A modified liquid chromatography/tandem mass spectrometry method for predominant disaccharide units of urinary glycosaminoglycans in patients with mucopolysaccharidoses

Abstract

BackgroundThe 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.MethodsA 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.ResultsOne 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).ConclusionsThe 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.

Analysis of Total Human Urinary Glycosaminoglycan Disaccharides by Liquid Chromatography–Tandem Mass Spectrometry

“…This study provides the first detailed look at the disaccharide composition and distribution of these GAGs. Most prior studies 14,29,31,33,35 have only evaluated GAGs present at greatly elevated levels in patients suffering from MPS. Moreover, since <1 μ g of GAG might be present in 1 mL of a normal urine sample, a more effcient method of GAG isolation, as well as a more sensitive method of GAG detection, than that used in previous studies of MPS patients with high urinary GAG levels was required.…”

The relationships between urinary glycosaminoglycan levels and phenotypes of mucopolysaccharidoses

“…The DMB ratio can provide useful information to evaluate the effectiveness of ERT from a biochemical point of view; however, it cannot directly reflect the efficacy of DS, HS, or KS degradation after ERT. Only a few studies have described changes in urine DS, HS, or KS after ERT for MPS I, II, IVA, and VI (Chuang et al, ; Ru et al, ). In the present study, the KS level decreased by 90% after ERT for MPS IVA compared with only a 31% decrease in the change of DMB ratio.…”

“…Seventy-nine patients diagnosed with different types of MPS (type I [n = 14], II mild form [n = 14], II severe form [n = 9], III [n = 11], IV [n = 23], and VI [n = 8]; 57 males and 22 females; age range: 0.7-49.3 years) were enrolled from January 2013 to December 2017 at Mackay Memorial Hospital, Taipei, Taiwan. The levels of three urinary GAGs (DS, HS, and KS) were evaluated using the LC-MS/MS method, and the non-specific total level of urinary GAGs (DMB/creatinine ratio) was evaluated using the DMB spectrophotometric method as previously described (Auray-Blais et al, 2016, 2011Chuang et al, 2001Chuang et al, , 2002Chuang et al, , 2014Chuang, Lin, & Lin, 2017;Kubaski et al, 2017;Martell et al, 2011;Oguma, Tomatsu, & Okazaki, 2007). The diagnosis of the type of MPS was confirmed by specific enzyme activity assays in serum, leukocytes and/or skin fibroblasts, and/or identification of a pathogenic mutation.…”

“…2-D EP is the most commonly used method to help determine specific MPS diseases; however, it is time-consuming and the interpretation is ambiguous and subjective, making the diagnosis unreliable. To overcome the limitations of these first-line MPS screening methods, the liquid chromatography/tandem mass spectrometry (LC-MS/MS) method has been used to determine MPS subgroups, and it has been demonstrated to be a valuable and important method (Auray-Blais et al, 2016, 2011Chuang et al, 2014;Mashima et al, 2016).…”

Development and validation of an LC–MS/MS Method for the quantitation of heparan sulfate in human urine

“…Despite the success of these methods, there exist some limitations. The lower limit of quantitation (LLOQ) of most of the methods ranges from high nanogram per milliliter (ng/mL) to low microgram per milliliter (μg/mL) and usually requires a large sample volume (Auray‐Blais et al, , ; Chuang et al, ; Trim et al, ; Zhang, Wood, Young, & Millington, ). Such sensitivity is not enough to detect low levels of excreted heparan sulfate from urine samples of healthy donors.…”