Determination Of Glycosaminoglycans Research Articles

Newborn Screening Program for Mucopolysaccharidosis Type II and Long-Term Follow-Up of the Screen-Positive Subjects in Taiwan.

Background: Mucopolysaccharidosis II (MPS II) is an X-linked disorder resulting from a deficiency in lysosomal enzyme iduronate-2-sulfatase (IDS), which causes the accumulation of glycosaminoglycans (GAGs) in the lysosomes of many tissues and organs, leading to progressive cellular dysfunction. An MPS II newborn screening program has been available in Taiwan since 2015. The aim of the current study was to collect and analyze the long-term follow-up data of the screen-positive subjects in this program. Methods: From August 2015 to April 2022, 548,624 newborns were screened for MPS II by dried blood spots using tandem mass spectrometry, of which 202 suspected infants were referred to our hospital for confirmation. The diagnosis of MPS II was confirmed by IDS enzyme activity assay in leukocytes, quantitative determination of urinary GAGs by mass spectrometry, and identification of the IDS gene variant. Results: Among the 202 referred infants, 10 (5%) with seven IDS gene variants were diagnosed with confirmed MPS II (Group 1), 151 (75%) with nine IDS gene variants were classified as having suspected MPS II or pseudodeficiency (Group 2), and 41 (20%) with five IDS gene variants were classified as not having MPS II (Group 3). Long-term follow-up every 6 months was arranged for the infants in Group 1 and Group 2. Intravenous enzyme replacement therapy (ERT) was started in four patients at 1, 0.5, 0.4, and 0.5 years of age, respectively. Three patients also received hematopoietic stem cell transplantation (HSCT) at 1.5, 0.9, and 0.6 years of age, respectively. After ERT and/or HSCT, IDS enzyme activity and the quantity of urinary GAGs significantly improved in all of these patients compared with the baseline data. Conclusions: Because of the progressive nature of MPS II, early diagnosis via a newborn screening program and timely initiation of ERT and/or HSCT before the occurrence of irreversible organ damage may lead to better clinical outcomes. The findings of the current study could serve as baseline data for the analysis of the long-term effects of ERT and HSCT in these patients.

Mass spectrometry and two-dimensional electrophoresis in prenatal diagnosis of mucopolysaccharidosis type VI

BackgroundMucopolysaccharidosis VI (MPS VI) or Maroteaux–Lamy syndrome is an autosomal recessive lysosomal storage disorder. Clinical manifestations are related to progressive accumulation of dermatan sulfate (DS). Two-dimensional electrophoresis has traditionally been used for the diagnosis of MPS disorders. The method is only qualitative and is time consuming. For prenatal diagnosis of MPS, 6–8 ml of amniotic fluid is required and 5 working days to complete. It needs personal experience to do the test and to interpret the results. Mass spectrometry (MS) is now available as a quantitative method and for prenatal diagnosis of MPS it needs less amniotic fluid and takes only 2 working days. It is more accurate, less person dependent, but it costs more. Our aim was to introduce quantitative determination of dermatan sulfate using mass spectrometry in the prenatal diagnosis of MPS VI in Egypt and to compare this technique to the classical qualitative diagnosis using two-dimensional electrophoresis (2-DEP) of the glycosaminoglycans (GAGs) in amniotic fluid. Thirty pregnant females each with single fetus were subjected to amniocentesis at 16 weeks gestation. Ten with a previously affected MPS VI infant and twenty served as controls. Prenatal diagnosis (PD) was done by both MS and 2-DEP.ResultsMS verified 2-DEP results which showed 5 affected and 5 non-affected fetuses with MPS VI.ConclusionTwo-dimensional electrophoresis of the GAGs in amniotic fluid is a good qualitative method and MS was an accurate quantitative method for prenatal diagnosis of MPS type VI. Quantitative determination of GAGs in AF by mass spectrometry is quicker. Where prenatal diagnosis is recommended for at risk pregnancies, mass spectrometry could be used more in the future as it gives rapid and accurate results.

Determination of glycosaminoglycans in biological matrices using a simple and sensitive reversed-phase HPLC method with fluorescent detection

This paper suggests a sensitive reversed-phase gradient HPLC method combined with fluorescence detection that has been developed, optimized and tested via the quantitative analysis of authentic biological material in an effort to determine and subsequently compare the total content of glycosaminoglycans (GAGs) in various collagen-based biomaterials intended for medical application.The proposed analytical method enabled the identification and separation of the GAGs present from the other components in the samples using commonly-available laboratory equipment; moreover, the very low detection limit of the method permits the determination of GAGs even for very small samples.This study describes the development of the method, including the isolation and processing of the collagen samples prior to HPLC analysis and the optimal parameters applied during the chromatographic analysis. The application of the method in laboratory practice is documented by means of several examples of the determination of GAGs employing both commercial standards and real collagen samples isolated from various animal tissues.

Study of Urinary Glycosaminoglycans among Essential Hypertensive Patients

Introduction: Essential hypertension is a systemic disease which affects endothelial basement membrane. Changes in Glycosaminoglycans (GAG) distribution pattern on glomerular basement membrane has been noted in hypertension. Hence it seems likely that an increase excretion of GAG levels may be an indicator of reduction in renal function. The qualitative or quantitative determination of urinary GAGs may be of value and could constitute a non invasive marker to assess the renal damage in essential hypertension. Aim: Differential analysis of urinary GAGs in essential hypertensive patients. Materials and Methods: This analytical case-control observational study which was conducted from November 2014 to June 2016 in Department of Biochemistry, SDM College of Medical Sciences and Hospital, Dharwad, Karnataka, India. The study group included 50 male patients with age group of 30-60 years, clinically diagnosed with essential hypertension. Control group included 50 healthy male individuals with age group of 30-60 years, blood donors visiting to hospital blood bank. Random urine sample was collected. Urine creatinine was estimated by Jaffe’s method, urine GAGs by Dimethyl methylene blue (DMMB) dye method and urine microalbumin by particle enhanced Turbidimetric inhibition immunoassay method. Correlation was tested with Spearman’s correlation coefficient. Level of significance was set for p-value <0.05 with confidence interval of 95%. Results: In present study, urinary GAGs levels in essential hypertensive study group was 14.57±10.16 mg/dL and in control group was 10.09±6.04 mg/dL. Urinary GAGs levels in essential hypertensive group was significantly high (p-value=0.890) when compared with normotensive control group. But no statistical significant correlation was found between urine GAGs and urine microalbumin in hypertensive study group. Conclusion: Estimation of urine glycosaminoglycans in essential hypertensive patients is a simple, rapid and cost effective test which asseses the glomerular function. It can be used as one of the early marker for diagnosis of nephropathy before microalbuminuria sets in.

Synthesis and Biological Uses of A3B Type Water‐Soluble Phthalocyanine Alternate to Alcian Blue

AbstractThis study describes the synthesis and characterization of new non‐symmetric cationic phthalocyanine derivatives and their capability for use in staining of microscopic normal and pathological tissue sections, polyacrylamide gel staining, and spectrophotometric analyses. These new synthesized compounds have specifically stained the glycosaminoglycans (GAGs) in the matrix of cartilage tissue of the trachea. In addition, the mucus produced by the stomach, the small intestine and the colon tissue were stained with non‐symmetrical cationic phthalocyanines comparable to the Alcian Blue 8GX. In addition, the determination of GAGs and glycoproteins in polyacrylamide gels has also been evaluated. Besides, the usage of these compounds to spectrophotometrically measure GAGs in biological fluids was also investigated. We have found that 6 and 7 are good alternatives to Alcian Blue 8GX due to comparable staining performance and interaction with biological molecules, especially acidic components such as acidic GAGs and mucin. These dyes have good solubility with quaternary ammonium groups effecting their binding performance to biological molecules.

Composition and structure of glycosaminoglycans in DBS from 2-3-day-old newborns for the diagnosis of mucopolysaccharidosis

Dried blood spot (DBS) technology is a cheap and easy method largely applied in newborn screening. Mucopolysaccharidoses (MPS) are characterized by the deficit of enzymes that degrade glycosaminoglycans (GAGs) characterized by progressive worsening of the conditions. For a possible early diagnosis of MPS, we developed a method of uronic acid (UA)-GAGs determination in DBS of 600 healthy newborns and from a small group of MPS subjects matched for age. Spotted blood UA-GAGs of the normal newborns are composed of 67.2% chondroitin sulfate (CS), 28.6% heparan sulfate (HS) and 4.4% hyaluronic acid with a CS/HS ratio of 2.35 and a total GAGs content of 0.43 μg/DBS. A chemical evaluation of CS and HS structure was performed by measuring their disaccharide composition, sulfation and the overall charge density. The DBS of four different MPS types presented an increase of total or single UA-GAGs content and/or modifications of the CS and HS disaccharide composition as well as chemical signature also related to the MPS enzymatic defect. The modifications of the UA-GAGs composition, parameters and structure of healthy newborns determined in DBS would be useful for a possible early diagnosis of various MPS types.

Neural cells generated from human induced pluripotent stem cells as a model of CNS involvement in mucopolysaccharidosis type II.

Mucopolysaccharidosis type II (MPSII) is a rare X-linked lysosomal storage disorder caused by mutations in the iduronate-2-sulfatase (IDS) gene (IDS, Xq28). MPSII is characterized by skeletal deformities, hearing loss, airway obstruction, hepatosplenomegaly, cardiac valvular disease, and progressive neurological impairment. At the cellular level, IDS deficiency leads to lysosomal storage of glycosaminoglycans (GAGs), dominated by accumulation of dermatan and heparan sulfates. Human induced pluripotent stem cells (iPSC) represent an alternative system that complements the available MPSII murine model. Herein we report on the reprogramming of peripheral white blood cells from male and female MPSII patients into iPSC using a non-integrating protocol based on the Sendai virus vector system. We differentiated the iPSC lines into IDS deficient and GAG accumulating β-Tubulin III+ neurons, GFAP+ astrocytes, and CNPase+ oligodendrocytes. The lysosomal system in these cells displayed structural abnormalities reminiscent of those previously found in patient tissues and murine IDS deficient neuronal stem cells. Furthermore, quantitative determination of GAGs revealed a moderate increase in GAG levels in IDS deficient neurons and glia. We also tested the effects of recombinant IDS and found that the exogenous enzyme was internalized from the culture media and partially decreased the intracellular GAG levels in iPSC-derived neural cells; however, it failed to completely prevent accumulation of GAGs. In summary, we demonstrate that this human iPSC based model expresses the cellular and biochemical features of MPSII, and thus represents a useful experimental tool for further pathogenesis studies as well as therapy development and testing.

FACE analysis as a fast and reliable methodology to monitor the sulfation and total amount of chondroitin sulfate in biological samples of clinical importance.

Glycosaminoglycans (GAGs) due to their hydrophilic character and high anionic charge densities play important roles in various (patho)physiological processes. The identification and quantification of GAGs in biological samples and tissues could be useful prognostic and diagnostic tools in pathological conditions. Despite the noteworthy progress in the development of sensitive and accurate methodologies for the determination of GAGs, there is a significant lack in methodologies regarding sample preparation and reliable fast analysis methods enabling the simultaneous analysis of several biological samples. In this report, developed protocols for the isolation of GAGs in biological samples were applied to analyze various sulfated chondroitin sulfate- and hyaluronan-derived disaccharides using fluorophore-assisted carbohydrate electrophoresis (FACE). Applications to biologic samples of clinical importance include blood serum, lens capsule tissue and urine. The sample preparation protocol followed by FACE analysis allows quantification with an optimal linearity over the concentration range 1.0–220.0 µg/mL, affording a limit of quantitation of 50 ng of disaccharides. Validation of FACE results was performed by capillary electrophoresis and high performance liquid chromatography techniques.

Biochemical diagnosis of mucopolysaccharidoses over 11 years

Aim of the study The aim of the study was to perform biochemical laboratory diagnosis of mucopolysaccharidoses (MPS) among clinically suspected patients who referred to our department and to find out the frequency of each type of MPS among the studied patients. Patients and methods The study included 1249 patients who referred to our Biochemical Genetics Laboratory during the last 11 years for the diagnosis or exclusion of MPS. Each patient was subjected to quantitative determination of total urinary glycosaminoglycans (GAGs). Patients with high concentrations and patients clinically suspected to have MPS type-IV (104 patients of the 1249) were subjected to electrophoretic separation of GAGs in urine. The activity of the specific enzymes was fluorometrically assayed in the patients proved to have MPS by electrophoresis and in those suspected clinically to have Morquio syndrome (MPS type-IV) even with normal levels of total urinary GAGs or normal electrophoretic separation. Results Of the 1249 patients screened for MPS, 548 (43.9%) patients had elevated total GAGs in urine. Using two-dimensional electrophoretic separation of GAGs extracted from urine in patients with high total GAGs and in patients suspected clinically to have Morquio syndrome, 278 (22.3% of the total 1249) patients proved to be affected by MPS. The specific enzyme assay in the 278 positive patients revealed the following distribution: MPS type-I (n=79) (28.5% of the 278), MPS type-II (n=46) (16.5% of the 278), MPS type-III B (n=18) (6.5% of the 278), MPS type-III, probable A, C, or D undetermined (n=25) (9% of the 278), MPS type-IV A (n=39) (13.9% of the 278), and MPS type-VI (n=71) (25.5% of the 278). Conclusion Quantitative determination of total GAGs in urine is a simple procedure to select patients for electrophoretic separation of GAGs. However, enzymatic assay is mandatory to confirm the MPS type, especially with the presence of enzyme replacement therapy for types-I, II, and VI and for prenatal diagnosis in coming pregnancies. The commonest MPS disorders in this study were MPS type-I followed by type-VI and then type-II.

Assessment of respiratory involvement in children with mucoplysaccharidosis using pulmonary function tests

BackgroundMucopolysaccharidosis (MPS) are classified into seven clinical types based on eleven known lysosomal enzyme deficiencies of glycosaminoglycan (GAG) metabolism. Respiratory involvement seen in most MPS types includes recurrent respiratory infections, upper and lower airway obstruction, tracheomalacia, restrictive lung disease, and sleep disturbances. Aim of the studyTo delineate the pattern of respiratory compromise and pulmonary function abnormalities in MPS patients. MethodsThis is a cross section observational study conducted on 30 patients recruited from the Neurometabolic Clinic, Children’s Hospital, Cairo University over a period of 18months. All patients were screened first by the quantitative determination of GAGs in urine, and diagnosis was confirmed by unidimensional electrophoresis for GAGs in urine and/or specific enzymatic assay in blood leucocytes. Infant pulmonary functions (IPFT) were done in twenty-two patients (<3years of age), while 8 cases performed impulse oscillometry (IOS) test (3–6years of age). ResultsAges at diagnosis ranged from 1 to 9years with a median of 2.3years. Male to female ratio was 4:1. Consanguinity was observed in 53.3% whereas similar family condition was present in 40% of cases. Lumbar kyphosis was detected in 60% of cases, while scoliosis was detected in 46.7%. Results of pulmonary functions were mainly obstructive in 20 (66.6%) cases; however, combined obstructive and restrictive were detected in only 6 (20%) of cases. Data showed no association between the presence of scoliosis or the presence of organomegaly and the pattern of pulmonary function abnormalities. ConclusionsEvaluation and follow up of patients with MPS using pulmonary function tests are essential to detect early involvement of respiratory system and hence start treatment for respiratory complications early in the course of the disease.

English

The significance of serum levels of glycosaminoglycans (GAGs) and insulin like growth factor-1 (IGF-1) in early screening of hepatocellular carcinoma in cirrhotic patients was evaluated. The effect of diabetes on GAGs and IGF-1 levels was also estimated. Fifty cirrhotic patients with early stage Hepatocellular carcinoma (HCC) (22 were diabetic), thirty control cirrhotic patients without HCC (11 were diabetic) and twenty normal control subjects, were enrolled to the study. Serum α-fetoprotein (AFP), the commonly used marker for HCC, was measured in all HCC patients. Serum GAGs increased significantly, while IGF-1 was reduced in patients with cirrhosis and early stage HCC compared to normal control (P < 0.001). There was a significant reduction in GAGs and IGF-1 levels in control cirrhotic group compared to HCC group (P < 0.05). HCC patients who had normal AFP showed significantly increased GAGs and reduced IGF-1 levels compared to normal control. In comparison with corresponding non-diabetic patients, diabetic patients showed a significant increase in serum GAGs in both cirrhotic control and HCC (P < 0.01), while a significant decrease was observed in serum IGF-1 only in HCC (P < 0.05). Concomitant determination and monitoring of serum GAGs and IGF-1 could be used as a simple, low cost and non-invasive marker for HCC in cirrhotic patients. Key words: Hepatocelluar carcinoma, liver cirrhosis, alpha fetoprotein, glycosaminoglycans, insulin like growth factor-1.

Reliable detection of mucopolysacchariduria in dried-urine filter paper samples

BackgroundThe mucopolysaccharidoses (MPS) are inherited metabolic disorders with bone, joint, and visceral abnormalities, leading to multi-organ dysfunction and, sometimes, neurological manifestations. These diseases are caused by storage of glycosaminoglycans (GAGs) and other complex molecules in tissues, among other pathogenic mechanisms. Definitive diagnosis of the affected individual is mainly based on the identification of the specific enzyme deficiency. New therapies are available or are in development for these pathologies, and early diagnosis seems to be important for the therapy outcomes. Almost all MPS patients have increased levels of GAGs in urine being their evaluation usually the first step in the screening of these conditions. Test on urine may be challenging as transportation of liquid urine samples in appropriate conditions for long distances, especially across international borders, could be difficult. MethodsWith the aim of overcoming the difficulties related to the use of liquid samples, we extended and validated previous studies about colorimetric determination of GAGs in dried-urine filter paper (DUFP) samples. ResultsIn the conditions we described, there are no differences in the concentration of GAGs between urine and DUFP samples. Untreated patients with MPS and normal controls were well discriminated using any of the samples. ConclusionsDried-urine filter paper is a suitable sample for the colorimetric quantitation of GAGs, and that its incorporation as an additional tool for screening of MPS should be considered by reference laboratories.

Progress in structural analysis of glycosaminoglycans and their applications in biomaterials

The rising interest in the application of glycosaminoglycans (GAGs) is one of the main reasons for exploring different species and optimizing the extracting conditions of various GAGs in the last decade. Recent research data on GAGs have suggested that they have many new biological functions such as anti-atherogenesis, anticoagulation, prevention and cure of arthritis, morphogenesis and cell division. They are widely applied in functional food, clinical medicine, cosmetics and biomaterial. Especially, in the biomaterials industry, an increasing number of new applications have been found for GAGs such as tissue-engineering material, biodegradable film-forming materials and micro-encapsulating agents. However, these bioactivities and applications are dependent on their fine structure with different monosaccharide unit and substitute group patterns. This review provides recent information on GAGs preparation, determination and structural assay, as well as their potential novel or improved applications such as tissue engineering biomaterial.

Analysis of Glycosaminoglycans Using Mass Spectrometry.

The glycosaminoglycans (GAGs) are linear polysaccharides expressed on animal cell surfaces and in extracellular matrices. Their biosynthesis is under complex control and confers a domain structure that is essential to their ability to bind to protein partners. Key to understanding the functions of GAGs are methods to determine accurately and rapidly patterns of sulfation, acetylation and uronic acid epimerization that correlate with protein binding or other biological activities. Mass spectrometry (MS) is particularly suitable for the analysis of GAGs for biomedical purposes. Using modern ionization techniques it is possible to accurately determine molecular weights of GAG oligosaccharides and their distributions within a mixture. Methods for direct interfacing with liquid chromatography have been developed to permit online mass spectrometric analysis of GAGs. New tandem mass spectrometric methods for fine structure determination of GAGs are emerging. This review summarizes MS-based approaches for analysis of GAGs, including tissue extraction and chromatographic methods compatible with LC/MS and tandem MS.

Reliability of a visual test for the rapid detection of mucopolysaccharidoses: GAG-test®

Mucopolysaccharidoses (MPS) are a group of lysosomal storage disorders, characterized by the deficiency/absence of one of the enzymes involved in the intralysosomal degradation of glycosaminoglycans (GAGs). The quantitative determination of urinary GAGs using dimethylmethylene blue (DMB) shows high reliability. However, the logistics and staff for this method are not always available in primary care centers. Sending urine samples to reference laboratories increases the cost and delays the diagnosis. Thus, the aim of this article is to develop and evaluate a simple and low-cost visual test (GAG-test(®)) for the screening of urine samples from patients under suspicion of suffering from MPS. The purpose is to narrow down the number of samples to be assayed through the quantitative method. A measure of 50 µl urine was added to 2 ml DMB solution. A color change from dark blue to purple indicates an excess of GAGs. The quantitative analyses showed a significant difference between controls' and patients' concentrations (P<0.05). After optimization of the composition, positive and negative results obtained with the qualitative test were able to discriminate between normal urines and those from patients suffering from mucopolysaccharidosis. Therefore, GAG-test(®) has proved to be a useful tool for the prior diagnosis of patients suffering from mucopolysaccharidosis, reducing the number of individuals with whom investigations should be continued.

Neonatal screening for mucopolysaccharidoses by determination of glycosaminoglycans in the eluate of urine‐impregnated paper: preliminary results of an improved DMB‐based procedure

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.

Noninvasive Assessment of Glycosaminoglycan Production in Injectable Tissue-Engineered Cartilage Constructs Using Magnetic Resonance Imaging

The glycosaminoglycan (GAG) content of engineered cartilage is a determinant of biochemical and mechanical quality. The ability to measure the degree to which GAG content is maintained or increases in an implant is therefore of importance in cartilage repair procedures. The gadolinium exclusion magnetic resonance imaging (MRI) method for estimating matrix fixed charge density (FCD) is ideally suited to this. One promising approach to cartilage repair is use of seeded injectable hydrogels. Accordingly, we assess the reliability of measuring GAG content in such a system ex vivo using MRI. Samples of the photopolymerizable hydrogel, poly(ethylene oxide) diacrylate, were seeded with bovine chondrocytes (approximately 2.4 million cells/sample). The FCD of the constructs was determined using MRI after 9, 16, 29, 36, 43, and 50 days of incubation. Values were correlated with the results of biochemical determination of GAG from the same samples. FCD and GAG were found to be statistically significantly correlated (R2 = 0.91, p < 0.01). We conclude that MRI-derived FCD measurements of FCD in injectable hydrogels reflect tissue GAG content and that this methodology therefore has potential for in vivo monitoring of such constructs.

Stability of urinary glycosaminoglycans in patients with mucopolysaccharidoses

Objective The aim of this work is to investigate the degradation of urinary glycosaminoglycans (GAGs) at different storage temperatures, in order to identify whether frozen transportation to reference laboratories is necessary. An improved method for the determination of total GAGs with 1,9-dimethylmethylene blue (DMB) is presented. Design and methods Urine samples of 37 patients suffering from mucopolysaccharidoses (MPS) were analyzed in this study (13 Hunter, 6 Maroteaux–Lamy, 6 Morquio, 6 Sanfilippo, 5 Hurler–Scheie, and 1 Sly). Stability was assayed at room temperature, 5 °C and − 30 °C, and analyses were repeated for at least 15 days. Spectrophotometric quantitation of GAGs with DMB was used for all determinations, using a variable wavelength for quantitation. Results The concentration of urinary GAGs was stable for 10 days at room temperature, but it was found to be stable for more than 15 days at 5 °C and − 30 °C. Conclusions The stability of GAGs allows urine samples to be sent for quantitation at a clinical laboratory without the need to freeze samples, as this would not affect results. This issue is important for the rapid detection of MPS at hospitals or primary health care centres, where GAGs determination is not performed.

Determination of endogenous glycosaminoglycans derived disaccharides in human plasma by HPLC: Validation and application in a clinical study

SB-424323 is a new, orally active anti-thrombotic agent presently in phase-II clinical development, with limited hemorrhagic risk and a unique mechanism of action involving the induction of glycosaminoglycans (GAGs) biosynthesis. The objective of the present study was to develop a simple and rapid high performance liquid chromatography (HPLC) method for determination of endogenous GAGs derived disaccharides in plasma samples from a phase-II clinical study of SB-424323. Sample preparation was a simple heat treatment of the diluted plasma followed by digestion of endogenous GAGs with chondroitinase ABC to yield unsaturated disaccharides, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-galactose (DeltaDi-0S), 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose (DeltaDi-4S), and 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose (DeltaDi-6S). These disaccharides were recovered and purified using centrifugal filtration through a filter with 3000 molecular weight cut-off along with externally added internal standard 2-acetamido-2-deoxy-3-O-(2-O-sulfo-beta-D-gluco-4-enepyranosyluronic acid)-D-galactose (DeltaDi-UA2S). A gradient reverse phase HPLC separation was developed on a Waters Symmetry C(18) column (4.6 mm x 150 mm, 5 microm) with a gradient mobile phase system consisting of 0.8 mM tetrabutylammonium hydrogen sulfate and 2mM sodium chloride and acetonitrile at a flow rate of 1.0 mL/min. The eluate was monitored with an ultraviolet detector set at 230 nm. Plasma standard curves were linear (r(2)> or =0.994) in the concentration range 1.0-20 microg/mL with a lower limit of quantification (LLOQ) of 1.0 microg/mL for each of the disaccharide. The mean measured quality control (QC) concentrations for the disaccharides deviated from the nominal concentrations in the range of -8.92 to 5.61% and -16.3 to 16.7%, for inter and intra-day, respectively. The inter and intra-day precision in the measurement of QC samples, were in the range of 3.21 to 18.2% relative standard deviation (R.S.D.) and 0.32 to 20.9% R.S.D., respectively. The inter and intra-day precision in the measurement of endogenous GAGs derived disaccharides in human control plasma, were in the range of 5.8 to 15.9% R.S.D. and 1.17 to 7.74% R.S.D., respectively. Stability of the processed samples was confirmed up to 48 h in the auto-sampler. The method is simple, reliable, and easily adaptable to analysis of large number of samples under logistics of a clinical study. The present method has been used to investigate the GAGs levels in the plasma of patients in a phase II clinical study of SB-424323.

Threshold effect of urinary glycosaminoglycans and the walk test as indicators of disease progression in a survey of subjects with Mucopolysaccharidosis VI (Maroteaux-Lamy syndrome)

A cross-sectional survey in individuals affected with the lysosomal storage disease Mucopolysaccharidosis VI (MPS VI) was conducted to establish demographics, urinary glycosaminoglycan (GAG) levels, and clinical progression of the disease. The survey evaluated 121 bona fide MPS VI-affected individuals over the age of 4 years from 15 countries across the Americas, Europe, and Australasia representing greater than 10% of the estimated world prevalence of the disease. A medical history, complete physical exam, urinary GAG determination, and assessment of several clinical measures related to physical endurance, pulmonary function, joint range of motion, strength, and quality of life were completed for each participant. Although a wide variation in clinical presentation was observed, several general findings were obtained reflecting progression of the disease. Impaired physical endurance, as measured by the distance achieved in a 6-min walk, could be demonstrated across all age groups of MPS VI-affected individuals. High urinary GAG values (>200 mug/mg creatinine) were associated with an accelerated clinical course comprised of age-adjusted short stature and low body weight, impaired endurance, compromised pulmonary function, and reduced joint range of motion. An unexpected result was the predominance of urinary GAG values <100 mug/mg creatinine for those participants over the age of 20 years. Pending the collection of longitudinal data, these results suggest that urinary GAG levels predict clinical morbidity, and longer-term survival is associated with urinary GAG levels below a threshold of 100 mug/mg creatinine.