly or in combination depending on the specific enzyme deficiency. There are 11 known enzyme deficiencies, resulting in seven distinct forms of MPS with a collective incidence of higher than 1 in 25,000 live births. Accumulation of undegraded metabolites in lysosomes gives rise to distinct clinical syndromes. Generally, the clinical conditions progress if untreated, leading to developmental delay, systemic skeletal deformities, and early death. MPS disorders are potentially treatable with enzyme replacement therapy or hematopoietic stem cell transplantation. For maximum benefit of available therapies, early detection and intervention are critical.
We recently developed a novel high-throughput multiplex method to assay DS, HS, and KS simultaneously in blood samples by using high performance liquid chromatography/tandem mass spectrometry for MPS. The overall performance metrics of HS and DS values on MPS I, II, and VII patients vs. healthy controls at newborns were as follows using a given set of cut-off values: sensitivity, 100%; specificity, 98.5–99.4%; positive predictive value, 54.5–75%; false positive rate, 0.62–1.54%; and false negative rate, 0%. These findings show that the combined measurements of these three GAGs are sensitive and specific for detecting all types of MPS with acceptable false negative/positive rates. In addition, this method will also be used for monitoring therapeutic efficacy. We review the history of GAG assay and application to diagnosis for MPS.
Analytical aspects of pharmaceutical grade chondroitin sulfates
2007, Journal of Pharmaceutical Sciences
Citation Excerpt :
Electrophoretic separation of polysaccharides on cellulose acetate strips has been used for a long time. Barium acetate has been used as a buffer to facilitate the analytical separation of CS from other GAGs, i.e. hyaluronic acid, DS and heparin.47 Since that first application, a rapid and sensitive method to separate CS from heparan sulfate, DS, hyaluronic acid, heparin, and keratan sulfate has been developed on cellulose acetate with a single monodimensional electrophoresis separation in a barium acetate buffer exploiting the differential sensitivity of polysaccharides to precipitation by ethanol.48
Chondroitin sulfate is a very heterogeneous polysaccharide in terms of relative molecular mass, charge density, chemical properties, biological and pharmacological activities. It is actually recommended by EULAR as a symptomatic slow acting drug (SYSADOA) in Europe in the treatment of knee osteoarthritis based on meta‐analysis of numerous clinical studies. Chondroitin sulfate is also utilized as a nutraceutical in dietary supplements mainly in the United States. On the other hand, chondroitin sulfate is derived from animal sources by extraction and purification processes. As a consequence, source material, manufacturing processes, the presence of contaminants, and many other factors contribute to the overall biological and pharmacological actions of these agents. The aim of this review is to evaluate new possible more specific analytical approaches to the determination of the origin and purity of chondroitin sulfate preparations for pharmaceutical application and in nutraceuticals, such as the evaluation of the molecular mass values, the constituent disaccharides, and the specific and sensitive agarose‐gel electrophoresis technique. Furthermore, a critical evaluation is presented, together with a discussion of the limits of these analytical approaches. Finally, the necessity for reference standards having high specificity, purity and well‐known physico‐chemical properties useful for accurate and reproducible quantitative analyses will be discussed. © 2007 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 96: 3168–3180, 2007
Electrophoretic approaches to the analysis of complex polysaccharides
2006, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Complex polysaccharides, glycosaminoglycans (GAGs), are a class of ubiquitous macromolecules exhibiting a wide range of biological functions. They are widely distributed as sidechains of proteoglycans (PGs) in the extracellular matrix and at cellular level. The recent emergence of enhanced analytical tools for their study has triggered a virtual explosion in the field of glycomics. Analytical electrophoretic separation techniques, including agarose-gel, capillary electrophoresis (HPCE) and fluorophore-assisted carbohydrate electrophoresis (FACE), of GAGs and GAG-derived oligosaccharides have been employed for the structural analysis and quantification of hyaluronic acid (HA), chondroitin sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS), heparan sulfate (HS), heparin (Hep) and acidic bacterial polysaccharides. Furthermore, recent developments in the electrophoretic separation and detection of unsaturated disaccharides and oligosaccharides derived from GAGs by enzymatic or chemical degradation have made it possible to examine alterations of GAGs with respect to their amounts and fine structural features in various pathological conditions, thus becoming applicable for diagnosis. In this paper, the electromigration procedures developed to analyze and characterize complex polysaccharides are reviewed. Moreover, a critical evaluation of the biological relevance of the results obtained by these electrophoresis approaches is presented.

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Analytical and preparative separation of acidic glycosaminoglycans by electrophoresis in barium acetate

Abstract

J. Biol. Chem

Anal. Biochem

J. Biol. Chem

J. Biol. Chem

Biochim. Biophys. Acta