Ceramide Dihexoside Separation

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Separation and Analysis of Lactosylceramide, Galabiosylceramide and Globotriaosylceramide by LC-MS/MS in Urine of Fabry Disease Patients Michel Boutin, Iskren Menkovic, Tristan Martineau, Vanessa Vaillancourt-Lavigueur, Amanda Toupin, and Christiane Auray-Blais Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03609 • Publication Date (Web): 03 Nov 2017 Downloaded from http://pubs.acs.org on November 4, 2017

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Separation and Analysis of Lactosylceramide, Galabiosylceramide and Globotriaosylceramide by LC-MS/MS in Urine of Fabry Disease Patients Michel Boutin, Iskren Menkovic, Tristan Martineau, Vanessa Vaillancourt-Lavigueur, Amanda Toupin, and Christiane Auray-Blais* Division of Medical Genetics, Department of Pediatrics, Faculty of Medicine and Health Sciences, Université de Sherbrooke, 3001, 12th Avenue North, Sherbrooke, Quebec, Canada J1H 5N4 Graphical abstract

Ceramide Dihexoside Separation (Urine from Fabry patient)

100

LacCer

Cer O

OH

HO O

O

O

OH

OH HO O

Cer O

0 4.5

OH OH

HO

%

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

5.0

5.5

HO

Ga2 OHO O

OH OH OH

OH

Time (min)

Abstract Fabry disease is an X-linked lysosomal storage disorder caused by alpha-galactosidase A (α-GAL A) deficiency. This enzyme contributes to the cellular recycling of glycosphingolipids such as galabiosylceramide (Ga2), globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3) by hydrolysing the terminal alpha-galactosyl moiety. Urine and plasma α-GAL A substrates are currently analyzed as biomarkers for the detection, monitoring and follow-up of Fabry disease patients. The sensitivity of the analysis of Ga2 is decreased by the co-analysis of its structural isomer, lactosylceramide (LacCer), which is not an α-GAL A substrate. A normal phase ultraperformance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) methodology, allowing the baseline separation of 12 Ga2 isoforms/analogs from their 1 ACS Paragon Plus Environment


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lactosylceramide counterparts, was developed and validated in urine. The method was multiplexed with the analysis of 12 Gb3 isoforms/analogs having the same fatty acid moieties as those of Ga2 for comparison, and with creatinine for sample normalization. Urine samples were studied from 34 untreated and 33 Fabry males treated by enzyme replacement therapy (ERT), 54 untreated and 19 ERT-treated Fabry females, along with 34 males and 25 female healthy controls. The chromatographic separation of Ga2 from LacCer increased the sensitivity of analysis, especially in women. One untreated Fabry female and 2 treated Fabry females presented abnormal levels of Ga2 but normal levels of Gb3, supporting the importance of analyzing Ga2, in addition to Gb3. Our results show that urine LacCer levels from females were significantly higher than males. Moreover, LacCer levels were not affected by Fabry disease for both males and females.

Key Words: Fabry disease, Lactosylceramide (LacCer), Galabiosylceramide (Ga2), Globotriaosylceramide (Gb3), Ceramide dihexoside (CD), Creatinine, Urine, Ultra-performance liquid chromatography (UPLC), Tandem mass spectrometry (MS/MS)

2 ACS Paragon Plus Environment

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INTRODUCTION

Fabry disease (OMIM 301500) is a panethnic, multisystemic X-linked lysosomal storage disorder caused by GLA mutations leading to decreased or absent alpha-galactosidase A (α-GAL A, EC 3.2.1.22) activity. The function of this enzyme is to cleave the terminal galactosyl moiety of glycosphingolipids such as galabiosylceramide (Ga2) (Figure 1A), globotriaosylceramide (Gb3) [Gal(α1→4)Gal(β1→4)Glc(β1→1)Cer] (Figure 1C), and globotriaosylsphingosine (lyso-Gb3) (Figure 1D) [Gal(α1→4)Gal(β1→4)Glc(β1→1)Sph]. α-GAL A deficiency causes the accumulation of these substrates in organs, tissues and biological fluids.1 The renal, cerebral and cardiovascular manifestations of Fabry disease often result in premature death of patients.2 Heterozygous Fabry females are usually less clinically affected than hemizygous Fabry males. However, some Fabry females may develop symptoms as severe as those experienced by males owing in part to random X-chromosome inactivation.3 The current primary treatment for Fabry disease is the enzyme replacement therapy (ERT), consisting of the intravenous infusion of recombinant α-GAL A.4 Other treatments such as chaperone therapy5, substrate reduction therapy6, and gene therapy7 are also being investigated.

The diagnosis, high-risk screening and monitoring of Fabry disease patients rely on the analysis of globotriaosylceramide (Gb3) (Figure 1C)8 or its deacylated form, globotriaosylsphingosine (lysoGb3) (Figure 1D),9-11 in urine and/or plasma. However, because of difficulties in the diagnosis of patients with residual enzyme activity, such as those with the Taiwanese late-onset IVS4+919G>A cardiac variant mutation,12 considerable effort has been devoted to the discovery of new Fabry disease biomarkers. Metabolomic studies performed with urine13 and plasma14 revealed 18 and 24



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Gb3 isoforms/analogs, respectively, as Fabry disease biomarkers. Isoforms are defined here as Gb3 molecules with different fatty acid chains, whereas analogs correspond to Gb3 molecules with modified sphingosine moieties.13-14 During these metabolomic studies, Gb3 isoforms with a methylated amide linkage were discovered.15 Other metabolomic studies revealed lyso-Gb3 analogs in urine16 and in plasma.17 Another Fabry disease biomarker, which has been less completely investigated, is galabiosylceramide (Ga2) [Gal(α1→4)Gal(β1→4)Cer] (Figure 1A). Some papers reported the analysis of ceramide dihexoside (CD)18-21 which is the sum of Ga2 and its structural isomer lactosylceramide (LacCer) [Gal(β1→4)Glc(β1→4)Cer] (Figure 1B); the latter is not an α-GAL A substrate. We noticed that for Fabry patients who excrete high levels of various biomarkers, such as untreated classic Fabry males, the levels of LacCer are negligible compared to the levels of Ga2. Urine samples from this specific Fabry disease patient population were compared to healthy controls during a metabolomic study which revealed 22 Ga2 isoforms and analogs as Fabry disease biomarkers.22 However, a similar study performed with urine from untreated Fabry females revealed no biomarkers (unpublished results) probably owing to the interference of LacCer.

The main objective of this study was to develop and validate an ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) methodology allowing the analysis of 12 Ga2 isoforms/analogs separated from their LacCer structural isomers in urine. To our knowledge, this is the first time that a liquid chromatography method has achieved the systematic separation of Ga2 from LacCer. This method also permits simultaneous comparative analysis of 12 Gb3-related isoforms/analogs, with normalization by measurement of creatinine for urine sample. The secondary objectives of the study were: (1) to evaluate the relative abundance of


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57 potential Ga2 isoforms/analogs and their Gb3 counterparts in urine from untreated Fabry males, and to focus on the 12 most abundant Ga2 isoforms/analogs to be analyzed; (2) to analyze Ga2, LacCer, and Gb3 in urine from large cohorts of Fabry disease patients (treated and untreated, males and females) and healthy controls; (3) to compare Ga2 with ceramide dihexoside (CD; Ga2+LacCer) and with Gb3 on the basis of their efficiency to diagnose Fabry disease patients; (4) to statistically evaluate the effect of Fabry disease on the urinary levels of LacCer; and (5) to compare LacCer urinary levels statistically between male and female patients.

A) Galabiosylceramide (Ga2)

α-GAL A

OH OH

HO O

α-Gal

HO O

O

H

OH

O OH

H OH

Sphingosine

Ceramide dihexosides (CD)

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β-Gal

OH

NH

Fatty acid

O

α-GAL A

B) Lactosylceramide (LacCer)

X H OH

OH

HO O

Sphingosine

H

O

β-Glc

OH OH

HO O

O

OH

β-Gal

OH

NH

Fatty acid

O

α-GAL A

C) Globotriaosylceramide (Gb3)

OH OH

HO O

α-Gal

H OH

OH

HO O

Sphingosine

O

H

β-Glc

OH

O OH

HO O

O

OH

β-Gal

OH

NH

Fatty acid

O

α-GAL A

D) Globotriaosylsphingosine (Lyso-Gb3)

OH OH

HO O

α-Gal

H OH

OH

HO

Sphingosine

O H2N H

O

β-Glc

OH

O OH

HO O

O

OH

β-Gal

OH

Figure 1: Structures of A) Galabiosylceramide (Ga2); B) Lactosylceramide (LacCer); C) Globotriaosylceramide (Gb3) and D) Globotriaosylsphingosine (Lyso-Gb3); Ga2, LacCer and Gb3



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are illustrated with a C22:0 fatty acid moiety. The cleavage sites of the α-galactosiadse A (α-GAL A) are indicated on the Figure. Glu = glucose, and Gal = galactose. EXPERIMENTAL SECTION Ethics approval. This project was approved by the Research Ethics Board (REB) of the Faculty of Medicine and Health Sciences and the Centre hospitalier universitaire de Sherbrooke (CHUS), and the REBs of all clinician collaborators. Urine samples. Random urine samples were collected after informed consent was obtained from 34 untreated Fabry males (UFM; 1 to 65 y; mean 25 y), 33 Fabry males treated by ERT (TFM; 4 to 60 y; mean 35 y), 34 healthy control males (CM; 19 to 69 y, mean 37 y), 54 untreated Fabry females (UFF; 4 to 78 y; mean 34 y), 19 Fabry females treated by ERT (TFF; 10 to 66 y, mean 45 y), and 25 healthy control females (CF; 19 to 59 y, mean 39 y). The diagnosis of Fabry disease was confirmed by enzyme

activity measurement in leucocytes or by mutation analysis. Treated patients were receiving ERT with either agalsidase-alfa at 0.2 mg/kg/2 weeks (Replagal, Shire Human Genetic Therapies Inc. Lexington, MA) or agalsidase-beta at 1.0 mg/kg/2 weeks (Fabrazyme, Sanofi-Genzyme, Cambridge, MA), the dosages recommended by the manufacturers. Fresh random urine specimens were stored at -20oC until analysis. Urine samples were not centrifuged or filtered since Ga2, LacCer and Gb3 are only slightly soluble in water, and are integral components of urinary sediments. Reagents. Deuterated N-omega-CD3-palmitoyl-lactosylceramide (LacCer(C16:0)D3) (>98%), Nheptadecanoyl-lactosylceramide (LacCer(C17:0)) (>98%), lactosylceramide (isoform mixture) from pork red blood cells (>98%), deuterated N-omega-CD3-octadecanoyl-ceramide trihexoside (Gb3(C18:0)D3) (>98%), N-heptadecanoyl ceramide trihexoside (Gb3(C17:0)), and ceramide trihexoside (isoform mixture) from pork red blood cells (>98%) were purchased from Matreya 6 ACS Paragon Plus Environment

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(Pleasant Gap, PA). Deuterated creatinine (methyl-D3) (99.9 atom % D) was supplied by CDN Isotopes (Pointe-Claire, Canada). HPLC grade methanol (MeOH) and acetonitrile (ACN) were from EMD Chemicals Inc. (Darmstadt, Germany). Optima LC/MS grade water and A.C.S. Reagent grade ammonium formate (Amm. Form.) were from Fisher Scientific (Fair Lawn, NJ). Formic acid (FA) (>99%) was from Acros Organics (Morris Plain, NJ). ReagentPlus® grade dimethylsulfoxide (DMSO) (≥99.5%), HPLC Plus grade tert-butyl methyl ether (MTBE) (99.9%), and creatinine (≥98%) were purchased from Sigma-Aldrich (Saint-Louis, MO).

Sample preparation. Urine samples (500 µL) were added to a 2 mL polypropylene tube followed by 25 µL of deuterated creatinine (10 mM in water), 310 µL of MeOH, 40 µL of sphingolipid internal standards (LacCer(C16:0)D3 0.963 µM and Gb3(C18:0)D3 0.790 µM in methanol), and 650 µL of MTBE. The samples were then vortexed 3 s and centrifuged 1 min at 9400 g. The organic phase was recovered. All samples were extracted a second time with 650 µL of MTBE. The two organic phases were combined, evaporated to dryness and resuspended in 500 µL of 20% DMSO/80% phase A (94.5% ACN/2.5% MeOH/2.5% H2O/0.5% FA/5 mM Amm. Form.). For the creatinine calibration curve, the 500 µL aliquot of urine was replaced by the same volume of standard creatinine solutions (Table 1). For the LacCer(C17:0) and Gb3(C17:0) calibration curves, 500 µL of urine from a healthy control was used as the matrix, 40 µL of the calibration point standard solution (Table 1) was added and the volume of MeOH was decreased to 270 µL to compensate for the MeOH contained in the calibration curve standards.



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Table 1: Concentrations of the stock solutions used for the calibration curves (P0 to P6) and for the spiked (S-) low (L), medium (M) and high (H) quality controls (QCs).

Stock solution P0 P1 P2 P3 P4 P5 P6 S-LQC S-MQC S-HQD

Creatinine mM 0.0 0.5 2.0 5.0 7.5 12.0 20.0 1.5 9.0 18.0

Concentration LacCer(C17:0) μM 0.00 0.05 0.13 0.63 1.25 3.13 6.25 0.15 1.88 5.00

Gb3(C17:0) μM 0.00 0.05 0.25 1.50 6.25 12.50 31.25 0.15 6.25 18.75

UPLC-MS/MS Analysis of LacCer, Ga2 and Gb3 isoforms/analogs, and creatinine in urine samples. The Acquity I-Class UPLC system from Waters Corp. (Milford, MA, USA) was used for the separation of the different molecules analyzed. The parameters of the isocratic normal-phase UPLC method are shown in Table 2 and were adapted from a method previously developed in our laboratory for the separation and analysis of glucosylceramide and galactosylceramide.23 The UPLC system was linked to a Xevo TQ-S (Waters) tandem mass spectrometer operated in positive electrospray using the multiple reaction monitoring (MRM) mode. The general mass spectrometry parameters are presented in Table 2. In an exploratory study, a total of 57 potential Ga2 isoforms/analogs (Table S-1) were analyzed in pooled urine from 5 untreated Fabry males to identify the 12 most abundant Ga2 isoforms/analogs. The relative abundance of the same 57 isoforms/analogs was also measured for Gb3 (Table S-2). The structure of Ga2(C22:0)OHMe, an isoform not revealed as a Fabry disease biomarker during the metabolomic study of Ga2 isoforms/analogs,22 was validated by tandem mass spectrometry using a quadrupole-time-of-flight (Q-Tof) mass spectrometer (Synapt G1, Waters) and a collision energy ramp of 20-40 V. Table S3 shows the transitions, dwell times, cone voltages and collision energies for the molecules


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analyzed in this study corresponding to creatinine, creatinine-D3, LacCer(C16:0)D3, Gb3(C18:0)D3, the 12 most abundant Ga2 isoforms/analogs and their LacCer and Gb3 counterparts (same fatty acid moieties). For the hydroxylated isoforms, the in-source fragmentation resulting from the loss of one water molecule was not negligible even after optimization of the ionization parameters. We therefore integrated the peaks corresponding to these in-source fragments and their areas were added to the areas of their precursors. For example, the in-source fragment of the isoform (C22:0)OH was recorded on the MRM channel of the isoform (C22:1). Thus, the MRM transitions corresponding to the isoforms (C20:1) and (C22:1)Me were added to the MRM method to monitor the in-source fragments of the isoforms (C20:0)OH and (C22:0)OHMe, respectively. For Ga2, LacCer, and Gb3 the fragment ions recorded corresponded to the dehydrated ceramide. Ga2 was analyzed from 0 to 7 min, Gb3 from 7 to 12 min, and creatinine from 12 to 16 min. The analysis of MRM results was performed using the QuanLynx V4.1 (SCN704) software (Waters). The quadratic calibration curves were forced to the origin and a 1/x weighing factor was applied.

Table 2: UPLC and MS/MS parameters for the analysis of LacCer, Ga2, Gb3 and creatinine in urine.

mobile phase A

UPLC Parameters Halo HILIC 2.7 Advanced Materials Technology (Wilmington, DE) length: 150 mm; internal diameter: 4.6 mm particle diameter: 2.7 μm ACN 94.5%/MeOH 2.5%/H2O 2.5%/FA 0.5%/5 mM Amm. Form.

MS Parameters ionization mode ESI polarity positive acquisition mode MRM capillary voltage 3.2 kV source offset voltage 50 V

mobile phase B mobile phases ratio weak wash solvent strong wash solvent flow rate injection volume

ACN 49.5%/H2O 45.0% MeOH 5.0%/FA 0.5%/5 mM Amm. Form. 90% A/10% B (isocratic) ACN ACN 0.5 mL/min 2 μL (partial loop with needle overfill)

desolvation temperature desolvation gas flow cone gas flow nebulized pressure collision gas flow span

column

250 o C 1000 L/h 150 L/h 6.3 Bar 0.15 mL/min 0.1 Da



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Table S-1: Relative abundance of 57 potential Ga2 isoforms/analogs in pooled urine samples from 5 untreated Fabry males. nd = not detected #

Isoform/analog

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 48 49 50 46 47 51 52 53 54 55 56 57

(d18:1)(C14:0) (d18:1)(C16:0) (d18:1)(C18:0) (d18:1)(C20:0) (d18:1)(C22:0) (d18:1)(C24:0) (d18:1)(C26:0) (d18:1)(C16:1) (d18:1)(C18:1) (d18:1)(C20:1) (d18:1)(C22:1) (d18:1)(C24:1) (d18:1)(C26:1) (d18:1)(C16:2) (d18:1)(C18:2) (d18:1)(C20:2) (d18:1)(C22:2) (d18:1)(C24:2) (d18:1)(C26:2) (d18:1)(C16:0)OH (d18:1)(C18:0)OH (d18:1)(C20:0)OH (d18:1)(C22:0)OH (d18:1)(C24:0)OH (d18:1)(C26:0)OH (d18:1)(C16:1)OH (d18:1)(C18:1)OH (d18:1)(C20:1)OH (d18:1)(C22:1)OH (d18:1)(C24:1)OH (d18:1)(C26:1)OH (d18:0)OH(C16:0) (d18:0)OH(C18:0) (d18:0)OH(C20:0) (d18:0)OH(C22:0) (d18:0)OH(C24:0) (d18:0)OH(C26:0) (d18:0)OH(C22:0)OH (d18:0)OH(C24:0)OH (d18:1)(C16:0)Me (d18:1)(C18:0)Me (d18:1)(C20:0)Me (d18:1)(C22:0)Me (d18:1)(C24:0)Me (d18:1)(C26:0)Me (d18:1)(C16:1)Me (d18:1)(C18:1)Me (d18:1)(C20:1)Me (d18:1)(C22:1)Me (d18:1)(C24:1)Me (d18:1)(C26:1)Me (d18:1)(C16:0)OHMe (d18:1)(C18:0)OHMe (d18:1)(C20:0)OHMe (d18:1)(C22:0)OHMe (d18:1)(C24:0)OHMe (d18:1)(C26:0)OHMe Total

Precursor ion Fragment ion m/z m/z 834,59 492,48 862,63 520,51 890,66 548,54 918,69 576,57 946,72 604,60 974,75 632,63 1002,78 660,67 860,61 518,49 888,64 546,53 916,67 574,56 944,70 602,59 972,74 630,62 1000,77 658,65 858,59 516,48 886,63 544,51 914,66 572,54 942,69 600,57 970,72 628,60 998,75 656,63 878,62 536,50 906,65 564,54 934,68 592,57 962,71 620,60 990,75 648,63 1018,78 676,66 876,60 534,49 904,64 562,52 932,67 590,55 960,70 618,58 988,73 646,61 1016,76 674,65 880,64 538,52 908,67 566,55 936,70 594,58 964,73 622,61 992,76 650,65 1020,79 678,68 980,72 638,61 1008,76 666,64 876,64 534,53 904,67 562,56 932,70 590,59 960,74 618,62 988,77 646,65 1016,80 674,68 874,63 532,51 902,66 560,54 930,69 588,57 958,72 616,60 986,75 644,63 1014,78 672,67 892,64 550,52 920,67 578,55 948,70 606,58 976,73 634,61 1004,76 662,65 1032,79 690,68

Ga2 Retention time min 5,71 5,56 5,44 5,32 5,22 5,11 5,05 5,58 5,45 5,33 5,22 5,16 5,08 nd nd nd 5,24 5,16 5,09 5,93 5,78 5,64 5,50 5,40 5,33 5,96 5,78 5,63 5,50 5,43 5,37 5,95 5,79 5,65 5,52 5,41 nd nd nd 5,49 5,38 5,26 5,16 5,09 nd nd nd 5,23 5,16 5,13 nd 5,74 5,62 5,50 5,41 5,33 nd

Area

%

Abundance Rank

246 54658 7998 39544 84929 90891 4712 1782 2006 12021 33769 106080 5330 nd nd nd 2826 46230 1994 27311 11449 37408 184413 181844 6288 944 1289 6017 27692 72522 5431 3105 1228 3834 24932 26541 nd nd nd 1115 1003 2791 10063 2087 nd nd nd 1651 5369 1775 nd 2027 2184 8804 68351 5874 nd 1230362

0,020 4,442 0,650 3,214 6,903 7,387 0,383 0,145 0,163 0,977 2,745 8,622 0,433 nd nd nd 0,230 3,757 0,162 2,220 0,931 3,040 14,989 14,780 0,511 0,077 0,105 0,489 2,251 5,894 0,441 0,252 0,100 0,312 2,026 2,157 nd nd nd 0,091 0,082 0,227 0,818 0,170 nd nd nd 0,134 0,436 0,144 nd 0,165 0,178 0,716 5,555 0,477 nd

46 8 21 10 5 4 28 38 36 17 12 3 27 nd nd nd 31 9 37 14 18 11 1 2 22 45 41 23 13 6 25 30 42 29 16 15 nd nd nd 43 44 32 19 34 nd 26 39 nd nd 40 nd 35 33 20 7 24 nd


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Table S-2: Relative abundance of 57 potential Gb3 isoforms/analogs in pooled urine samples from 5 untreated Fabry males. nd = not detected Gb3 #

Isoform/analog

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 48 49 50 46 47 51 52 53 54 55 56 57

(d18:1)(C14:0) (d18:1)(C16:0) (d18:1)(C18:0) (d18:1)(C20:0) (d18:1)(C22:0) (d18:1)(C24:0) (d18:1)(C26:0) (d18:1)(C16:1) (d18:1)(C18:1) (d18:1)(C20:1) (d18:1)(C22:1) (d18:1)(C24:1) (d18:1)(C26:1) (d18:1)(C16:2) (d18:1)(C18:2) (d18:1)(C20:2) (d18:1)(C22:2) (d18:1)(C24:2) (d18:1)(C26:2) (d18:1)(C16:0)OH (d18:1)(C18:0)OH (d18:1)(C20:0)OH (d18:1)(C22:0)OH (d18:1)(C24:0)OH (d18:1)(C26:0)OH (d18:1)(C16:1)OH (d18:1)(C18:1)OH (d18:1)(C20:1)OH (d18:1)(C22:1)OH (d18:1)(C24:1)OH (d18:1)(C26:1)OH (d18:0)OH(C16:0) (d18:0)OH(C18:0) (d18:0)OH(C20:0) (d18:0)OH(C22:0) (d18:0)OH(C24:0) (d18:0)OH(C26:0) (d18:0)OH(C22:0)OH (d18:0)OH(C24:0)OH (d18:1)(C16:0)Me (d18:1)(C18:0)Me (d18:1)(C20:0)Me (d18:1)(C22:0)Me (d18:1)(C24:0)Me (d18:1)(C26:0)Me (d18:1)(C16:1)Me (d18:1)(C18:1)Me (d18:1)(C20:1)Me (d18:1)(C22:1)Me (d18:1)(C24:1)Me (d18:1)(C26:1)Me (d18:1)(C16:0)OHMe (d18:1)(C18:0)OHMe (d18:1)(C20:0)OHMe (d18:1)(C22:0)OHMe (d18:1)(C24:0)OHMe (d18:1)(C26:0)OHMe Total

Precursor ion Fragment ion m/z m/z 996,65 492,48 1024,68 520,51 1052,71 548,54 1080,74 576,57 1108,77 604,60 1136,80 632,63 1164,83 660,67 1022,66 518,49 1050,69 546,53 1078,73 574,56 1106,76 602,59 1134,79 630,62 1162,82 658,65 1020,65 516,48 1048,68 544,51 1076,71 572,54 1104,74 600,57 1132,77 628,60 1160,80 656,63 1040,67 536,50 1068,70 564,54 1096,74 592,57 1124,77 620,60 1152,80 648,63 1180,83 676,66 1038,66 534,49 1066,69 562,52 1094,72 590,55 1122,75 618,58 1150,78 646,61 1178,81 674,65 1042,69 538,52 1070,72 566,55 1098,75 594,58 1126,78 622,61 1154,81 650,65 1182,85 678,68 1142,78 638,61 1170,81 666,64 1038,69 534,53 1066,73 562,56 1094,76 590,59 1122,79 618,62 1150,82 646,65 1178,85 674,68 1036,68 532,51 1064,71 560,54 1092,74 588,57 1120,77 616,60 1148,80 644,63 1176,83 672,67 1054,69 550,52 1082,72 578,55 1110,75 606,58 1138,78 634,61 1166,81 662,65 1194,85 690,68

Retention time min 9,35 9,27 8,97 8,70 8,42 8,20 8,00 9,29 9,05 8,74 8,47 8,30 8,09 nd nd nd 8,55 8,36 nd 9,39 9,34 9,29 9,05 8,75 8,50 9,40 9,36 9,30 9,13 8,80 nd 9,38 9,30 nd nd nd nd 9,34 9,28 9,15 8,85 8,56 8,29 8,14 8,00 nd nd nd 8,39 8,20 nd 8,92 8,63 8,37 8,11 7,99 nd

Area

%

Abundance Rank

5415 933128 600941 1089189 2730781 3366085 49905 11245 58864 212489 707985 3985576 82738 nd nd nd 89833 762989 nd 21026 5464 19237 59190 151159 6618 371 586 1642 3279 63659 nd 4936 4706 nd nd nd nd 25236 29121 31820 45267 111114 319505 83739 637 nd nd nd 168788 87615 nd 92161 153559 383124 407364 8199 nd 16976284

0,032 5,497 3,540 6,416 16,086 19,828 0,294 0,066 0,347 1,252 4,170 23,477 0,487 nd nd nd 0,529 4,494 nd 0,124 0,032 0,113 0,349 0,890 0,039 0,002 0,003 0,010 0,019 0,375 nd 0,029 0,028 nd nd nd nd 0,149 0,172 0,187 0,267 0,655 1,882 0,493 0,004 nd nd nd 0,994 0,516 nd 0,543 0,905 2,257 2,400 0,048 nd

36 5 8 4 3 2 25 32 24 12 7 1 21 nd nd nd 18 6 nd 30 35 31 23 15 34 43 42 40 39 22 nd 37 38 nd nd nd nd 29 28 27 26 16 11 20 41 nd nd nd 13 19 nd 17 14 10 9 33 nd



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Table S-3: Multiple reaction monitoring transitions for the analysis of LacCer, Ga2, Gb3, and creatinine. IS = Internal standard; STD = Standard for the calibration curve


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Method validation. Urine samples from an untreated Fabry female and an untreated male with classic Fabry disease were separated in 100 µL aliquots and used as low (L) and high (H) quality controls (QCs), respectively, for the method validation. Pooled control urine samples were also spiked (S-) with standards of LacCer(C17:0) and Gb3(C17:0) at low (L), medium (M) and high concentrations (H) according to Table 1. Spiked QCs (S-QCs) of creatinine were also prepared (Table 1). The QCs and S-QCs were used to evaluate intraday (n = 5) and interday (n = 5) precision assays. The QCs were also used to evaluate sample stability for 6, 24 and 48 h at room temperature (22oC) (n = 2); 24 and 48 h at 4oC (n = 2); 10 weeks and 6 months at -20oC (n=2); and after 4 freeze-thaw cycles (n = 2). The stability of the reconstituted QCs was also evaluated after 24 h in the autosampler at 20oC (n = 2) and their adsorption to glass- and plasticware was measured after 5 transfers (n = 1). S-QCs were used to evaluate intraday (n=5) and interday (n = 5) accuracy assays. QCs were prepared as samples and S-QCs were prepared as calibration curve points. Matrix effects (suppression or enhancement) were evaluated by post-column infusion of LacCer(C17:0) and Gb3(C17:0) at a concentration of 200 nM and at a flow rate of 15 µL/min. Water and control urine samples with creatinine levels of 3.1, 9.8, and 12.1 were injected to test different matrix concentrations. The extraction recovery was evaluated by spiking 40 µL of a solution containing 4 µg/mL of LacCer isoform mixture and 32 µg/mL of Gb3 isoform mixture in control urine before and after the extraction (n = 3). In both cases, the internal standard was added after the extraction and before the evaporation. The same control urine was also analyzed without the addition of the standard mixture to correct for the endogenous concentration of LacCer and Gb3 isoforms/analogs (n = 3).



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Statistical analysis. PASW Statistics 18 software (SPSS, Quarry Bay, Hong Kong) was used for all the statistical analyses. The levels of ceramide dihexoside (CD) were obtained by adding the LacCer and Ga2 levels. For different molecule groups (LacCer, Ga2, CD, and Gb3), statistical analyses were performed on the total levels of their 12 isoforms/analogs analyzed. The statistical tests were also applied to a Ga2 subset, the hydroxylated Ga2 (Ga2OH) isoforms corresponding to Ga2(C20:0)OH, Ga2(C22:0)OH, Ga2(C22:0)OHMe, and Ga2(C24:0)OH. A previous metabolomic study22 revealed that the proportion of fatty acids with a hydroxyl group was significantly higher for Ga2 compared to Gb3 and thus suggested a different behaviour for this subclass of Ga2 isoforms/analogs. The comparison between biomarker levels from different patient groups and healthy control groups were done using the non-parametrical Mann-Whitney test and the areas under the ROC curves. The normal values for Ga2, Ga2OH, CD, and Gb3 were set to the 95th percentile of the biomarker levels measured in urine from the healthy controls. Males (n = 34) and females (n = 25) were confounded to evaluate the Ga2, Ga2OH and Gb3 normal values and were separated to evaluate the normal values of CD. Normal values were used to calculate the sensitivity (true positive/(true positive + false negative) and the specificity (true negative/(true negative + false positive) of the biomarkers under study.

RESULTS AND DISCUSSION UPLC-MS/MS Analysis of LacCer, Ga2 and Gb3 isoforms/analogs, and creatinine in urine samples. The use of normal phase liquid chromatography allowed the separation of Ga2 isoforms/analogs from their LacCer structural isomers, which are differentiated only by the conformation of two asymmetric carbon atoms involved in the glycosidic linkage (i.e. β-galactose from Ga2 is a C-4 epimer of β-glucose from LacCer, and α-galactose from Ga2 is a C-1 anomer of


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β-galactose from LacCer). (Figure 1A and B). Figure 2 shows an example of the separation of Ga2(C24:0) from LacCer(C24:0) in urine. Since Ga2 standards are not commercially available, the attribution of the Ga2(C24:0) peak was validated by the analysis of samples with different α-Gal activities. As expected, taking into account the normalization with creatinine, the Ga2(C24:0) urinary level was higher for the untreated classic hemizygous Fabry male (no α-Gal activity) (Figure 2A) than for the untreated heterozygous Fabry female (residual α-Gal enzyme activity) (Figure 2B), which was also higher than the healthy control female (normal α-Gal activity) (Figure 2C). Whereas, the attribution of the LacCer(C24:0) peak was confirmed by comparing its retention time with a commercial standard (Figure 2D). The LacCer, Ga2 and Gb3 isoforms/analogs with a hydroxyl group on the fatty acid chain are significantly more susceptible to the in-source loss of one water molecule than the other LacCer, Ga2 and Gb3 isoforms/analogs. Even after optimization of the ionization parameters, the in-source fragmentation of the LacCer and Ga2 hydroxylated isoforms/analogs was ~23%, and for Gb3 hydroxylated isoforms/analogs it was ~4.5%. The insource fragments of these molecules were therefore also recorded and their peak areas were added to the peak areas of their precursors in order to provide more reliable results. Since the in-source fragments of the hydroxylated isoforms/analogs were recorded in the same MRM transition than isoforms/analogs with an extra double-bond, a chromatographic separation of these peaks was required. As example, Figure 3A shows the peaks corresponding to LacCer(C22:0)OH and Ga2(C22:0)OH, whereas Figure 3B shows the peaks corresponding to their in-source fragments, and to LacCer(C22:1) and Ga2(C22:1). A total of 57 possible Ga2 isoforms/analogs were analyzed in pooled urine sample from 5 untreated Fabry males to evaluate their relative abundance (Table S-2). Among the 57 molecules analyzed, 46 Ga2 isoforms/analogs were detected. Since the analysis of all these molecules on a



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regular basis would be too cumbersome, the method focussed on the 12 most abundant isoforms/analogs, accounting for 81% of all Ga2 molecules. The UPLC-MS/MS method was also multiplexed with the analysis of their LacCer and Gb3 counterparts for a comparison purpose, and with creatinine to normalize the concentration of urine samples. Table S-3 shows the relative abundance of 43 Gb3 isoforms/analogs detected in the pool of urine samples from untreated Fabry males. Moreover, the 12 Gb3 isoforms/analogs analyzed with this method account for 84% of all Gb3 molecules. Among the 12 most abundant Ga2 isoforms/analogs analyzed, there is the Ga2(C22:0)OHMe isoform, which was has not been reported previously in the literature. Therefore, its structural elucidation was performed by tandem mass spectrometry (Figure S-1). The presence of the methylation of the amide linkage was confirmed by the presence of fragments at m/z 278.3 (methylated and di-dehydrated sphingosine), 352.4 (methylated and dehydrated fatty acid (C22:0)OH as the amide), and 370.4 (methylated fatty acid (C22:0)OH as the amide). This latter fragment also confirmed the presence of the hydroxyl group on the fatty acid chain. Unfortunately, the position of the hydroxyl group on the fatty acid chain was not revealed by the fragmentation spectrum.


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LacCer (C24:0)

Ga2 (C24:0)

100 A) Untreated Fabry male

1.58e5 cps

%

X10

0 100 B) Untreated Fabry female %

1.36e4 cps

0 100 C) Control female %

1.58e4 cps

0 100 D) Standard LacCer

1.67e5 cps

%

0 4.0

4.5

5.0

Time (min)

5.5

Figure 2: Ion chromatograms corresponding to the analysis of LacCer(C24:0) and Ga2(C24:0) structural isomers (m/z 974.75 → 632.63) in: A) Urine from an untreated Fabry male (Creatinine = 23.7 mM); B) Urine from an untreated Fabry female (Creatinine = 3.3 mM); C) Urine from a control female (creatinine = 8.3 mM); and D) LacCer commercial mixture standard. Cps = counts per second.

100 A)

1.41e4 cps

%

Ga2(C22:0)OH LacCer(C22:0)OH

0 LacCer(C22:1)

100 B)

Ga2(C22:1)

5.41e3 cps Ga2(C22:0)OH (in-source)

%

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


LacCer(C22:0)OH (in-source)

0 4.0

4.4

4.8

5.2

5.6

6.0

Time (min)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figure 3: Ion chromatograms corresponding to: A) the m/z 962.71 → 620.60 transition showing LacCer(C22:0)OH and Ga2(C22:0)OH peaks; and B) the m/z 944.70 → 602.59 transition showing the LacCer(C22:1) and Ga2(C22:1) peaks, and the in-source fragments of LacCer(C22:0)OH and Ga2(C22:0)OH peaks in a urine sample from an untreated Fabry female. Cps = counts per second


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634.7

100 Sphingosine

408 Cps

-H2O

-2H2O˥H+

Sphingosine -2H2O+CH2˥H+

616.7

Sphingosine –H2O˥H+

-Gal

(C22:0)OHMe-H2O˥H+ (C22:0)OHMe˥H+

%

Relative abundance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


-H2O

264.3

796.7

-Gal

958.8

-H2O 278.3

604.7

352.4 370.4 282.3

0

300

400

MH+ 976.7

652.7

500

600

700

800

900

m/z

Figure S-1: Tandem mass spectrometry analysis of Ga2(C22:0)OHMe in urine from an untreated Fabry male using a Q-Tof mass spectrometer. Cps = counts per second; MH+ = molecular ion; Gal = galactose; (C22:0)OHMe˥H+ = protonated amide fragment of the C22:0 fatty acid with an hydroxylation of the carbon chain and a methylation of the amide.

No commercial standard of Ga2 was available. Therefore, LacCer(C17:0) and LacCer(C16:0)D3 were used for the calibration curve and as the internal standard for all LacCer and the Ga2 isoforms/analogs, respectively. Whereas, Gb3(C17:0) and Gb3(C18:0)D3 were used for the calibration curve and as the internal standard for all Gb3 isoforms/analogs, respectively. Finally, creatinine-D3 was used as the internal standard for the analysis of creatinine. Since only the response factors of LacCer(C17:0) and of Gb3(17:0) were used for the quantification for all sphingolipids under study, an isocratic elution was chosen because of the major influence of the mobile phase composition on the response factors of the different molecules analyzed.



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Method validation. Figure S-2 shows the results of the post-column infusion matrix effect analysis. The matrix effect was evaluated by infusion of LacCer(C17:0) and Gb3(C17:0). Water and control urine samples with creatinine levels of 3.1, 9.8, and 12.1 mM were injected with a sample preparation concentration factor (CF) of 1, to test different matrices. The most important matrix effect observed was an ion suppression of -8% occurring at an elution time of 5.8 min inside the segment corresponding to the elution of Ga2 isoforms/analogs, for the urine sample with a creatinine of 12.1 mM. This matrix effect was also evaluated with the control urine sample with a creatinine level of 12.1 mM for sample CF between 0.5 and 5 (Figure S-3). Ion suppression effects ranging from -2 to -37% were measured. The CF of 1 was chosen to minimize ion suppression without sacrificing too much sensitivity.


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Figure S-2: Matrix effect evaluation of 3 urine samples from healthy controls with different creatinine concentrations (3.1 to 12.1 nM) on the LacCer, Ga2, and Gb3 isoform responses. A 200 nM mixture of LacCer(C17:0) and Gb3(C17:0) was infused at 15 µL/min combined with the UPLC elution of the urine samples. A) Ion signal of infused LacCer(C17:0) in the retention time interval of LacCer isoforms/analogs; B) Ion chromatograms of LacCer isoforms/analogs in the LacCer standard mixture (2 µg/mL); C) Ion signal of infused LacCer(C17:0) in the retention time interval of Ga2 isoforms/analogs; D) Ion chromatograms of Ga2 isoforms in urine from an untreated Fabry male; E) Ion signal of infused Gb3(C17:0) in the retention time interval of the Gb3 isoforms/analogs; and F) Ion chromatograms of Gb3 isoforms in urine from an untreated Fabry male. Concentration factor = 1; Injection volume = 2 µL.



100

No matrix CF = 0.5 CF = 1.0 CF = 2.5 CF = 5.0

-8% %

Relative Abundance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0

5.3

-30%

-37%

5.4

5.5

5.6

5.7

5.8

5.9

Time (min)

Figure S-3: Matrix effect evaluation in the Ga2 retention time interval at different urine concentration factors (CF) and an injection volume of 2 µL. LacCer(C17:0) 200 nM infused at 15 µL/min combined with the UPLC elution of urine from a healthy control male (creatinine 12.2 mM). CF = Urine volume/Sample reconstitution volume.


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Table 3 shows the LOD, LOQ endogenous concentrations in LQC and HQC, and the intra- and interday precision assays for creatinine, and the 12 isoforms/analogs analyzed for LacCer, Ga2 and Gb3. Precision (RSD%) were ≤ 13.9 % for all the molecules with concentrations > LOQ analyzed in the LQC and HQC. Table 4 reviews the precision and accuracy results for the quality controls spiked with creatinine, LacCer (C17:0) and Gb3(C17:0). All biases (%) measured were ≤ 10.9%. Table S-4 summarises the stability results for LQC and HQC. Ga2 and Gb3 isoforms/analogs were stable (bias ≤ 19.3%) for at least 48 h at 22oC, 48 h at 4oC, 6 months at -20oC, and for 4 freezethaw cycles when the concentrations were over the LOQ. Prepared samples were also stable (bias ≤ 17.1%) for 24 h at 20oC in the autosampler, and for 5 transfers in glass- or plasticware when the concentrations were over the LOQ. Table S-5 shows the extraction recovery results for 12 LacCer and 12 Gb3 isoforms/analogs analyzed. For LacCer, recoveries between 81 and 93% were measured for concentrations ranging from 2.1 to 77.0 nM, and for Gb3, recoveries between 81 and 92 were obtained for concentrations ranging from 4.5 to 746.9 nM.



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Table 3: Limits of detection (LOD) and of quantification (LOQ), endogenous concentrations, and intra- and interday precision assays (RSD%) measured in low (L) and high (H) quality control (QC) Fabry urine samples.

Precision (RSD%) Concentration Intraday (n = 5) Interday (n = 5) LQC HQC LQC HQC LQC HQC Creatinine (mM) 0,09 0,30 3,15 4,87 0,91 0,95 2,4 1,7 LacCer(C16:0) (nM) 0,5 1,7 53,4 2,0 4,2 7,4 4,9 8,8 LacCer(C20:0) (nM) 0,4 1,3 0,8 0,2 10,6 nd 11,4 nd LacCer(C20:0)OH (nM) 0,4 1,3 0,2 0,1 nd nd nd nd LacCer(C22:0) (nM) 0,5 1,6 5,1 0,5 7,6 nd 8,6 nd LacCer(C22:1) (nM) 0,4 1,3 2,6 0,1 13,6 nd 9,9 nd LacCer(C22:0)OH (nM) 0,5 1,7 0,3 0,2 nd nd nd nd LacCer(C22:0)OHMe (nM) 0,4 1,3 nd 0,0 nd nd nd nd LacCer(C24:0) (nM) 0,6 2,0 8,4 0,5 3,4 nd 5,6 nd LacCer(C24:1) (nM) 1,0 3,3 25,6 0,7 5,0 nd 5,0 nd LacCer(C24:0)OH (nM) 1,0 3,3 0,4 0,1 nd nd nd nd LacCer(C24:1)OH (nM) 0,6 2,0 0,3 nd nd nd nd nd LacCer(C24:2) (nM) 0,6 2,0 6,5 0,1 4,6 nd 7,0 nd 1,1 3,6 6,9 7,3 5,7 4,5 5,5 6,7 Ga2(C16:0) (nM) Ga2(C20:0) (nM) 1,1 3,6 1,0 1,7 nd 12,5 nd 13,3 Ga2(C20:0)OH (nM) 1,0 3,2 0,8 1,9 nd 3,5 nd 12,3 Ga2(C22:0) (nM) 0,9 2,9 3,3 4,9 9,3 13,3 6,2 6,4 Ga2(C22:1) (nM) 1,2 4,0 2,2 2,8 6,8 17,5 4,5 9,7 Ga2(C22:0)OH (nM) 1,2 4,1 3,8 8,1 10,6 6,1 8,7 8,5 Ga2(C22:0)OHMe (nM) 1,4 4,8 2,2 3,8 6,1 7,3 11,2 14,9 Ga2(C24:0) (nM) 1,7 5,7 3,6 7,1 4,7 8,0 9,4 6,1 Ga2(C24:1) (nM) 1,7 5,6 7,3 8,4 8,4 13,9 5,1 8,0 Ga2(C24:0)OH (nM) 1,7 5,5 4,7 10,9 7,4 5,5 15,9 13,3 Ga2(C24:1)OH (nM) 1,6 5,3 2,5 4,2 11,2 10,3 16,1 11,1 Ga2(C24:2) (nM) 1,3 4,4 5,8 4,4 3,4 7,9 6,5 10,8 Gb3(C16:0) (nM) 1,0 3,3 20,8 226,7 5,0 3,4 6,1 3,8 Gb3(C20:0) (nM) 1,4 4,5 14,6 281,1 10,0 4,9 5,2 2,6 Gb3(C20:0)OH (nM) 0,9 3,0 1,0 1,9 17,6 12,6 12,6 13,4 Gb3(C22:0) (nM) 1,7 5,7 28,9 590,2 4,6 5,5 3,8 3,2 Gb3(C22:1) (nM) 1,0 3,3 10,9 156,1 8,8 3,2 6,7 4,0 1,1 3,8 2,1 5,3 9,5 10,8 10,4 6,1 Gb3(C22:0)OH (nM) Gb3(C22:0)OHMe (nM) 1,1 3,8 0,9 2,1 nd 10,5 nd 7,9 Gb3(C24:0) (nM) 1,2 3,9 26,2 652,4 4,8 5,0 2,8 5,2 Gb3(C24:1) (nM) 1,3 4,3 31,6 660,0 6,9 5,0 5,8 5,5 2,0 6,7 3,6 14,2 6,1 5,2 8,0 11,0 Gb3(C24:0)OH (nM) Gb3(C24:1)OH (nM) 1,7 5,5 1,7 6,1 13,2 5,7 13,9 9,9 Gb3(C24:2) (nM) 1,0 3,4 8,3 126,4 6,5 6,0 4,1 3,6 nd = not detected (Bias(%) for the molecules with a level under the limit of detection(LOD)) Bias(%) for molecules with levels over de LOD but under the limit of quantification (LOQ) are written in bold blue. Analyte

LOD

LOQ

Table 4: Intra- and interday precision and accuracy assays for low (L), medium (M), and high (H) quality controls (QC) spiked (S-) with LacCer(C17:0) and Gb3(C17:0) or creatinine.

Analyte Creatinine LacCer(C17:0) Gb3(C17:0)

Precision (RSD%) Intraday (n = 5) Interday (n = 5) S-LQC S-MQC S-HQC S-LQC S-MQC S-HQC 0,9 2,0 2,2 2,9 1,9 3,1 7,7 4,2 2,2 3,8 5,0 2,2 5,4 3,8 2,9 5,6 3,1 1,3

Accuracy (Bias%) Intraday (n = 5) S-LQC S-MQC S-HQC 5,3 1,4 -1,3 -2,4 1,7 2,2 -3,3 1,7 3,7

Accuracy range (Bias %) Interday (n = 5) S-LQC S-MQC S-HQC 0,4 to 7,0 0,7 to 5,0 -5,9 to 2,0 -4,7 to 5,0 -1,9 to 10,3 0,9 to 8,6 -3,3 to 10,9 -4,3 to 3,5 1,3 to 4,6


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Table S-4: Stability of creatinine, lactosylceramide (LacCer), galabiosylceramide (Ga2), and globotriaosylceramide (Gb3) in urine samples at 22oC, 4oC, -20oC and after 4 freeze-thaw cycles, and of reconstituted samples after 24 hours in the autosampler (20oC), 5 transfers in plasticware and 5 transfers in glassware. 22oC (n = 2) 4oC (n = 2) 24 h 48 h 24 h 48 h LQC HQC LQC HQC LQC HQC LQC HQC LQC HQC Bias(%) Bias(%) Bias(%) Bias(%) Bias(%) Bias(%) Bias(%) Bias(%) Bias(%) Bias(%) -1,6 -3,9 -0,6 -2,9 -2,2 -1,6 1,0 -2,4 1,3 -3,3 LacCer(C16:0) -3,2 25,5 -10,5 172,3 -2,2 -3,2 -5,2 -12,4 2,2 -3,0 -15,4 nd -15,1 nd -25,4 nd -3,5 nd -11,7 nd LacCer(C20:0) LacCer(C20:0)OH nd nd nd nd nd nd nd nd nd nd LacCer(C22:0) -5,4 nd -3,8 nd 7,4 nd -10,1 nd 9,3 nd LacCer(C22:1) -1,7 nd -18,5 nd -1,7 nd -4,6 nd -0,6 nd LacCer(C22:0)OH nd nd nd nd nd nd nd nd nd nd LacCer(C22:0)OHMe nd nd nd nd nd nd nd nd nd nd LacCer(C24:0) -14,0 nd 0,8 nd 7,6 nd -4,1 nd -3,8 nd LacCer(C24:1) -20,3 nd -8,0 nd -4,0 nd -4,2 nd -8,4 nd LacCer(C24:0)OH nd nd nd nd nd nd nd nd nd nd LacCer(C24:1)OH nd nd nd nd nd nd nd nd nd nd LacCer(C24:2) -7,5 nd -7,8 nd -0,5 nd -2,6 nd -3,3 nd Ga2(C16:0) -6,2 -4,9 -8,0 -3,3 -0,6 -9,7 -5,8 -10,7 -2,7 -17,9 nd 18,6 nd 0,5 nd 1,3 nd -9,6 nd 7,5 Ga2(C20:0) Ga2(C20:0)OH nd -14,2 nd -9,9 nd -4,4 nd -5,6 nd 5,6 Ga2(C22:0) -3,4 3,6 -7,6 5,6 -10,7 0,1 -3,2 0,1 -1,3 -12,3 Ga2(C22:1) -4,3 19,6 -6,1 17,4 -14,6 -0,9 -9,1 -2,0 4,4 -4,2 Ga2(C22:0)OH -2,5 -0,8 -6,7 4,7 3,2 0,6 7,2 0,2 -6,8 -5,7 Ga2(C22:0)OHMe -14,7 -1,9 -5,0 -3,2 -6,0 1,1 -7,3 -0,4 15,8 -22,8 Ga2(C24:0) -9,5 6,2 -4,7 6,7 -5,3 1,1 -6,9 0,4 -10,8 -1,9 -13,0 7,5 -4,7 0,3 -11,7 2,2 -4,1 -1,6 -14,8 -2,2 Ga2(C24:1) Ga2(C24:0)OH -26,4 1,8 -3,9 0,3 -7,3 1,6 -4,0 -5,5 -9,4 -16,1 Ga2(C24:1)OH -16,2 -6,8 -10,5 -7,9 -8,4 -4,7 -5,7 -10,1 1,6 -14,7 Ga2(C24:2) -6,2 10,0 -8,0 8,1 3,7 -9,7 -3,4 -7,2 0,2 -4,7 Gb3(C16:0) -4,0 4,1 -8,2 7,1 0,0 -3,0 0,1 -2,9 2,1 -1,4 Gb3(C20:0) -6,2 2,4 -4,5 4,9 2,5 2,0 -2,6 -1,9 2,0 -1,9 Gb3(C20:0)OH -28,7 13,7 -15,7 -7,6 3,1 17,6 -7,8 -4,9 -25,7 3,6 Gb3(C22:0) -14,5 1,6 -4,0 4,7 -7,6 0,6 -2,8 -2,3 -1,6 -5,6 Gb3(C22:1) -10,1 3,2 -7,4 4,0 0,9 1,1 -1,0 -2,1 -3,5 -0,9 Gb3(C22:0)OH -8,3 4,2 -2,7 7,6 5,7 -2,4 6,4 2,1 1,1 -1,7 Gb3(C22:0)OHMe nd 11,0 nd 13,1 nd 8,3 nd -17,8 nd -6,8 Gb3(C24:0) -19,3 3,8 -5,4 6,0 -8,2 1,3 -5,3 -1,4 -4,7 -5,9 -11,0 5,2 -4,5 4,0 2,4 2,6 -3,7 -2,5 0,7 -0,9 Gb3(C24:1) Gb3(C24:0)OH -13,4 5,9 -17,8 0,1 -1,4 6,9 -15,6 -6,4 -0,8 -3,9 Gb3(C24:1)OH 7,4 19,0 -10,4 -0,3 10,7 15,2 8,0 4,4 -3,2 12,7 Gb3(C24:2) -18,7 2,5 -4,2 2,9 -6,1 0,4 -0,9 -3,4 -1,2 -3,7 nd = not detected (Bias(%) for the molecules with a level under the limit of detection(LOD)) Bias(%) for molecules with levels over de LOD but under the limit of quantification (LOQ) are written in bold blue. Analyte

6h

-20oC (n = 2) 10 weeks 6 months LQC HQC LQC HQC Bias(%) Bias(%) Bias(%) Bias(%) 2,3 3,1 2,4 4,5 -6,7 -16,0 -10,9 -22,7 -15,2 nd -23,6 nd nd nd nd nd -7,4 nd -9,1 nd -8,9 nd -15,3 nd nd nd nd nd nd nd nd nd -5,4 nd -12,5 nd -4,9 nd -12,5 nd nd nd nd nd nd nd nd nd -9,8 nd -12,7 nd -9,8 -12,8 -7,3 -17,8 nd -14,2 nd -12,8 nd -1,5 nd -13,7 -2,4 -5,0 -9,0 -9,1 nd 3,3 -23,4 -19,1 -0,2 1,4 -13,2 -13,0 -13,3 -6,1 -17,2 -21,0 -4,9 -0,4 -9,9 -2,9 -3,3 0,7 -9,2 -10,2 -14,4 -10,1 -20,5 -17,4 -19,0 -11,9 -13,7 -19,3 0,5 -3,9 -12,9 -11,2 -1,4 -1,7 -13,1 -10,3 -4,9 -0,2 -10,1 -8,6 1,4 -11,6 -23,2 -27,6 -1,4 2,8 -9,2 -7,2 -3,7 0,2 -9,9 -11,2 11,5 5,6 -1,3 -4,6 nd 6,0 nd 17,2 -0,9 6,9 -4,4 -2,8 1,6 7,0 -6,5 -6,0 -13,0 nd -19,8 14,6 0,2 2,8 -24,6 -7,4 -5,9 -1,1 -7,1 -10,8

Freeze Thaw (n = 2) 4 cycles LQC HQC Bias(%) Bias(%) 2,5 12,3 -5,7 -4,9 -7,9 nd nd nd -8,5 nd -10,0 nd nd nd nd nd -5,5 nd -2,4 nd nd nd nd nd 0,2 nd 6,9 1,2 nd 1,7 nd -8,2 -3,4 -8,9 1,4 -13,4 4,4 -2,4 -3,1 6,2 -7,3 -3,8 -2,7 0,4 -3,9 -3,7 5,3 1,9 0,7 3,3 -3,0 -2,8 -3,9 1,1 -15,3 -55,9 -8,2 -1,9 -7,3 -2,0 -10,7 8,2 nd -7,1 -11,1 -1,9 -8,0 -3,0 -11,9 3,3 -7,6 0,0 -9,4 -3,9

20oC in autosampler (n = 5) 24 h LQC HQC Bias(%) Bias(%) -0,5 1,3 7,0 3,5 21,3 nd nd nd 2,8 nd 16,6 nd nd nd nd nd 4,6 nd 7,0 nd nd nd nd nd 10,3 nd 9,6 1,0 nd 10,1 nd 10,9 5,5 7,0 24,4 12,0 -7,2 5,0 10,4 7,8 -8,2 3,0 9,4 7,6 20,3 17,1 7,8 4,2 15,1 13,1 -1,3 -4,3 -2,8 -3,9 4,6 17,6 -8,7 -4,8 -5,8 -4,3 22,0 -1,3 nd 4,4 -6,3 -3,0 -8,5 -2,1 -12,1 0,1 -12,7 11,1 -10,2 -4,7

Glass adsorption (n = 1) 5 transfers LQC HQC Bias(%) Bias(%) ---1,8 -2,6 -7,6 41,6 nd nd nd 6,3 nd -4,9 nd nd nd nd nd -9,3 nd -11,7 nd nd nd nd nd 4,0 nd 15,6 14,1 nd 15,8 nd 5,5 -7,8 11,3 19,0 -4,8 22,6 -0,5 4,9 7,3 32,9 1,9 4,1 -8,6 16,3 -0,4 5,9 -6,5 -0,6 -4,5 1,8 2,2 1,4 -4,8 -4,6 -6,6 -6,4 -5,9 4,3 -4,9 0,5 -7,4 nd 11,7 4,0 -4,1 0,9 -4,5 -11,6 -1,5 1,7 1,6 -5,6 -3,5

Plastic adsorption (n = 1) 5 transfers LQC HQC Bias(%) Bias(%) ----1,2 -0,1 9,7 -12,4 nd nd nd -8,7 nd -14,2 nd nd nd nd nd -5,6 nd -4,5 nd nd nd nd nd 11,9 nd 17,1 -8,7 nd 54,5 nd -6,3 6,5 16,8 -0,4 -5,6 5,9 -8,2 -6,3 -2,5 5,8 7,8 -4,4 7,6 2,8 -4,1 19,5 0,4 3,2 -9,4 7,9 -0,2 -7,5 6,3 -53,5 -6,3 -5,6 2,3 -8,0 2,7 -5,6 -8,4 -16,4 -18,9 -11,4 5,3 -5,6 5,9 13,2 -5,0 -11,2 -7,8 4,5 1,7

Table S-5: Extraction recoveries for 12 LacCer and Gb3 isoforms/analogs in urine spiked with a commercial standard mixture of LacCer and Gb3 isoforms/analogs before and after extraction (n = 3) nd = not detected



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LacCer Gb3 Conc. Recovery Conc. Recovery nM % nM % C16:0 4,8 84 28,7 90 C20:0 8,4 84 38,9 91 (C20:0)OH 0,5* 72 6,6 92 C22:0 52,0 89 438,0 90 C22:1 2,1 92 4,5 81 (C22:0)OH 7,7 83 87,7 89 (C22:0)OHMe nd nd 16,4 90 C24:0 77,0 90 746,9 91 C24:1 19,8 93 97,0 88 (C24:0)OH 39,3 83 428,8 89 (C24:1)OH 29,0 81 236,2 92 C24:2 3,4 91 18,1 86 * Concentration under the limit of quantification (LOQ) Isoform


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LacCer, Ga2 and Gb3 isoforms/analogs urinary levels. Table S-6 shows the age, the α-GAL A mutations, the creatinine concentrations, and the biomarker levels (LacCer, Ga2, and Gb3 isoforms/analogs) normalized to creatinine for all samples analyzed (patients and controls) subdivided according to gender and enzyme replacement therapy status (treated or untreated). Figure 4 shows the box-plot generated from these results. For LacCer, Ga2, CD, and Gb3, the 12 isoforms/analogs analyzed were added together to simplify the Figure. The outliers * and extreme outliers (*) were more than 1.5 and 3 times, respectively, the interquartile range over the higher quartile. As expected, the total Ga2 (Figure 4A) and total Gb3 (Figure 4D) were higher for Fabry patients (males and females) compared to controls, higher for Fabry males compared to Fabry females, and higher for untreated Fabry patients compared to treated Fabry patients. Surprisingly, the total LacCer levels were significantly higher for females compared to males (Figure 4B). The Mann-Whitney non-parametrical statistical test confirmed significant differences (p < 0.05) between the 4 Fabry patient groups and the gender-matched healthy controls for total Ga2, Ga2OH, CD and Gb3 (Table S-7). However, no significant differences were observed for total LacCer levels between the Fabry groups and the healthy controls indicating that this latter sphingolipid, which is not an α-GAL substrate, is not affected by the pathophysiology of Fabry disease even if it is involved in the same biological pathways as Gb3 which is a Fabry disease biomarker. The Mann-Whitney U test also confirmed higher levels of LacCer and CD for females compared to males (p < 0.001) which was not the case for Ga2, Ga2OH and Gb3 (p > 0.34). The areas under the ROC curves (AUCs) were also calculated to evaluate the efficiency of the biomarkers under study to discriminate the patients from the control groups. For the 4 Fabry patient groups studied (UFM, TFM, UFF, TFF), the AUCs were slightly higher (from 0.88 to 0.95) for total Gb3 compared to total Ga2 (from 0.86 to 0.90). Total Ga2 was more reliable than its



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subset, total Ga2OH (AUCs from 0.78 to 0.85), to discriminate between patients and controls. According to AUCs, total Ga2 is significantly more efficient as a Fabry disease biomarker than total CD (Ga2 + LacCer), especially for women. Indeed, total Ga2 presented AUCs of 0.86 for both UFF and TFF which were significantly higher than the AUCs of 0.70 and 0.71 obtained for the same groups with total CD, thus proving again the importance to separate the structural isomers. When, the control males were compared to the control females, no statistical differences were obtained for total Ga2, Ga2OH, and Gb3 (AUCs between 0.50 et 0.57), but significant statistical differences were detected for total LacCer and CD (AUCs of 0.80 and 0.78) due to higher levels of LacCer measured for females. These results might be explained by the fact that LacCer is the principal neutral glycosphingolipid of peripheral blood leucocytes24 and that females are more prone to urinary tract infections than males. For the biomarkers studied, the normal values were set to the 95th percentile measured in the control groups (Table 5). For total Ga2, Ga2OH, and Gb3, CM and CF groups were confounded since no differences were detected between these groups, but for CD different normal values were established for males and females due to higher levels of LacCer concentrations for females compared to males. These normal values were used to calculate the sensitivity and specificity of the different biomarkers for patient cohorts under study. The sensitivity of total Ga2 was higher than the sensitivity of total CD, especially for the UFF group where it increased from 9.3% for total CD to 70.4% for total Ga2. For the 4 Fabry patient groups, the sensitivity of total Gb3 was equal or higher than total Ga2. However, when both biomarkers (total Ga2 and total Gb3) were taken in account, the sensitivity increased for the UFF and the TFF groups compared to total Gb3 because one UFF and two TFF presented abnormal levels of total Ga2, but normal levels of total Gb3. The specificity was similar


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for the 4 biomarkers studied and for the combination of total Ga2 and Gb3 with values ranging from 94.9 to 97.1%.

Table S-8 shows the mean and median values for total LacCer, Ga2, Ga2OH, CD, and Gb3 for different patient groups according to the age of patients (< 18 or ≥ 18 years). Our results show that total LacCer was higher for adult males (≥ 18 years) compared to male children (< 18 years); whereas, the reverse was observed for females. Levels of total Ga2, Ga2OH, and CD were higher for male children compared to adult males. For these biomarkers, no trend was observed for adult females and female children. Total Gb3 biomarker levels were similar for adult males and male children, and were higher for untreated adult females compared to untreated female children. Nevertheless, to establish unequivocally differences based on the age of patients, the pediatric cohort should be mutation-matched with the adult cohort.

Table S-9 summarizes the relative abundance of Fabry disease biomarkers (total Ga2, Ga2OH and Gb3) for the most common mutations (n ≥ 3) in the untreated Fabry disease male and female groups under study. Total Ga2 and Ga2OH were significantly lower (9.5 and 5.2%, respectively) for untreated Fabry males with the mutation A143P compared to the same mutation for untreated Fabry women (38.2 and 25.5%, respectively), as well as for other mutations studied (N215S, R118C, P293T, A348P, R220X) (22.5-34.0% and 7.7-19.7%, respectively). Untreated Fabry males with the A143P mutation had biomarker levels significantly higher compared to other patients in Table S-9. These results suggest that the relative abundance of Fabry disease biomarkers may vary according to mutations and might correlate differently with clinical manifestations of the disease.



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As demonstrated in this study, Ga2 and Gb3 isoforms/analogs in urine are good complementary biomarkers to diagnose Fabry disease. These biomarker analyses are significantly less expensive than mutation analysis and they allow the diagnosis of heterozygous patients which might not be detected by enzyme activity analysis.25 Fabry disease high-risk screening can be performed for patients with cardiac hypertrophy,26 renal insufficiency,27 or ocular manifestations such as corneal opacity and vascular tortuosities of the upper eyelid.28-30

Table S-6: Age, α-GAL A mutation, creatinine concentration, and lactosylceramide (LacCer), galabiosylceramide (Ga2) and globotriaosylceramide (Gb3) concentrations normalized with creatinine for A) Untreated Fabry males; B) Treated Fabry males; C) Healthy control males; D) Untreated Fabry Females; E) Treated Fabry females, and F) Healthy control females. Concentrations under the limit of detection (LOD) were considered as zero and concentrations between LOD and the limit of quantification (LOQ) are indicated in red.


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A) Total Ga2 100

B) Total LacCer

M=32.9 M=1.01 M=11.9 (nd-90.4) (nd-6.08) (nd-51.0) M=16.7 M=10.9 M=0.96 (nd-59.4) (nd-76.3) (nd-3.26)

35

90

M=0.84 M=1.18 M=8.49 (nd-2.61) (nd-7.12) (0.16-36.8) M=1.03 M=12.6 M=9.51 (0.05-4.74) (nd-155) (0.11-37.0) *

30

70

nM/mM creat.

nM/mM creat.

80

60 50 40 **

30

25 (*) (*)

* **

20

*

15 10

*

20 5 10

**

C) Total CD 100

M=33.7 M=2.19 M=20.4 (nd-91.7) (nd-9.30) (1.50-55.8) M=17.8 M=23.5 M=10.5 (0.10-61.8) (nd-155) (0.22-37.0)

1000

80

800

70

700

60 50 40

(*) (*)

* **

30

nM/mM creat.

900

*

M=329 M=1.69 M=4.60 (nd-1406) (nd-5.67) (0.70-16.4) M=113 M=25.4 M=2.04 (0.51-743) (0.83-195) (nd-6.71) *

600 500 400 300 200

20

0

** * *

D) Total Gb3

90

10

**

0

0

nM/mM creat.

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(*) ** ***

100 0

*** **

**

Figure 4: Urinary levels of: A) Total galabiosylceramide (Ga2); B) Total lactosylceramide (LacCer); C) Total ceramide dihexoside (CD, Ga2+LacCer); and D) Total globotriaosylceramide (Gb3) normalized with creatinine (creat.) for the six sample groups. Untreated Fabry males (n = 34); Treated Fabry males (n = 33); Control males (n = 34); Untreated Fabry females (n = 54); 31 ACS Paragon Plus Environment


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Treated Fabry females (n = 19); Control females (n = 25). For each group, green dash line = median; box = higher and lower quartiles; whiskers = higher and lower non outlier values; M = mean; minimum and maximum values in brackets; * = outliers; (*) = extreme outliers.


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Table S-7: Statistical comparison of the studied biomarkers between the 4 Fabry patient sample groups and the healthy control groups, and between control males (CM) (n = 34) and control females (CF) (n = 25). Mann-Whitney U test p-values and the areas under the ROC curves (AUCs) with their asymptotic 95% confidence interval in brackets. UFM = Untreated Fabry males (n = 34); TFM = Treated Fabry males (n = 34); UFF = Untreated Fabry females (n = 54); TFF = Treated Fabry females (n = 19). Statistically significant differences (p-values < 0.05 and AUC > 0.5) written in bold blue. Comparison UFM/CM TFM/CM UFF/CF TFF/CF CM/CF

Total Ga 2 p -value/AUC

Ceramide Dihexoside Separation

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