Quantification of sulfatides and lysosulfatides in tissues and body fluids by liquid chromatography-tandem mass spectrometry

Sulfatides are found in brain as components of myelin, oligodendrocytes, and neurons but are also present in various visceral tissues. Metachromatic leukodystrophy (MLD) is an inherited lysosomal storage disorder caused by a deficiency of arylsulfatase A, leading to severe white matter disease due to the accumulation of sulfatides and lysosulfatides. To study the physiological role of sulfatides, accessible and sensitive quantitative methods are required. We developed a sensitive LC/MS/MS method to quantify total sulfatide and lysosulfatide content as well as individual molecular species in urine and plasma from MLD patients and plasma and tissues from an MLD mouse model. Our results demonstrate that the method can quantify a wide range of sulfatide concentrations and can be used to quantify total sulfatide content and levels of individual molecular species of sulfatides in tissues, cells, and body fluids. Even though plasma sulfatides and lysosulfatides would seem attractive candidate biomarkers that could possibly correlate with the severity of MLD and be of use to monitor the effects of therapeutic intervention, our results indicate that it is unlikely that the determination of these storage products in plasma will be useful in this respect. Sulfatides are found in brain as components of myelin, oligodendrocytes, and neurons but are also present in various visceral tissues. Metachromatic leukodystrophy (MLD) is an inherited lysosomal storage disorder caused by a deficiency of arylsulfatase A, leading to severe white matter disease due to the accumulation of sulfatides and lysosulfatides. To study the physiological role of sulfatides, accessible and sensitive quantitative methods are required. We developed a sensitive LC/MS/MS method to quantify total sulfatide and lysosulfatide content as well as individual molecular species in urine and plasma from MLD patients and plasma and tissues from an MLD mouse model. Our results demonstrate that the method can quantify a wide range of sulfatide concentrations and can be used to quantify total sulfatide content and levels of individual molecular species of sulfatides in tissues, cells, and body fluids. Even though plasma sulfatides and lysosulfatides would seem attractive candidate biomarkers that could possibly correlate with the severity of MLD and be of use to monitor the effects of therapeutic intervention, our results indicate that it is unlikely that the determination of these storage products in plasma will be useful in this respect. Sulfatides are anionic sulfoglycolipids mainly found in the outer leaflet of the plasma membranes of cells (1Eckhardt M. The role and metabolism of sulfatide in the nervous system.Mol. Neurobiol. 2008; 37: 93-103Crossref PubMed Scopus (137) Google Scholar, 2Takahashi T. Suzuki T. Role of sulfatide in normal and pathological cells and tissues.J. Lipid Res. 2012; 53: 1437-1450Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Sulfatides are particularly abundant in the brain, where they constitute an important component of myelin. Oligodendrocytes are responsible for the synthesis of sulfatides and the maintenance of sulfatide levels in myelin (1Eckhardt M. The role and metabolism of sulfatide in the nervous system.Mol. Neurobiol. 2008; 37: 93-103Crossref PubMed Scopus (137) Google Scholar). However, recent studies in mice indicate that their presence is not restricted to oligodendrocytes and Schwann cells; they are also present in neurons and astrocytes (3Wittke D. Hartmann D. Gieselmann V. Lullmann-Rauch R. Lysosomal sulfatide storage in the brain of arylsulfatase A-deficient mice: cellular alterations and topographic distribution.Acta Neuropathol. 2004; 108: 261-271Crossref PubMed Scopus (53) Google Scholar). Sulfatides are also components of extraneural tissues, and high concentrations are found in selected organs, including kidney, colon, pancreas, and gall bladder. Increased concentrations of sulfatides have been reported in renal cell carcinoma, adenocarcinoma of colon and lung, and ovarian cancer (2Takahashi T. Suzuki T. Role of sulfatide in normal and pathological cells and tissues.J. Lipid Res. 2012; 53: 1437-1450Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 4Liu Y. Chen Y. Momin A. Shaner R. Wang E. Bowen N.J. Matyunina L.V. Walker L.D. McDonald J.F. Sullards M.C. et al.Elevation of sulfatides in ovarian cancer: an integrated transcriptomic and lipidomic analysis including tissue-imaging mass spectrometry.Mol. Cancer. 2010; 9: 186Crossref PubMed Scopus (103) Google Scholar). Sulfatides have been proposed to play a role as ion barriers to osmotic stress and as counterions of ammonium in the renal adaptation to chronic metabolic acidosis (5Niimura Y. Nagai K. Metabolic responses of sulfatide and related glycolipids in Madin-Darby canine kidney (MDCK) cells under osmotic stresses.Comp. Biochem. Physiol. 2008; 149 (B Biochem. Mol. Biol.): 161-167Crossref Scopus (12) Google Scholar, 6StettnerP.BourgeoisS.MarschingC.Traykova-BrauchM.PorubskyS.NordstromV.HopfC.KostersR.SandhoffR.WiegandtH.. Sulfatides are required for renal adaptation to chronic metabolic acidosis. Proc. Natl. Acad. Sci. U S A., 110: 9998–10003.Google Scholar) Sulfatides are synthesized from their precursor galactosylceramide in the Golgi apparatus by 3-O-sulfation of the galactose residue by the enzyme 3′-phosphoadenosine-5′-phosphosulfate: cerebroside sulfotransferase (CST) (EC2.8.2.11). Galactosylceramide is synthesized in the endoplasmic reticulum from ceramides and UDP-galactose by the enzyme UDP-galactose:ceramide galactosyltransferase (CGT) (EC 2.4.1.45) and is then transported to the Golgi apparatus prior to sulfation to sulfatides. Sulfatides consist of many molecular species, with structures differing in acyl chain length and hydroxylation and sphingoid base. These sulfatide molecular species are cell and tissue specific, which may be explained by the cell- and tissue-specific expression of ceramide synthases and fatty acid 2-hydroxylase (1Eckhardt M. The role and metabolism of sulfatide in the nervous system.Mol. Neurobiol. 2008; 37: 93-103Crossref PubMed Scopus (137) Google Scholar, 7Ishizuka I. Chemistry and functional distribution of sulfoglycolipids.Prog. Lipid Res. 1997; 36: 245-319Crossref PubMed Scopus (222) Google Scholar). It is difficult to assign specific functions to the different molecular species of sulfatides, but sulfatides with C16:0 fatty acids were reported to be involved in the regulation of insulin secretion in rat pancreatic β-cells (8Buschard K. Blomqvist M. Mansson J.E. Fredman P. Juhl K. Gromada J. C16:0 sulfatide inhibits insulin secretion in rat beta-cells by reducing the sensitivity of KATP channels to ATP inhibition.Diabetes. 2006; 55: 2826-2834Crossref PubMed Scopus (28) Google Scholar), and elevated levels of C18:0 sulfatides have been implicated as a cause of audiogenic seizers in mice overexpressing CGT (9van Zyl R. Gieselmann V. Eckhardt M. Elevated sulfatide levels in neurons cause lethal audiogenic seizures in mice.J. Neurochem. 2010; 112: 282-295Crossref PubMed Scopus (20) Google Scholar). There is also heterogeneity of the sphingoid base composition of sulfatides. Most of the sulfatides contain 4-sphingenine (sphingosine, d18:1), but the presence of sphingadienine (d18:2) has been reported in mammalian brain (10Colsch B. Afonso C. Popa I. Portoukalian J. Fournier F. Tabet J.C. Baumann N. Characterization of the ceramide moieties of sphingoglycolipids from mouse brain by ESI-MS/MS: identification of ceramides containing sphingadienine.J. Lipid Res. 2004; 45: 281-286Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Degradation of sulfatides takes place in the lysosomes and is catalyzed by the lysosomal enzyme arylsulfatase A (ASA) (EC 3.1.6.8). An accessory protein saposin B, a so-called “sphingolipid activator protein,” is required for the lysosomal degradation of sulfatides by ASA. A deficiency of ASA or of saposin B leads to intralysosomal storage of sulfatides and is the cause of the lysosomal storage disease metachromatic leukodystrophy (MLD) (11Von FiguraK. G. V.JaekenJ.2001. Metachromatic leukodystrophy. In The metabolic and molecular basis of inherited disease. C. R. Scriver, A. L. Beaudet, D. Valle, and W. S. Sly, editors. McGraw Hill, New York. 3695–3724.Google Scholar). Lysosulfatide, the deacylated form of sulfatides, also accumulates in tissues of patients with MLD (12Toda K. Kobayashi T. Goto I. Kurokawa T. Ogomori K. Accumulation of lysosulfatide (sulfogalactosylsphingosine) in tissues of a boy with metachromatic leukodystrophy.Biochem. Biophys. Res. Commun. 1989; 159: 605-611Crossref PubMed Scopus (24) Google Scholar). The accumulation of sulfatides in kidney leads to an increased excretion of sulfatides in urine. Determination of sulfatides in urine is a convenient diagnostic tool to confirm MLD in patients with arylsulfatase A deficiency or saposin B deficiency (13Cui Y. Colsch B. Afonso C. Baumann N. Tabet J.C. Mallet J.M. Zhang Y. Synthetic sulfogalactosylceramide (sulfatide) and its use for the mass spectrometric quantitative urinary determination in metachromatic leukodystrophies.Glycoconj. J. 2008; 25: 147-155Crossref PubMed Scopus (16) Google Scholar, 14Natowicz M.R. Prence E.M. Chaturvedi P. Newburg D.S. Urine sulfatides and the diagnosis of metachromatic PubMed Scopus Google Scholar, P. Characterization of urinary sulfatides in metachromatic leukodystrophy mass spectrometry.Mol. PubMed Scopus Google Scholar). In to analysis of the ASA it is also used to confirm a of arylsulfatase A. is an important of MLD in (11Von FiguraK. G. V.JaekenJ.2001. Metachromatic leukodystrophy. In The metabolic and molecular basis of inherited disease. C. R. Scriver, A. L. Beaudet, D. Valle, and W. S. Sly, editors. McGraw Hill, New York. 3695–3724.Google Scholar). A mouse of arylsulfatase A deficiency by of the ASA B. P. Hartmann D. R. Lullmann-Rauch R. M. K. R. G. et of arylsulfatase A-deficient mice: to metachromatic Natl. Acad. Sci. PubMed Scopus Google Scholar). and many of including storage of sulfatides and lysosulfatide (3Wittke D. Hartmann D. Gieselmann V. Lullmann-Rauch R. Lysosomal sulfatide storage in the brain of arylsulfatase A-deficient mice: cellular alterations and topographic distribution.Acta Neuropathol. 2004; 108: 261-271Crossref PubMed Scopus (53) Google Scholar, M. Gieselmann V. Mansson J.E. of lysosulfatide in the brain of arylsulfatase A-deficient PubMed Scopus (16) Google Scholar). However, in to the this mouse of MLD not (3Wittke D. Hartmann D. Gieselmann V. Lullmann-Rauch R. Lysosomal sulfatide storage in the brain of arylsulfatase A-deficient mice: cellular alterations and topographic distribution.Acta Neuropathol. 2004; 108: 261-271Crossref PubMed Scopus (53) Google Scholar, B. P. Hartmann D. R. Lullmann-Rauch R. M. K. R. G. et of arylsulfatase A-deficient mice: to metachromatic Natl. Acad. Sci. PubMed Scopus Google Scholar). could be in ASA mice overexpressing by the in the lysosomes that the to sulfatides Lullmann-Rauch R. C. Gieselmann V. Eckhardt M. sulfatide synthesis in cells of arylsulfatase A-deficient mice and of metachromatic PubMed Scopus Google Scholar). methods have been used to quantify sulfatides, including L. B. E. B. J. Determination of urinary sulfatides and by of and Biochem. PubMed Scopus Google Scholar), M.R. Prence E.M. Chaturvedi P. Newburg D.S. Urine sulfatides and the diagnosis of metachromatic PubMed Scopus Google Scholar, G. Y. determination of levels of and and sulfatide by high Biophys. PubMed Scopus Google Scholar), Y. Chen Y. Momin A. Shaner R. Wang E. Bowen N.J. Matyunina L.V. Walker L.D. McDonald J.F. Sullards M.C. et al.Elevation of sulfatides in ovarian cancer: an integrated transcriptomic and lipidomic analysis including tissue-imaging mass spectrometry.Mol. Cancer. 2010; 9: 186Crossref PubMed Scopus (103) Google Scholar, Y. Colsch B. Afonso C. Baumann N. Tabet J.C. Mallet J.M. Zhang Y. Synthetic sulfogalactosylceramide (sulfatide) and its use for the mass spectrometric quantitative urinary determination in metachromatic leukodystrophies.Glycoconj. J. 2008; 25: 147-155Crossref PubMed Scopus (16) Google Scholar, P. Characterization of urinary sulfatides in metachromatic leukodystrophy mass spectrometry.Mol. PubMed Scopus Google Scholar, M. to disease severity in metachromatic 2010; PubMed Scopus Google Scholar, A. J. mass spectrometric analysis of Determination of and of molecular species in brain and in pancreatic Biophys. PubMed Scopus Google Scholar, L. B. J. F. J. mass spectrometric of sulfatides in urinary for of metachromatic Scholar, M. J. mass for determination of sulfatides in from patients with metachromatic Commun. PubMed Scopus Google Scholar, M. F. of sulfatides in and urine from metachromatic leukodystrophy patients by mass PubMed Scopus Google Scholar). a LC/MS/MS method to quantify sulfatides and their molecular species a wide range of concentrations in normal and tissues, and urine. In a sensitive and LC/MS/MS method for the of lysosulfatides in the tissues. and were from ammonium from ammonium and from and for from Lysosulfatide, and were from brain sulfatides were from and ASA mice the to to arylsulfatase A were used in this study F. E. Lullmann-Rauch R. R. C. C. J. Gieselmann V. of to arylsulfatase A in a mouse of metachromatic PubMed Google Scholar). Urine and plasma were from patients with MLD diagnostic or from patients or their of the not required of the of the The diagnosis of MLD been by a deficiency of arylsulfatase A in and in the ASA concentrations of and from of metabolic disease were to a in urine by the the Sulfatides were from urine by a of the method of J. M. A method for the and of total from tissues.J. 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Full Text PDF PubMed Google Scholar) as for a The a and the of and for The and and then to The to a to which and were and for The to a and the with of which with the The were and by as for the urine has a the of molecular species of sulfatides in which increased by a of to as a of ion An is in where the precursor of in plasma of a MLD with and of the In the of the of due to leads to an in the of the of the sulfatide from plasma and mouse tissue as for by a of the method of and A method of total and J. Biochem. Physiol. 37: PubMed Scopus Google Scholar). a in an in E. A. K. A quantitative analysis of sulfatide by mass with ion Biochem. PubMed Scopus Google Scholar) and the and were with of The were for for to the were to and and were the were for to the the to a The by the of and which with the The were under a tissues, this a may be by the in and the with of The are for and the is The lysosulfatides and the are in the The residue in for and for in a The were for in the to The of lysosulfatide and were the sulfatides, from the under a and in prior to LC/MS/MS were in for and for in a The were for to prior to LC/MS/MS or were in ammonium in and for to An to a mass used for were with for the identification and of sulfatides in used the Lipid P. K. V. M. P. for the analysis of and in molecular Google Scholar). The for the of sulfatides and lysosulfatides were by and by the mass for were for and for sulfatides and for lysosulfatide and are in and molecular species of sulfatides were by precursor ion of of of urine from MLD patients or mouse brain and kidney were with P. K. V. M. P. for the analysis of and in molecular Google Scholar). These were used to to monitor LC/MS/MS Sulfatides were a by a from with ammonium to and ammonium the a from to B in and B to B The to and and to the mass of spectrometric were and The of the individual molecular species are in Sulfatides in of molecular species were to chain length of the fatty with molecular species with the chain length chain length of the fatty the molecular species of the sulfatides in the fatty acids containing molecular species, then fatty acid containing molecular species, and the fatty acid containing molecular were the and as for the sulfatides. A a from to B in and B to B The The to and and to the of were and The of the lysosulfatides and the are in urine with sulfatides from brain of and and were The the of the from of sulfatides the from the of the of sulfatides in the A the range this the concentrations of sulfatides as of patients with the form of patients with the and patients with the form of MLD (11Von FiguraK. G. V.JaekenJ.2001. Metachromatic leukodystrophy. In The metabolic and molecular basis of inherited disease. C. R. Scriver, A. L. Beaudet, D. Valle, and W. S. Sly, editors. McGraw Hill, New York. 3695–3724.Google Scholar) were and with the sulfatide in of of of MLD patients increased concentrations of sulfatides The of is The of of the and the of of the plasma with sulfatides from brain of and A the range of sulfatides from the In to in MLD plasma sulfatides were as with The range of sulfatide concentrations in plasma The concentrations of sulfatide of patients and were the that of the patients the The range of sulfatide concentrations in plasma of MLD patients The of is and plasma sulfatides were a the of the from molecular species to that of the the results of sulfatide analysis of selected tissues and plasma from normal mice and ASA mice the F. E. Lullmann-Rauch R. R. C. C. J. Gieselmann V. of to arylsulfatase A in a mouse of metachromatic PubMed Google Scholar). increased levels of sulfatides were found in brain kidney and from the ASA These results the of the method to quantify sulfatides a wide range of concentrations in tissue in of ASA The levels of sulfatides in the ASA mice were increased in in mouse tissues and arylsulfatase in a arylsulfatase The use of LC/MS/MS of individual molecular species of sulfatides. the composition of the molecular species of sulfatides in brain, kidney, and from mice and ASA mice A of sulfatide species found for mainly of and and fatty species for a the composition is tissue An is sulfatide which found to be a species for kidney in ASA in The not in the of for the of total sulfatides in tissues leading to a of the sulfatide content in quantitative in the composition are the molecular species in tissues from mice and ASA mice An the C16:0 species of sulfatides, which were in plasma from ASA mice with the composition of the molecular species of sulfatide in plasma and urine. The and molecular species were in MLD plasma with fatty acids containing sulfatides were in MLD In to the molecular species in in plasma and urine a sulfatide species with and a of species could not be with but with a sulfatide The of this sulfatide species in in MLD in and in MLD in mouse tissue and arylsulfatase are the composition from or in a composition plasma and metachromatic not are the sulfatide composition in plasma or urine from MLD patients or and or in a arylsulfatase are the composition from or metachromatic not are the sulfatide composition in plasma or urine from MLD patients or and or tissue and plasma lysosulfatide were a to the of the The method has sensitivity to quantify lysosulfatide in normal mouse The lysosulfatide levels in brain of the ASA mice are in the increased levels of lysosulfatide were also in kidney and of ASA lysosulfatide could be in the normal kidney and their levels were the of In to lysosulfatide with a base a lysosulfatide with a molecular mass and a of the presence of a lysosulfatide with a sphingadienine (d18:2) base. species present of the lysosulfatide in brain and kidney and in in of ASA could not be in plasma from normal and ASA mice or in plasma from MLD patients and its the of in mouse but of arylsulfatase not but of in a arylsulfatase not We developed an LC/MS/MS method for identification and of sulfatides and lysosulfatides in different sulfatide used prior to analysis by LC/MS/MS analysis of the total of urine and of and from plasma in to the and the mass of that may with the precursor ion of of Y. Chen Y. Momin A. Shaner R. Wang E. Bowen N.J. Matyunina L.V. Walker L.D. McDonald J.F. Sullards M.C. et al.Elevation of sulfatides in ovarian cancer: an integrated transcriptomic and lipidomic analysis including tissue-imaging mass spectrometry.Mol. Cancer. 2010; 9: 186Crossref PubMed Scopus (103) Google Scholar, 7Ishizuka I. Chemistry and functional distribution of sulfoglycolipids.Prog. Lipid Res. 1997; 36: 245-319Crossref PubMed Scopus (222) Google Scholar), and of effects and ion in containing of sulfatides in the presence of of and to LC/MS/MS and for a sensitive of sulfatides in tissues, cells and body that contain of sulfatides. of sulfatides in different a wide range of MLD mice and patients can be from In to methods the of sulfatides in and urine M. J. mass for determination of sulfatides in from patients with metachromatic Commun. PubMed Scopus Google Scholar, M. F. of sulfatides in and urine from metachromatic leukodystrophy patients by mass PubMed Scopus Google Scholar) from MLD patients and normal our method could quantify sulfatides in normal plasma and urine with a to of for of its of a urine for the analysis of MLD and normal of the or urine which are methods or are used M.R. Prence E.M. Chaturvedi P. Newburg D.S. Urine sulfatides and the diagnosis of metachromatic PubMed Scopus Google Scholar, L. B. E. B. J. Determination of urinary sulfatides and by of and Biochem. PubMed Scopus Google Scholar, G. Y. of sulfatides, and in different of rat and distribution in of and white matter of Biophys. PubMed Scopus Google Scholar). An of the is that of ceramides and in sulfatides and glycolipids in and in in tissue are The analysis of sulfatides species to which the study of in the tissue content of individual molecular species of sulfatides in insulin and cancer and and Y. Chen Y. Momin A. Shaner R. Wang E. Bowen N.J. Matyunina L.V. Walker L.D. McDonald J.F. Sullards M.C. et al.Elevation of sulfatides in ovarian cancer: an integrated transcriptomic and lipidomic analysis including tissue-imaging mass spectrometry.Mol. Cancer. 2010; 9: 186Crossref PubMed Scopus (103) Google Scholar, K. Blomqvist M. Mansson J.E. Fredman P. Juhl K. Gromada J. C16:0 sulfatide inhibits insulin secretion in rat beta-cells by reducing the sensitivity of KATP channels to ATP inhibition.Diabetes. 2006; 55: 2826-2834Crossref PubMed Scopus (28) Google Scholar, Y. T. I. Sulfatides as a of in patients with PubMed Scopus Google Scholar, S. of sulfatide from rat and white matter by ion mass Biophys. PubMed Scopus Google Scholar). A sulfatide levels in plasma from and ASA mice In MLD patients and and patients increased levels of plasma sulfatides, and patients that the determination of sulfatides in plasma has sensitivity to MLD patients from in and may have as a to monitor the of from MLD patients high levels of C16:0 and sulfatides. A for the C16:0 species reported for sulfatides in from MLD patients M. J. mass for determination of sulfatides in from patients with metachromatic Commun. PubMed Scopus Google Scholar, M. F. of sulfatides in and urine from metachromatic leukodystrophy patients by mass PubMed Scopus Google Scholar) In to the sulfatide the determination of the deacylated form of the storage by LC/MS/MS for the methods used methods for and of lysosulfatide (12Toda K. Kobayashi T. Goto I. Kurokawa T. Ogomori K. Accumulation of lysosulfatide (sulfogalactosylsphingosine) in tissues of a boy with metachromatic leukodystrophy.Biochem. Biophys. Res. Commun. 1989; 159: 605-611Crossref PubMed Scopus (24) Google Scholar, M. Gieselmann V. Mansson J.E. of lysosulfatide in the brain of arylsulfatase A-deficient PubMed Scopus (16) Google Scholar). In to sensitivity and our method has the of a with is due to the quantitative to the in the and to the to the of its of the present in the not and are to The presence of lysosulfatide has been in normal tissues, in the Our method lysosulfatide in tissue of mice were found in normal kidney and The content of lysosulfatide in ASA mice increased in brain and kidney, but increased levels were also found in an not in Our of a lysosulfatide species with an of that this may from sulfatide species with a sphingadienine (d18:2) base to be present in and sulfatides from mouse brain (10Colsch B. Afonso C. Popa I. Portoukalian J. Fournier F. Tabet J.C. Baumann N. Characterization of the ceramide moieties of sphingoglycolipids from mouse brain by ESI-MS/MS: identification of ceramides containing sphingadienine.J. Lipid Res. 2004; 45: 281-286Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). by the presence of in plasma from mice and patients J.M. J.E. S. A. R. C. M. et is a of Natl. Acad. Sci. 2008; PubMed Scopus Google Scholar, M. N. M. J. J.E. et of in plasma and urine of patients by mass PubMed Scopus Google Scholar) and in plasma from mice and patients J. M. T. J. D. Zhang K. J. et mouse disease cellular and molecular the Natl. Acad. Sci. 2010; PubMed Scopus Google Scholar, N. L. S. K. J.E. M. et plasma in to storage cell and therapeutic PubMed Scopus Google Scholar), for the presence of lysosulfatide in plasma from the and ASA the presence of lysosulfatide in in plasma from normal and ASA mice as well as normal plasma and plasma from MLD it could not be in to and where and are in MLD lysosulfatide may not be a useful to monitor the of the disease and the effects of may be explained by the of visceral in the storage in metachromatic leukodystrophy as with and disease. These visceral organs, the tissue may be an important of plasma J.M. J.E. S. A. R. C. M. et is a of Natl. Acad. Sci. 2008; PubMed Scopus Google Scholar, J. M. T. J. D. Zhang K. J. et mouse disease cellular and molecular the Natl. Acad. Sci. 2010; PubMed Scopus Google Scholar). The V. Gieselmann of for the mouse tissues, the for plasma and urine from patients with metachromatic P. and P. of for and The for with arylsulfatase A UDP-galactose:ceramide galactosyltransferase cerebroside sulfotransferase Lipid metachromatic leukodystrophy