Extraction completeness and signal suppression in DBS and DUS sample matrix

Transcription

1 Extraction of sulfatides We have compared previously published extraction protocols (1,2)and also proposed new protocols to improve extraction yields of sulfatides. The detailed procedure for each protocol is given in Supplementary Table 1. The extraction efficiencies were tested for each protocol using 1.5 pmol of C17:0 sulfatide from a stock solution and C18:0 and C24:0 sulfatides from bovine brain sulfatide mix. The protocols using water and methanol or isopropanol showed the best extraction efficiencies of >80% from filter paper blank and >68% from DBS (Supplementary Table 2). Therefore protocol P1 (water and methanol) with extraction yields of % sulfatides in filter paper blank and 68-79% in DBS was further used for the MLD patient study. Extraction completeness and signal suppression in DBS and DUS sample matrix We have tested whether sulfatides are completely extracted from DBS and DUS sample matrix by applying the extraction protocol twice on the same samples. We applied 5 pmol of C17:0 sulfatide or 7 pmol of bovine brain sulfatide mix in methanol on blank filter paper, DBS or DUS sample and dried the sample under a stream of nitrogen for 1 hour. Then we have extracted samples according to protocol P1 (Supplementary Table 2), removed residual solvent and applied the same protocol again. Recoveries calculated from the response of tested sulfatides in neat non-extracted solution were between % and % in DBS and DUS respectively for all sulfatide species tested during the first extraction. The second extraction yields were ranging between 1-8% and 3-7% in DBS and DUS respectively, which illustrates nearly complete yield of all extractable sulfatides during the first extraction (Supplementary Table 3). Next we have evaluated signal suppression caused by DBS and DUS matrix by comparing the response measured in spiked DBS and DUS samples with the response measured in spiked blank filter paper. The results in Supplementary Table 3 show an average 25% signal suppression across tested sulfatide species by the DBS matrix and no significant signal suppression by the DUS matrix. Normalization of sultatides in DUS to the concentration of creatinine and sphingomyelin Sulfatides were extracted from a 10 mm DUS according to the protocol described in the main text, but the Millipore water used for pre-extraction was internally standardized with N-palmitoyl-d31-D-erythrosphingosylphosphorylcholine (d31-16:0-sm; Avanti Polar Lipids Inc., cat. # ) and (methyld3)-creatinine (Sigma Aldrich, Cat. # ) at concentrations of 0.5 µm and 95 µm, respectively. Fifty µl of the water with 25 pmol of 16:0-d31 SM and 4.75 nmol of (methyl-d3)-creatinine was added to a DUS punch, incubated at 37 C for 30 min, then 500 µl of methanol was added, and 200 µl was transferred to a new well plate for MS/MS analysis. The analysis was performed in triplicate (duplicate in cases where the material was insufficient) to calculate the CV%. We measured responses of 16:0-SM; d31-16:0-sm; creatinine and (methyl-d3)-creatinine to use them as normalization parameters. Suppl. Table 11 shows raw sulfatide responses as well as the response normalized to i) creatinine, ii) SM, iii) creatinine/(methyl-d3)-creatinine ratio and iv) 16:0-SM/d31-16:0-SM ratio. Suppl. Table 12 presents the effect of different normalization parameters to the discriminatory power between the control and MLD patient groups.

2 Synthesis of the reagent for lysosulfatide derivatization 5-(bis(3-(dimethylamino)propyl)amino)-5-oxopentanoic acid (1) A solution of 3,3ʹ-Iminobis(N,Ndimethylpropylamine) (500 mg, 2.66 mmol) in DMF (5 ml) was cooled on an ice bath. To the cooled solution glutaric anhydride (760 mg, 6.66 mmol) was added in small portions and allowed to warm to room temperature and let to stir for 24 hours. If the reaction does not go to completion as indicated by mass spectrometric (ESI) analysis, another portion of glutaric anhydride (760 mg, 6.66 mmol) was added in small portions and allowed to stir for 24 hours. The reaction mixture thus obtained was purified by ion-exchange using Amberlite CG-50. Amberlite CG-50 was soaked in 1N HCl for 1 hour and loaded on to a column and the resin bed was washed with DI water for three times. The reaction mixture was diluted with 15 ml of DI water and loaded on the Amberlite CG-50 column. After pushing the reaction mixture through, the column was washed with DI water (6 times). The desired compound was eluted from the column by using dilute aqueous ammonium hydroxide solution (with 3% ammonia content). The fractions with the desired compound were combined and concentrated under reduced pressure to one third of volume. The remaining water was removed by lyophilization to get the title compound 1 (603 mg, 75%). 1 H NMR (300 MHz, MeOD) δ 3.42 (dd, J = 15.2, 8.2 Hz, 4H), 2.75 (t, J = 7.4 Hz, 2H), (m, 2H), 2.61 (s, 6H), 2.53 (s, 6H), (m, 2H), 2.24 (t, J = 6.8 Hz, 2H), (m, 6H). (ESI) m/z: [M + H] + calcd for C 15 H 32 N 3 O , found ,5-dioxopyrrolidin-1-yl 5-(bis(3-(dimethylamino)propyl)amino)-5-oxopentanoate (2) To the solution of 1 (300 mg, mmol) in DMF (2.5 ml), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (332 mg, 1.73 mmol), potassium phosphate monobasic (0.8 g) and N-hydroxysuccinimide (200 mg, 1.74 mmol) were added and left stirring at room temperature for 16 hours. The reaction mixture was extracted between a solution of 10% methanol in chloroform and 10% brine in water. The organic layer was dried with anhydrous sodium sulfate and concentrated under reduced pressure to yield 2 (61 mg, 15%). MS (ESI) m/z: [M + H] + calcd for C 19 H 35 N 4 O , found Linearity and Precision of Analytical Methods. The titration series of C-17:0, C-24:0 and isotopically labeled C-16:0-d 5 and C-18:0-d 5 sulfatide standards was prepared methanol at concentration levels 0.1; 0.5; 1.0; 5.0; 10.0 and 50.0 nm and 300 ul of mixed standard solution (C17:0, C-24:0, C16:0-d 5 and

3 C18:0-d 5 ) was added to 30 ul of water (neat-solution calibration curve) or 3 0uL of water solubilized DBS (matrix-matched calibration curve). LC-MS/MS response (SRM) was measured in five repetitions and linearity was evaluated using linear regression analysis. Standard deviations at each concentration level as well as the slope, intercept, r 2, standard deviation of residuals, and the standard deviations of the slope and intercept are given in Supplementary table 4. Inter-day precision for two consecutive days (n=10) at concentration levels 0.5; 5.0 and 50.0 nm was determined for each sulfatide and results are shown in Supplementary table 4. References: 1. Barcenas M, Suhr TR, Scott CR, Turecek F, Gelb MH. Quantification of sulfatides in dried blood and urine spots from metachromatic leukodystrophy patients by liquid chromatography/electrospray tandem mass spectrometry. Clin Chim Acta 2013; 433, Han M, Jun S-H, Song SH, Park H-D, Park KU, Song J. Ultra-performance liquid chromatography/tandem mass spectrometry for determination of sulfatides in dried blood spots from patients with metachromatic leukodystrophy. Rapid Commun Mass Spectrom. 2014;28, Baum H, Dodgson KS, Spencer B. The assay of arylsulfatase A and B in human urine. Clin Chim Acta 1959;4, Whitfield PD, Sharp PC, Johnson DW, Nelson P, Meikle PJ. Characterization of urinary sulfatides in metachromatic leukodystrophy using electrospray ionization-tandem mass spectrometry. Clin. Chim. Acta 2001;73,

4 Supplementary Table 1. Detailed procedure of five extraction protocols compared for the extraction efficiency. Supplementary Table 2. Extraction recoveries [%] and CV values (n=5) for C17:0; C18:0 and C24:0 sulfatide species using different extraction protocols. The extraction recovery was calculated based on a response of not extracted standard solution. Supplementary Table 3. The recovery [%] of the first and second extraction step, and the SRM response of sulfatide standard measured in filter paper, DBS sample matrix and DUS sample matrix with the signal suppression expressed in percent. The negative value indicates suppression and positive value indicates increase of SRM signal. The experiment was performed in triplicate. Supplemental Table 4. Standard curves for the LC-MS/MS response to standard amounts of sulfatide standards. Supplementary Table 5. Settings of mass spectrometer source and analyzer for positive and negative ion detection mode. Supplementary Table 6. Transition list for SRM analysis of sulfatides in positive and negative ion detection mode. Supplementary Table 7. List of analyzed MLD patient samples indicating the age of onset. Supplementary Table 8. List of analyzed MLD pseudodeficiency samples. Supplementary Table 9. Concentrations in the blood (µg/ml) of the panel of 19 sulfatides determined in DBS from MLD patients (n=14), MLD pseudodeficiencies (n=12), and normal controls (n=50). The experimental limit of quantification for DBS was determined approx. at 3.1 ng/ml of whole blood. The concentrations of all sulfatide species was obtained using external callibration with C17:0-sulfatide and assuming an average MW of 870 Da for the species. Analysis was done using two 3 mm DBS punches per sample Supplementary Table 10. Concentrations in the blood (µg/ml) of 5 DBS from newborns who went on to receive a diagnosis of MLD. These were obtained as stored DBS from the CA state newborn screening program. The concentrations of all sulfatide species was obtained using external callibration with C17:0- sulfatide and assuming an average MW of 870 Da for the species. Analysis was done using two 3 mm DBS punches per sample Supplementary Table 11. Concentrations in the urine (µg/ml) of the panel of 19 sulfatides determined in DUS from MLD patients (n=14) and normal controls (n=104). The experimental limit of quantification for DBS was determined approx. at 1.5 ng/ml of urine. The concentrations of all sulfatide species was obtained using external callibration with C17:0-sulfatide and assuming an average MW of 870 Da for the species. Analysis was done using two 3 mm DUS punches per sample

5 Supplementary Table 12. Effects of various normalization parameters on CV% for replicate DUS analysis. Comparison shows total sulfatide raw response and the response normalized to i) creatinine, ii) SM, iii) creatinine/(methyl-d3) creatinine ratio and iv) 16:0 SM/d31 16:0 SM ratio. Supplementary Table 13. Effects of various normalization parameters on discriminatory power between controls and MLD patients for raw response of total sulfatides and the response normalized to i) creatinine, ii) SM, iii) creatinine/(methyl-d3) creatinine ratio and iv) 16:0 SM/d31 16:0 SM ratio. Supplementary Table 14. Optimization of SCDase enzyme on a control DBS sample spiked with bovine brain sulfatide mix, the amount of lysosulfatide [pmol] generated through enzymatic hydrolysis does not differ significantly when 2, 4 and 6 mu of SDCase are used. Supplementary Table 15. SDCase hydrolytic conversion yields measured in DBS from MLD patients for predominant sulfatides. Supplementary Table 16. Relative abundances in positive and negative ion detection mode, detecting lysosulfatide-derivative, underivatized lysosulfatide and total sulfatides. The data show comparable discriminatory power of all four assays. However determination of total sulfatides in the negative ion mode and lysosulfatide-derivative in the positive ion mode are the preferred due to higher sensitivity. Supplementary Figure 1 Typical SRM chromatograms depicting ion traces of 19 monitored sulfatides in DBS sample from (a) MLD patient, (b) healthy control and DUS sample from (c) MLD patient, (d) heathy control. Supplementary Figure 2 The total sulfatide concentrations in urine (µg/ml) in DUS from MLD patients (n=14) and controls (n=104) to demonstrate clear differentiation of both groups. The patients with less severe form of MLD (juvenile/adult) based on onset of clinical syndromes are the green data series. Supplementary Figure 3 Box plots visualizing data from Supplematary Table 15 and graphically illustrating the discriminatory power of assays based on determination of i) total sulfatides or ii) lysosulfatide in negative and iii) lysosulfatide or iv) lysosulfatide derivative in positive MS/MS. Although all assays show comparable performance, increased variance is evident in case of ii) and iii) due to low signal intensity.

6 Supplementary Table 1 # Protocol Ref. P1 add 30 µl H 2 O (2 h, 37 C) -> add 300 µl MeOH -> spin down (3000 rpm, 5 min, RT) and transfer 200 µl of supernatant P2 add 30 µl H 2 O (2 h, 37 C) -> add 300 µl IPA -> spin down (3000 rpm, 5 min, RT) and transfer 200 µl of supernatant-> dry under stream of N 2 gas -> re-dissolve in 200 µl MeOH P3 add 30 µl 0.8% Triton-X-100 buffer (2 h, 37 C) -> add 300 µl IPA -> spin down (3000 rpm, RT) and transfer 200 µl -> dry under stream of N 2 gas-> re-dissolve in 200 µl MeOH P4 add 50 µl 0.5 M KH 2 PO 4 buffer; 100 µl MeOH and 300 µl CHCl 3 (vortex and sonicate for 10 (2) min, centrifuge at 15,000 g for 2 min) -> transfer 250 µl of the bottom organic layer and dry under stream of N 2 gas-> re-dissolve in 200 µl of MeOH P5 add 30 µl H 2 O (2 h, 37 C) -> add 300 µl EthylAcetate -> spin down (3000 rpm, 5 min, RT) and transfer 250 µl of the upper organic layer-> dry under stream of N 2 gas -> re-dissolve in 200 µl MeOH (1) Supplementary Table 2 C-17:0 C-18:0 C-24:0 FP DBS FP DBS FP DBS R[%] CV[%] R[%] CV[%] R[%] CV[%] R[%] CV[%] R[%] CV[%] R[%] CV[%] P P P P P

7 Supplementary Table 3 Recovery (%) C-16 d5 C17-S C-18-0 d5 C-18-0-OH C-20-0 C-22-0 C-24-1 DBS 1st extraction DBS 2nd extraction DUS 1st extraction DUS 2nd extraction Response (peak area) C-16 d5 C17-S C-18-0 d5 C-18-0-OH C-20-0 C-22-0 C-24-1 Blank filter paper DBS matrix Suppression (%) DUS matrix Suppression (%) The matrix effects were measured by adding known amounts of standard sulfatides to a DBS or DUS punch from a stock solution in methanol using a Hamilton 10 ul syringe, after air drying, the punch was extracted and subjected to LC-MS/MS analysis.

8 Supplemental Table 4 The indicated concentrations of sulfatide standards were subjected to LC-MS/MS analysis in the absesnce (top) and presence of DBS extract (matrix effect). Each sample was analyzed by 5 injections into the UHPLC-MS/MS, and the standard deviation is given. Linearity data is given at the bottom of each table.

9 Supplementary Table 5 Parameter (units) Polarity ES- ES+ Capillary voltage (V) Extractor (V) Source temperature ( C) Desolvation temperature ( C) Cone Gas Flow (L/h) Desolvation Gas Flow (L/h) LM 1 Resolution HM 1 Resolution Ion Energy Collision Cell Entrance Potential (V) Collision Cell Exit Potential (V) LM 2 Resolution HM 2 Resolution Ion Energy Multiplier (V) Collision Gas Argon Argon

10 Supplementary Table 6 Name Formula SRM (-) CV CE SRM (+) CV CE precursor product (V) (ev) precursor product (V) (ev) LysoS C24H47NO10S LysoS-der. C39H76N4O12S C-16-0 C40H77NO11S C-16-1-OH C40H75NO12S C-16-0-OH C40H77NO12S C-18-0 C42H81NO11S C-18-0-OH C42H81NO12S C-20-0 C44H85NO11S C-20-0-OH C44H85NO12S C-22-0 C46H89NO11S C-22-1-OH C46H87NO12S C-22-0-OH C46H89NO12S C-24-1 C48H91NO11S C-24-0 C48H93NO11S C-23-0-OH C47H91NO12S C-24-1-OH C48H91NO12S C-24-0-OH C48H93NO12S C-26-1 C50H95NO11S C-26-0 C50H97NO11S C-26-1-OH C50H95NO12S C-26-0-OH C50H97NO12S

11 Supplementary Table 7 Patient MLD Form Onset Age Age at Age at DBS Sex Diagnosis Collection MLD 1 Juvenile 8 yrs 14 yrs 32 yrs F MLD 2 Late Infantile 19 mos 29 mos 13 yrs F MLD 3 Late Infantile 16 mos 22 mos 10 yrs F MLD 4 Adult 24 yrs 22 yrs 36 yrs M MLD 5 Late Infantile 17 mos 26 mos 6.5 yrs M MLD 6 Late Infantile 12 mos 23 mos 4.5 yrs F MLD 7 Late Infantile 18 mos 35 mos 8.5 yrs M MLD 8 Late Infantile 12 mos 27 mos 4.5 yrs F MLD 9 Juvenile 8 yrs 10 yrs 23 yrs M MLD 10 Late Infantile 14 mos 26 mos 3 yrs F MLD 11 Late Infantile 2 yrs 26 mos 2 yrs F MLD 12 Late Infantile 13 mos 27 mos 9 yrs F MLD 13 Late Infantile 18 mos 27 mos 2.7 yrs F MLD 14 Late Infantile 26 mos 31 mos 4.7 yrs M MLD 15 Adult no data no data no data M Newborn 1 Late Infantile 20 mos newborn F Newborn 2 Late Infantile 22 mos newborn M Newborn 3 Late Infantile 12 mos newborn F Newborn 4 Juvenile 7 yrs newborn F Newborn 5 Juvenile 8 yrs newborn F MLD patients 1-14 were identified via the MLD Foundation. The patient's onset age and age at diagnosis is the age when symptoms first appeared and diagnosis of MLD was made, respectively, as stated by the parents. Age at DBS collection is the age when the DBS was obtained for this study. Newborns 1-5 are DBS stored in the CA state newborn screening lab. These individuals were diagnosed with MLD after birth. Late-infantile MLD is defined as symptoms occuring between the ages of months. Some literaure uses months, and older literature uses 12 months to 5 years. The problem is that is is really a continuum and the age breaks have been somewhat arbitrary. See The patients had confirmed diagnosis of either late-infantile, juvenile or adult MLD based on age at onset of symptoms and in some cases mutations as well. There are known common late-infantile and adult mutations. Patients received their diagnosis from the Lysosomal Lab run by Dr. David Wenger at Thomas Jefferson University in Philadelphia.

12 Supplementary Table 8 Patient ARSA Activity (nmol/min/mg protein) Genotype Age at DBS collection PD yrs PD yrs PD c.465+1g>a/c.1178c>g 13 yrs PD yrs PD yrs PD yrs PD yrs PD yrs PD yrs PD yrs PD yrs PD c.511g>a,c.1055a>g/c.511g>a,c.1055a>g 4 yrs Pseudodeficiency patients PD 1-12 were identifed in the laboratory of Dr. Samantha Stark, National Referral Laboratory, Genetics and Molelcular Pathology, South Australia Pathology, Women's and Children's Hospital, Adelaide, Australia. Blood from these patients displayed arylsulfatase A activity below 1 nmole/min/mg protein when plasma was submittted to the published enzyme assay (3). These patients had normal urinary sulfatides when analyzed as described (4) and lack the symptoms of MLD. For two patients, pseudodeficiency was confirmed by genotyping as indicated.

13 Group No. C-16-0 C-16-1-OH C-16-0-OH C-18-0 C-18-0-OH C-20-0 C-20-0-OH C-22-0 C-22-1-OH C-22-0-OH C-23-0-OH C-24-1 C-24-0 C-24-1-OH C-24-0-OH C-26-1 C-26-0 C26-1-OH MLD-inf MLD <LOQ <LOQ <LOQ <LOQ (n=11) MLD <LOQ <LOQ <LOQ MLD <LOQ <LOQ <LOQ <LOQ MLD <LOQ <LOQ <LOQ <LOQ <LOQ MLD <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ MLD <LOQ <LOQ <LOQ <LOQ <LOQ MLD <LOQ <LOQ <LOQ 0.011, <LOQ <LOQ <LOQ MLD <LOQ <LOQ <LOQ <LOQ MLD <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ MLD <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ MLD <LOQ <LOQ <LOQ <LOQ Mean n/a n/a n/a Min n/a n/a n/a Max n/a n/a n/a SD n/a n/a n/a Ratio n/a n/a n/a MLD-juv MLD <LOQ <LOQ <LOQ <LOQ (n=3) MLD <LOQ <LOQ <LOQ <LOQ 0.01 <LOQ <LOQ <LOQ <LOQ <LOQ MLD <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ Mean n/a n/a n/a n/a Min n/a n/a n/a n/a Max n/a n/a n/a n/a SD n/a n/a n/a n/a Ratio n/a n/a n/a n/a MLD-PD PD <LOQ <LOQ <LOQ (n=12) PD <LOQ <LOQ <LOQ PD <LOQ <LOQ <LOQ PD <LOQ <LOQ <LOQ PD <LOQ <LOQ <LOQ PD <LOQ <LOQ <LOQ PD <LOQ <LOQ <LOQ PD <LOQ <LOQ <LOQ PD <LOQ <LOQ <LOQ PD <LOQ <LOQ <LOQ PD <LOQ <LOQ <LOQ PD <LOQ <LOQ <LOQ Mean n/a n/a n/a Min n/a n/a n/a Max n/a n/a n/a SD n/a n/a n/a Ratio n/a n/a n/a Control Mean n/a n/a n/a (n=50) Min n/a n/a n/a Max n/a n/a n/a SD n/a n/a n/a

14 Supplemental Table 10 Group No. C16-1-OH C-16-0 C-16-0-OH C-18-1 C-18-0 C-18-0-OH C-20-0 C-20-0-OH C-22-0 C-22-1-OH C-22-0-OH C-24-1 C-24-0 C-23-0-OH C-24-1-OH Total Ratio MLD-late infantile MLD-NB (n=3) MLD-NB MLD-NB MLD-juvenile MLD-NB (n=2) MLD-NB

15 Sup$ Supplemental$Table$11$ Supplemental*Table*10 * Sulfatide concentration in urine (µg/ml) Group No. C-16-0 C-16-1-OH C-16-0-OH C-18-0 C-18-0-OH C-20-0 C-20-0-OH C-22-0 C-22-1-OH C-22-0-OH C-23-0-OH C-24-1 C-24-0 C-24-1-OH C-24-0-OH C-26-1 C-26-0 C-26-1-OH C-26-0-OH Total MLD-Inf MLD # <LOQ <LOQ <LOQ (n=11) MLD # MLD # <LOQ <LOQ <LOQ MLD # <LOQ MLD # <LOQ <LOQ <LOQ <LOQ MLD # MLD # <LOQ <LOQ <LOQ <LOQ MLD # MLD # MLD # MLD # <LOQ Mean Min 0.01 <LOQ <LOQ <LOQ <LOQ <LOQ Max SD Ratio 66.7 n/a n/a n/a n/a 84.3 MLD-Juv MLD # <LOQ <LOQ <LOQ (n=3) MLD # <LOQ <LOQ MLD # <LOQ Mean n/a Min n/a Max n/a SD 0.08 n/a Ratio 17.3 n/a n/a n/a n/a 28.2 Control Mean n/a n/a n/a n/a (n=108) Min n/a n/a n/a n/a 0 Max n/a n/a n/a n/a SD n/a n/a n/a n/a 0.037

16 Supplementary Table 12 Total Sulfatides Creatinine_Norm SM C16:0_Norm Creatinine/d3IS_Norm SM/d31IS_Norm Response CV NormR CV NormR CV NormR CV NormR CV (n=3) % (n=3) % (n=3) % (n=3) % (n=3) % MLD#01-Juv MLD#02-Inf MLD#03-Inf MLD#04-Adult MLD#05-Inf MLD#06-Inf MLD#07-Inf MLD#08-Inf MLD#09-Juv MLD#10-Inf MLD#11-Inf MLD#12-Inf MLD#13-Inf MLD#14-Inf Ctrl2008# Ctrl2008# Ctrl2008# Ctrl2008# Ctrl2008# Ctrl2008# Ctrl2008# Ctrl2008# Ctrl2008# Ctrl2008# Ø CV%

17 CV% min CV% max Supplementary Table 13 Summary: Response Creat_Norm SM_Norm Creat/d3IS_Norm SM/d31IS_Norm Ø MLD-Inf Ø MLD-Juv Ø Control Ø MLD-Inf:Ctrl Ø MLD-Juv:Ctrl MLD-Inf min MLD-Juv min CTRL max MLD- Inf/CTRLmax MLD- Juv/CTRLmax

18 Supplementary Table 14 SDCase [activity] LysoS [pmol] 2 mu ± mu 9.72 ± mu 9.61 ± 1.60 Supplementary Table 15 MLD MLD MLD MLD MLD MLD MLD MLD MLD MLD MLD MLD MLD MLD #1 #2 #3 #4 #5 #6 #7 #8 #9 #10 #11 #12 #13 #14 C-16:1-OH [%] C-16:0-OH [%] C-24:1 [%]

19 Supplementary Table 16 Relative Abundance (+) Relative Abundance (-) LysoS-Der LysoS LysoS Total Sulfatides MLD Mean (Early) Min n=11 Max :Norm MLD Mean (Late) Min n=3 Max :Norm Controls Mean n=20 Min Max Figure S1 following pages:

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