Papers in Press. Published November 19, 2015 as doi:10.1373/clinchem.2015.245159 The latest version is at http://hwmaint.clinchem.org/cgi/doi/10.1373/clinchem.2015.245159 Clinical Chemistry 62:1 Automation and Analytical Techniques 000–000 (2016) Sulfatide Analysis by Mass Spectrometry for Screening of Metachromatic Leukodystrophy in Dried Blood and Urine Samples Zdenek Spacil,1* Arun Babu Kumar,1 Hsuan-Chieh Liao,1 Christiane Auray-Blais,2 Samantha Stark,3 Teryn R. Suhr,4 C. Ronald Scott,5 Frantisek Turecek,1 and Michael H. Gelb1,6* BACKGROUND: Metachromatic leukodystrophy (MLD) for newborn screening of MLD and sets the stage for a is an autosomal recessive disorder caused by deficiency larger-scale newborn screening pilot study. in arylsulfatase A activity, leading to accumulation of © 2015 American Association for Clinical Chemistry sulfatide substrates. Diagnostic and monitoring proce- dures include demonstration of reduced arylsulfatase Aactivityinperipheralbloodleukocytesordetection Metachromatic leukodystrophy (MLD),7 caused by de- of sulfatides in urine. However, the development of a ficiency of arylsulfatase A (EC 3.1.6.8), is a severe clinical screening test is challenging because of instability of condition categorized into infantile, late infantile, juve- the enzyme in dried blood spots (DBS), the wide- nile, and adult subtypes according to age of onset. New- spread occurrence of pseudodeficiency alleles, and the born screening (NBS) of MLD by direct measurement of lack of available urine samples from newborn screen- arylsulfatase A activity or protein abundance is not likely ing programs. to be feasible because of a severe pseudodeficiency prob- lem (1) and relative instability of the enzyme in dried METHODS: We measured individual sulfatide profiles in blood spots (DBS) (2). Sulfatides, the natural substrates DBS and dried urine spots (DUS) from MLD patients for arylsulfatase A, have been shown to be highly in- with LC-MS/MS to identify markers with the discrimi- creased in urine from MLD patients compared with natory power to differentiate affected individuals from healthy individuals (3–7). However, NBS programs typ- controls. We also developed a method for converting all ically use DBS, and dried urine samples (DUS) are usu- sulfatide molecular species into a single species, allowing ally not available. Recent studies have shown that sulfati- quantification in positive-ion mode upon derivatization. des are increased in DBS from MLD patients (8, 9), but the discriminatory power of sulfatide concentrations in RESULTS: In DBS from MLD patients, we found up to DBS between MLD patients and controls is less pro- 23.2-fold and 5.1-fold differences in total sulfatide con- nounced than in DUS, raising questions about the po- centrations for early- and late-onset MLD, respectively, tential for NBS. The interest in screening for MLD is compared with controls and pseudodeficiencies. Corre- timely owing to new treatment options being investi- sponding DUS revealed up to 164-fold and 78-fold dif- gated in the clinic (10). ferences for early- and late-onset MLD patient samples Sulfatides occur as an ensemble of molecular species compared with controls. The use of sulfatides converted caused by variation in the structure of the fatty acyl chain to a single species simplified the analysis and increased attached to the sphingosine amino group (3). In the detection sensitivity in positive-ion mode, providing a study reported here, we developed and optimized new second option for sulfatide analysis. tandem mass spectrometry (MS/MS) methods to detect sulfatides in DBS and DUS as disease markers of MLD. CONCLUSIONS: This study of sulfatides in DBS and DUS The first method covers the entire panel of sulfatide mo- suggests the feasibility of the mass spectrometry method lecular species with ultra-high performance liquid chro- 1 Department of Chemistry, 5 Department of Pediatrics, and 6 Department of Biochemis- Received June 16, 2015; accepted November 5, 2015. try, University of Washington, Seattle, WA; 2 Clinical Research Center, Centre Hospitalier Previously published online at DOI: 10.1373/clinchem.2015.245159 Universitaire de Sherbrooke, and Universite´de Sherbrooke, Sherbrooke, Quebec, Can- © 2015 American Association for Clinical Chemistry ada; 3 National Referral Laboratory, Genetics and Molecular Pathology, South Australia 7 Nonstandard abbreviations: MLD, metachromatic leukodystrophy; NBS, newborn Pathology, Women’s and Children’s Hospital, Adelaide, Australia; 4 MLD Foundation, screening; DBS, dried blood spots; DUS, dried urine samples; MS/MS, tandem mass West Linn, OR. spectrometry;UHPLC,ultra-highperformanceliquidchromatography;SCDase,sphingo- * Address correspondence to these authors at: Department of Chemistry, Campus Box lipid ceramide N-deacylase; SM, sphingomyelin. 351700, University of Washington, Seattle, WA 98195. Fax 206-685-8665; e-mail [email protected] or [email protected]. 1 Copyright (C) 2015 by The American Association for Clinical Chemistry matography (UHPLC) and MS/MS in negative ion 10-mm DUS punch, except with 50 ␮L water and 500 mode without the need to derivatize the analyte. The ␮L methanol. second method involves the enzymatic conversion of all sulfatide molecular species into a single species (lysosul- ENZYMATIC CONVERSION AND DERIVATIZATION OF fatide), after hydrolysis of fatty acid. Lysosulfatide is then SULFATIDES converted into a derivative that can be readily detected by DBS were incubated in a deep (1-mL) 96-well plate for UHPLC-MS/MS in positive-ion mode. We compared 16 h (37 °C with orbital shaking) in 30 ␮Lof50 these methods by analyzing DBS and DUS from Յ100 mmol/L sodium acetate buffer, pH 6.0, containing control samples and 14 MLD patients with various ages 0.8% taurodeoxycholate and 2 mU sphingolipid cer- of onset of clinical symptoms. amide N-deacylase (SCDase) (cat. no. S2563, Sigma Aldrich). The reaction was quenched with 300 ␮L Materials and Methods methanol, and 200 ␮Lwastransferredtoashallow (350 ␮L) 96-well plate. For direct lysosulfatide analy- All studies with human samples were carried out with sis, samples were transferred and subjected to LC- institutional review board approval at the University MS/MS analysis. For the analysis of lysosulfatide de- of Washington. DUS and DBS from MLD patients rivative, the methanol was evaporated under a stream were collected and provided by the MLD Foundation of nitrogen, and 50 ␮Lof5mmol/Lsuccinylester (http://www.mldfoundation.org/). We obtained DBS reagent (see Supplemental Material, which accompa- from MLD pseudodeficient patients from the Na- nies the online version of this article at http://www. tional Referral Laboratory, Genetics and Molecular clinchem.org/content/vol62/issue1) in ethanol was Pathology, South Australia Pathology, Women’s and added to each well. The plate was sealed with a silicone Children’s Hospital, Adelaide, Australia. Anonymized mat and incubated for2hat37°Cwith orbital shak- control DUS and DBS from healthy newborns were ing. Methanol (150 ␮L) was added to each well, and obtained from the Centre Hospitalier Universitaire de the sample was subjected to LC-MS/MS analysis. Sherbrooke before being discarded. DBS from MLD patients were collected by puncturing the fingertip UHPLC-MS/MS ANALYSIS with a lancet and letting the blood drip onto Whatman For UHPLC separation, we used an Acquity system 903 filter paper, which was air-dried for approximately equipped with an analytical column, HSS T3 C18 (50 ϫ 2handthenmailedatambienttemperatureoverafew 2.1 mm, 1.8 ␮m), and a guard column, VanGuard HSS days to the University of Washington. DUS from T3 (5 ϫ 2.1 mm, 1.8 ␮m), all from Waters Corp. The MLD patients were collected on Whatmann-GE 903 UHPLC solvents were water/acetonitrile (50:50) with paper disks (70 mm), which were allowed to air-dry for 0.1% formic acid (solvent A) and 2-propanol/acetonitrile approximately2hatroom temperature, then placed in (80:20) with 0.1% formic acid (solvent B). The elution azip-lockplasticbagforshipmentatambienttemper- solvents were prepared from LC-MS Optima grade ace- ature. After receipt, DBS and DUS were stored at tonitrile Ն99.9% (cat. no. 34967, Fisher), water, Ϫ20 °C in a closed jar with desiccant. DBS were man- 2-propanol, methanol Ն99.9% (cat. no. 34860), and ually punched with a 3-mm-diameter perforator, and a formic acid approximately 98% (cat. no. 06440). The single 3-mm punch was used for analysis in microtiter gradient elution program started with 50% solvent B, plate format. DUS were punched with a 3- or 10-mm- 0–1 min to 95% solvent B (linear), 1–1.5 min to 100% diameter perforator, and a single 10-mm punch or two solvent B (linear), 1.5–2 min to 100% solvent B (hold), 3-mm punches were used for sulfatide analysis. C17:0, and 2–2.5 min back to 50% solvent B for reequilibration. C24:0, C16:0-day5,andC18:0-day5 sulfatides were Total injection-to-injection time was 2.5 min, and the obtained as described (8). injection volume was 10 ␮L. A triple-quadrupole tandem mass spectrometer (Xevo TQ, Waters Corp.) was used in SULFATIDE EXTRACTION AND SAMPLE PROCESSING negative- and positive-ion detection modes. Details for To extract sulfatides from DBS and DUS, 30 ␮L water MS/MS methods are provided in online Supplementary was added to a 3-mm punch in a deep (1-mL) 96-well Tables 5 and 6. An external calibration curve was ac- plate and incubated for 2 h (37 °C with orbital shaking). quired for each sample batch with C17:0 sulfatide stan- We added 300 ␮L methanol, pipetted the mixture up dard (Avanti Polar Lipids). The use of internal standard- and down approximately 10 times, and centrifuged the ization with C17:0 was not suitable because of the plate for 5 min at 3000 rpm at room temperature. A isobaric molecular mass with C16:1-OH sulfatide and portion of the supernatant (200 ␮L) was transferred to a coelution of isobaric sulfatides at UHPLC gradient con- shallow (350-␮L) 96-well plate and directly used for LC- ditions.