A modified liquid chromatography/tandem mass spectrometry method for predominant disaccharide units of urinary glycosami
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RESEARCH
Open Access
A modified liquid chromatography/tandem mass spectrometry method for predominant disaccharide units of urinary glycosaminoglycans in patients with mucopolysaccharidoses Chih-Kuang Chuang1,4,5†, Hsiang-Yu Lin1,2,6,7,8†, Tuen-Jen Wang3, Chia-Chen Tsai1, Hsuan-Liang Liu4 and Shuan-Pei Lin1,2,6,7,9*
Abstract Background: The identification of acid mucopolysaccharide by the liquid chromatography/tandem mass spectrometry method (LC-MS/MS) of the predominant disaccharide units of glycosaminoglycans (GAGs) (chondroitin sulfate, CS; dermatan sulfate, DS; heparan sulfate, HS) after methanolysis is validated and applicable for mucopolysaccharidosis (MPS) type determination. Methods: A total of 76 urine samples were collected and analyzed, from nine MPS I patients, 13 MPS II patients, seven MPS III patients, eight MPS VI patients, and 39 normal controls. Urinary GAG was first precipitated by the Alcian blue method followed by a treatment of 3 N HCl methanol. The protonated species of the methylated disaccharide products were detected by using a multiple reaction monitoring experiment. Internal standards, [2H6] CS, [2H6] DS and [2H6] HS, were prepared in-house by deuteriomethanolysis of CS, DS and HS. Results: One particular disaccharide for each GAG was selected, in which the parent ion and its daughter ion after collision were m/z 426.1 → 236.2 for DS (m/z 432 → 239 for dimers derived from [2H6] CS and [2H6] DS) and m/z 384.2 → 161.9 for HS (m/z 390.4 → 162.5 for the [2H6] HS dimer). The quantities of DS and HS were determined, which varied from one MPS type to the other. The results can be used to evaluate the severity of MPS subgroups, as well as urinary GAG amelioration at follow-up after enzyme replacement therapy (ERT). Conclusions: The modified LC–MS/MS method for MPS type determination is specific, sensitive, validated, accurate, and applicable for simultaneous quantifications of urinary DS and HS. This method can help to make correct diagnosis of MPS patients and evaluate the effectiveness of ERT. Keywords: Enzyme replacement therapy, Glycosaminoglycans, Liquid chromatography/tandem mass spectrometry, Methanolysis, Mucopolysaccharidosis
Introduction The mucopolysaccharidoses (MPS), known as a group of lysosomal storage disorders, are caused by the deficiency of specific enzymes that catalyze the stepwise degradation of glycosaminoglycans (GAGs; mucopolysaccharides). There are at least 11 known enzymes involved in the * Correspondence: [email protected] † Equal contributors 1 Division of Genetics and Metabolism, Department of Medical Research, Mackay Memorial Hospital, Taipei, Taiwan 2 Department of Pediatrics, Mackay Memorial Hospital, Taipei, Taiwan Full list of author information is available at the end of the article
catabolism of dermatan sulfate (DS), heparan sulfate (HS), keratan sulfate (KS), chondroitin sulfate (CS), and hyaluronic acid (HA) [1-3]. Any enzyme deficiency will block the GAG degradation, which results in GAG macromolecules with a specific carbohydrate or sulfated carbohy
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