= Emerging. More than 5 years before clinical availability.
= Expected to be clinically available in 1 to 4 years.
= Clinically available now.
MSACL 2018 EU : Boutin

MSACL 2018 EU Abstract

Topic: Metabolomics

Discovery of Novel Galabiosylceramide-Related Biomarkers of Fabry Disease by Semi-Targeted Metabolomics: the Complex Issue of Structural Isomer Interferences

Michel Boutin (Presenter)
Université de Sherbrooke

Presenter Bio: Michel Boutin is a mass spectrometry specialist. He received his master’s and Ph.D. degrees from Montreal University for research projects related to airborne monitoring of contaminants in the workplace. He did a first postdoctorate in industrial hygiene at McGill University (Montreal, QC), a second postdoctorate at the Université Pierre et Marie Curie (Paris, France) on the analysis of proteins related to cancer by mass spectrometry (ESI and MALDI), and a third postdoctorate at the Institute for Research in Immunology and Cancer (Montreal, QC) in metabolomics. He worked for 2 years as a scientific assistant for a proteomic platform (Université Laval, QC). Since 2011, he is the technical director of the Water-CHUS Expertise Centre in Clinical Mass Spectrometry (Clinical Research Centre-CHUS, Sherbrooke, QC).

Authors: Michel Boutin, Iskren Menkovic, Tristan Martineau, Vanessa Vaillancourt-Lavigueur, Amanda Toupin, Christiane Auray-Blais
Université de Sherbrooke, Sherbrooke, Québec, Canada

Short Abstract

Fabry disease is an X-linked lysosomal storage disorder causing severe cardiac, renal and cerebrovascular complications. A metabolomic study targeting lipids in urine revealed 22 galabiosylceramide (Ga2) isoforms/analogs as Fabry disease biomarkers. Unfortunately, the efficiency of these biomarkers was significantly compromised by the co-analysis of their lactosylceramide (LacCer) structural isomers differing only by the conformation of one glycosidic linkage. A normal phase chromatography was developed to separate Ga2 isoforms/analogs from their LacCer counterparts. The removal of the LacCer interferences significantly increased the sensitivity of Ga2 biomarkers, especially for untreated Fabry females (from 9.3% to 70.4%.).

Long Abstract

Introduction

Fabry disease is an X-linked lysosomal storage disorder caused by the alpha-Galactosidase A deficiency. This latter enzyme is responsible of the recycling of cellular sphingolipids, such as galabiosylceramide (Ga2), globotriaosylceramide (Gb3) and globotriaosylsphingosine (Lyso-Gb3). Reduced enzyme activity leads to the accumulation of substrates in organs and biological fluids resulting in renal, cardiac and cerebrovascular manifestations often causing the premature death of Fabry patients. The development of new Fabry disease treatments, such as gene therapy, enzyme replacement therapy, chaperon therapy, and substrate reduction therapy renders the discovery of very sensitive and reliable Fabry disease biomarkers for diagnosis and monitoring purposes essential. A semi-targeted metabolomic study performed in our laboratory in urine from untreated Fabry males revealed 22 Ga2 isoforms/analogs as Fabry disease biomarkers. Unfortunately, these biomarkers were not sensitive and reliable especially for Fabry females and Fabry males having residual enzyme activity. These disappointing results were due to the co-analysis of lactosylceramide (LacCer), a structural isomer of Ga2 which is not a substrate of alpha-Galactosidase A or a biomarker for Fabry disease.

Methods

For the metabolomic study leading to the discovery of Ga2 isoforms/analogs as Fabry disease biomarkers, urine samples from 16 untreated Fabry males and 16 healthy control males were compared. The specimens were purified by liquid-liquid extraction, separated by reverse phase liquid chromatography (Acquity, Waters) and analyzed using a time-of-flight mass spectrometer (QTof, Synapt G1, Waters) in the mass range corresponding to Ga2 isoforms/analogs (m/z 820-1020). The results were analyzed by orthogonal partial least-square-discriminant analysis (OPLS-DA) (EZinfo, Umetrics, Sweden) to identify molecules with higher abundance in urine from Fabry patients compared to healthy controls. All theses molecules corresponded to Ga2 isoforms/analogs.

A normal phase UPLC method was developed to separate Ga2 isoforms/analogs from their LacCer isomeric interferences (Acquity I-Class, Waters). The multiple reaction monitoring analysis were performed on a triple-quadrupole mass spectrometer (Xevo TQ-S, Waters).

Results

The semi-targeted metabolomic study revealed 22 different Ga2 isoforms/analogs as Fabry disease biomarkers. More specifically, Ga2 isoforms correspond to Ga2 molecules with different fatty acid chains, and analogs correspond to Ga2 molecules with modified sphingosine moieties. To our knowledge, various Ga2 isoforms/analogs detected during this metabolomic study, such as Ga2 molecules with a hydrated sphingosine, an extra double bond on the sphingosine, or a methylated amide linkage were not reported in the literature before. Unfortunately, when analyzed by reverse phase chromatography, the Ga2 isoforms/analogs were not reliable biomarkers for the diagnosis of Fabry disease due to the co-analysis of LacCer with Ga2.

Therefore, a normal phase UPLC method was developed to analyze Ga2 isoforms/analogs separated from their LacCer counterparts. Urine samples from untreated Fabry patients (34 males and 54 females), treated Fabry patients (33 males and 19 females), and healthy controls (34 males and 25 females) were evaluated based on their Ga2 levels.

Conclusions & Discussion

The sensitivity and reliability of the 22 Ga2 isoforms/analogs detected during the metabolomic study were significantly increased by separating them from their structural isomers (LacCer). For untreated Fabry females, the sensitivity of total Ga2 (22 isoforms/analogs) increased from 9.3 to 70.4% by removing LacCer interferences. One untreated Fabry female and two treated Fabry females presented abnormal levels of Ga2, but normal levels of Gb3 (a clinically used Fabry disease biomarker) showing the importance to include Ga2 in the panel of Fabry disease biomarkers for the diagnosis and monitoring of the disease. Significantly, higher levels of LacCer were observed for females compared to males. This observation might be explained by the presence of LacCer in leukocytes and the fact that females can be more frequently affected by urinary tract infections than males.


References & Acknowledgements:

(1) M. Boutin, C. Auray-Blais, Metabolomic Discovery of Novel Urinary Galabiosylceramide Analogs as Fabry Diease Biomarkers, J. Am. Soc. Mass Spectrom., 26, 499-510, 2015.

(2) M. Boutin, I. Menkovic, T. Martineau, V. Vaillancourt-Lavigueur, A. Toupin, C. Auray-Blais, Separation and Analysis of Lactosylceramide, Galabiosylceramide, and Globotriaosylceramide by LC-MS/MS in Urine of Fabry Disease Patients, Anal. Chem., 89, 13382-13390, 2017.

We are grateful to Waters Corporation for their continued scientific support and partnership, and to Dr Joe T. R. Clarke for his scientific expertise. We would like to acknowledge the dedicated collaboration of colleagues and coordinating-nurses at the Canadian Fabry Disease Initiative. We thank all Fabry patients and healthy reference controls who generously provided urine samples for this study.


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