Neonatal Mass Urine Screening Approach for Early Detection of Mucopolysaccharidoses by UPLC-MS/MS

Abstract

Mucopolysaccharidoses (MPSs) are lysosomal storage disorders caused by deficiencies of enzymes involved in the catabolism of glycosaminoglycans (GAGs). Various treatments such as enzyme replacement therapy and/or hematopoietic stem cell transplant are available for MPSs. Early initiation of treatment improves the outcome and delays the onset of symptoms, highlighting the need for newborn screening for MPSs. The main objective of this project was to devise and validate a multiplex urine filter paper method for GAG analysis using a tandem mass spectrometry (MS/MS) approach to screen newborns for MPSs. Eluted urine samples from 21-day-old newborns were evaporated and a methanolysis reaction was performed. Samples were resuspended and analyzed using a UPLC-MS/MS system. A one-minute chromatographic method allowed the absolute quantification of heparan sulfate (HS), dermatan sulfate (DS), and creatinine. Method validation revealed high precision (< 9% relative standard deviation) and accuracy (< 7% bias) for all analytes. The reference values normalized to creatinine obtained by the analysis of five hundred 21-day-old newborn urine samples were 34.6 +/-6.2 mg/mmol of creatinine and 17.3 +/-3.9 mg/mmol of creatinine for HS and DS, respectively. We present a rapid and efficient method for populational newborn urine screening using MS/MS, which could also be applied to high-risk screening.

High-Throughput Liquid Chromatography–Tandem Mass Spectrometry Quantification of Glycosaminoglycans as Biomarkers of Mucopolysaccharidosis II

“…However, to our surprise, butanolysis yielded two HS-derived peaks with the same mass instead of a single peak as was expected. DS hydrolysis, via acid methanolysis, yielded at least five distinct peaks with the same mass in both DS standards as well as in biological samples such as CSF ( Figure S2 ), which was observed by Menkovic et al [ 8 ] in MPS patients’ urine samples. Additionally, we performed an MS/MS experiment that confirmed that those isomeric peaks were identical molecules (although, MS/MS spectra do not provide the stereochemical structural confirmation).…”

“…GAGs are a major class of extracellular matrix biomolecules [ 5 ] that are secreted into the circulatory system (blood), urine and CSF, and thus have been widely evaluated as a biomarker of primary storage in preclinical models of MPS as well as in MPS patients. There is a growing body of evidence to support the use of urinary GAGs as predictive biomarkers of MPS, as well as treatment responsive biomarkers in MPS II patients on enzyme replacement therapy (ERT) [ 3 , 6 , 7 , 8 ]. The field has previously acknowledged that there is a lack of relationship between GAG levels across different organs or biofluids [ 9 ].…”

“…This advancement has been directly enabled by the technological advances in liquid chromatography mass spectrometry (LC-MS/MS). Over the past decade, LC-MS/MS has become the preferred platform to analyze chemically or enzymatically degraded oligosaccharides from broad classes of GAGs [ 3 , 8 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 ]. Acid hydrolysis of GAGs coupled with LC-MS/MS quantification is a highly sensitive method that has been successfully applied to biological samples from mouse models of MPS and MPS patient samples [ 19 ].…”

“…Acid hydrolysis of GAGs coupled with LC-MS/MS quantification is a highly sensitive method that has been successfully applied to biological samples from mouse models of MPS and MPS patient samples [ 19 ]. The acid hydrolysis chemical reaction, via methanol [ 8 , 13 , 28 , 29 ], ethanol or butanol [ 22 ], depolymerizes the complex GAGs into discrete units detected via mass spectrometry. This method has the ability to simplify the complex and heterogeneous GAGs into reduced disaccharide species which can be helpful for analysis but potentially oversimplifies the chemical biology.…”

Mucopolysaccharidosis Type II: One Hundred Years of Research, Diagnosis, and Treatment

“…In addition, other approaches based on the measurement of GAGs in urine and blood samples through liquid chromatography-MS/MS have been developed in order to be potentially used as newborn screening tools for MPSs [85,86], also in combination with enzyme activity assays [87].…”