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J. Biol. Chem., Vol. 277, Issue 23, 20386-20398, June 7, 2002

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Kidney Sulfatides in Mouse Models of Inherited Glycosphingolipid Disorders

DETERMINATION BY NANO-ELECTROSPRAY IONIZATION TANDEM MASS SPECTROMETRY^*

Roger Sandhoff^§, Stefan T. Hepbildikler^¶, Richard Jennemann, Rudolf Geyer, Volkmar Gieselmann^**, Richard L. Proia, Herbert Wiegandt, and Hermann-Josef Gröne

From the  Deutsches Krebsforschungszentrum Heidelberg, Abteilung für Zelluläre und Molekulare Pathologie, INF 280, 69120 Heidelberg, Germany, the ^¶ Kekulé-Institut für Organische Chemie und Biochemie, Universität Bonn, Gerhard-Domagk-Str. 1, 53121 Bonn, Germany, the  Biochemisches Institut am Klinikum der Justus-Liebig-Universität Giessen, 35392 Giessen, Germany, the ^** Physiologisch Chemisches Institut, Rheinische Friedrich Wilhelms Universität, 53115 Bonn, Germany, and the  Genetics of Development and Disease Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, November 6, 2001, and in revised form, March 14, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Sulfatides show structural, and possibly physiological similarities to gangliosides. Kidney dysfunction might be correlated with changes in sulfatides, the major acidic glycosphingolipids in this organ. To elucidate their in vivo metabolic pathway these compounds were analyzed in mice afflicted with inherited glycosphingolipid disorders. The mice under study lacked the genes encoding either -hexosaminidase -subunit (Hexa/), the -hexosaminidase -subunit (Hexb/), both -hexosaminidase  and -subunits (Hexa/ and Hexb/), GD3 synthase (GD3S/), GD3 synthase and GalNAc transferase (GD3S/ and GalNAcT/), GM2 activator protein (Gm2a/), or arylsulfatase A (ASA/). Quantification of the sulfatides, I³SO-GalCer (SM4s), II³SO-LacCer (SM3), II³SO-Gg₃Cer (SM2a), and IV^3, II³-(SO)₂-Gg₄Cer (SB1a), was performed by nano-electrospray tandem mass spectrometry. We conclude for the in vivo situation in mouse kidneys that: 1) a single enzyme (GalNAc transferase) is responsible for the synthesis of SM2a and GM2 from SM3 and GM3, respectively. 2) In analogy to GD1a, SB1a is degraded via SM2a. 3) SM2a is hydrolyzed to SM3 by -hexosaminidase S (Hex S) and Hex A, but not Hex B. Both enzymes are supported by GM2-activator protein. 4) Arylsulfatase A is required to degrade SB1a. It is probably the sole sphingolipid-sulfatase cleaving the galactosyl-3-sulfate bond. In addition, a human Tay-Sachs patient's liver was investigated, which showed accumulation of SM2a along with GM2 storage. The different ceramide compositions of both compounds indicated they were probably derived from different cell types. These data demonstrate that in vivo the sulfatides of the ganglio-series follow the same metabolic pathways as the gangliosides with the replacement of sulfotransferases and sulfatases by sialyltransferases and sialidases. Furthermore, a novel neutral GSL, IV⁶GlcNAc-Gb₄Cer, was found to accumulate only in Hexa/ and Hexb/ mouse kidneys. From this we conclude that Hex S also efficiently cleaves terminal 1-6-linked HexNAc residues from neutral GSLs in vivo.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Sulfatides, (designating all sulfated glycosphingolipids) such as galactosylceramide I³-sulfate, occur enriched in the myelin sheets of the central and peripheral nervous system and in glandular epithelial tissues of mammals. Sulfatides of more complex structure have been found in the kidney (1). In the human renal cell carcinoma cell line SMKT-R3 high levels of sulfatides including gangliotriaosylceramide-II³ sulfate (SM2a)¹ were observed (2) that they may modulate the metastatic potential of these cells (3). In addition, complex sulfatides have been recognized to rank among the strongest ligands for NKR-P1. This membrane protein, with an extracellular Ca²⁺-dependent lectin domain, is expressed on natural killer cells that display innate immunity (4, 5). Other proteins involved in innate immunity, properdin and factor H, have also been reported to bind specifically to the sulfatides (6). More recently it has been shown that intracellular sulfation of lactosylceramide suppresses the expression of integrins (7).

In mice, complex kidney sulfatides belong to the ganglio-series glycosphingolipids (GSL) and thus show structural similarity to "brain type"-gangliosides (Fig. 1). This relationship is further suggested by largely identical carbohydrate substitution positions for sulfate and sialic acid. These mouse kidney sulfatides include lactosylceramide-II³ sulfate (SM3), SM2a, and gangliotetraosylceramide-II^3, IV³ bis-sulfate (SB1a) (8). In general, GSLs are synthesized from a ceramide core by modification with glycosyl- and sulfotransferases in the endoplasmic reticulum and Golgi. Degradation of GSL takes place at the surface of intra-lysosomal vesicles by the action of exoglycosidases, sulfatases, and sialidases. For several of these degradation steps, the presence of one of the five known lysosomal activator proteins is required (9). Defects in the lysosomal enzymes or activator proteins that degrade GSLs are the cause of severe human inherited diseases such as metachromatic leukodystrophy and the different forms of GM2 gangliosidosis.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Metabolic and structural comparison of gangliosides and sulfatides of the ganglio-series. Enzymes that are boxed in the figure were affected by the investigated mouse models. For enzyme abbreviations see the abbreviation footnote.

Deficiency in arylsulfatase A in metachromatic leukodystrophy leads to lethal demyelination in the central and peripheral nervous systems. This disease is characterized by the lysosomal accumulation of the sulfatides SM4s, SM4g, and SM3 (10, 11).

Defects in the -hexosaminidase isozymes (Hex S, (/); Hex A, (/); and Hex B, (/)) or in the GM2 activator protein lead to GM2 gangliosidosis, in which ganglioside GM2 and related glycolipids accumulate in lysosomes mainly of neuronal cells. The GM2 gangliosidoses include Tay-Sachs disease (B-variant), due to mutations in the HEXA gene encoding the -subunit of -hexosaminidase, Sandhoff disease (0-variant), due to mutations in the HEXB gene encoding the -subunit, and the AB-variant characterized by mutations in the GM2A gene. For all three enzyme defects, the severe infantile forms result in rapidly progressing neurodegeneration, culminating in death before age 4 years (12).

For the study of these human disorders, mouse models have been established that lack enzymes of GSL biodegradation (13-18). In addition, mutant mice deficient in enzymes of GSL biosynthesis have been generated (19-23).

The aim of the present investigation was to determine how mouse kidney sulfatides were affected by genetically transmitted deficiencies in the metabolism affecting the brain type gangliosides and sulfatide SM4s (Fig. 1). Mouse models of the four GM2-gangliosidoses (Hexa/, Hexb/, Hexa/ and Hexb/, and Gm2a/), metachromatic leukodystrophy (ASA/), as well as, UDP-GalNAc: -1,4-GalNAc transferase deficiency (GalNAcT/), were compared for their kidney sulfatide component profiles by nano-electrospray ionization tandem mass spectrometry (nano-ESI-MS/MS).

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Mutant mice used: Hexa/ (13), Hexb/ (15), Gm2a/ (17), ASA/ (14) and GD3S/ as well as GD3S/ and GalNAcT/ ("GM3-only") mice (23). Hexa/ and Hexb/ were produced by interbreeding Hexa/ and Hexb/ mice as described (16). PCR was employed for genotyping (24).

The human Tay-Sachs liver material was from a girl that died at the age of 2 years and 10 months. The girl was affected with the classical form of Tay-Sachs and symptoms were apparent from 8 months of age including hyperaccusis. The control liver material was from a healthy 42-year-old male donor who died in an accident.

Materials

All chemicals and solvents were of p.A. grade. Gold-sputtered borosilicate glass capillaries, type D, were purchased from Teer Coatings Ltd. (Worcestershire, UK). Lyso-sulfatide (lyso-SM4s), sulfatide (SM4s), GlcCer, GalCer, LacCer, GM3, Gg₃Cer, and bovine brain gangliosides were obtained from Matreya Inc. (Chalfont, PA), sphingosyl-phosphorylcholine, Forssman glycolipid, triethylamine, myristic acid, nonadecanoic acid, heptacosanoic acid, N-hydroxysuccinimide, N,N'-dicyclohexylcarbodiimide, anthrone, taurodesoxycholate, orcinol, absolute tetrahydrofuran, as well as, absolute N,N-dimethylformamide, both over molecular sieve from Sigma-Aldrich (Deisenhofen, Germany), SCDase (sphingolipid ceramide N-deacylase) from Takara Shuso (Otsu, Shiga, Japan), DEAE-Sephadex A-25 from Amersham Biosciences (Uppsala, Sweden), ammonia solution 25% p.A., LiChroprep RP-18 and LiChroprep Si 60 Silica Gel from Merck (Darmstadt, Germany), and dialysis tubes, Visking type 27/32 from Roth (Karlsruhe, Germany). GM2 was isolated from human GM2 gangliosidosis brain.

Methods

Purification of Sulfated GSL from Kidney Tissue-- SM3 was extracted from human kidney. SM2a and SB1a were isolated from rat kidney. In general, 100 g of tissue was homogenized on ice in 100 ml of distilled water with an Ultra Turrax T25 basic from IKA Labortechnik (Staufen, Germany) (6 × 2 min of homogenizing at 24,000 rpm with pauses of 2 min in between). The homogenate was freeze-dried and subsequently extracted with acetone. The freeze-dried tissue then was extracted for GSL, 2 times with chloroform/methanol/water (C/M/W) (10/10/1) and once with C/M/W (30/60/8). The combined C/M/W extracts were concentrated and dialyzed against 5 × 5 liters of distilled water. The dialyzed extract was lyophilized, dissolved in C/M/W (30/60/8) and loaded on DEAE A-25 column to separate neutral and acidic lipids. Elution was with a stepwise gradient of 20, 80, 200, 500, and 1000 mM methanolic potassium acetate (KAc). SM3 was eluted with the 200 mM, SM2a with the 80 mM, and SB1a in the 500 mM KAc fraction. The fractions were desalted by dialysis with 5 × 5 liters of distilled water and lyophilized. From the corresponding fractions, the sulfated GSLs were further purified by repeated silica gel flash column chromatography with the appropriate mixtures of n-hexane/isopropyl alcohol/water or C/M/W as running solvent systems.

Quantification of Purified GSL TLC Standards by Anthrone Reaction (25)-- Commercially available, or from tissue isolated and purified, GSLs were dissolved in C/M/W (10/10/1). Aliquots in the range of 5-20 nmol were dried in an 1.5-ml test tube with a gentle stream of nitrogen. 100 µl of water and 500 µl of anthrone reagent were added. Then the cups were sealed and clamped in between two metal plates so that the lids could not open. One of the plates had appropriate holes for the lower part of the cups to fit through. The cups were incubated for 15 min at 100 °C and then cooled in a water bath at room temperature for further 20 min. Calibration curves were obtained with samples containing a mixture of the free sugars in the appropriate equimolar ratio equaling the ratio of the individual GSL to be quantified (as an example: for SB1a quantification, Glc, Gal, and GalNAc were mixed in the ratio 1:2:1). 300 µl of each sample was placed into a flat-bottomed transparent 96-well microtiter plate. Absorption was measured at 620 nm.

Synthesis of GSL Standards for Nano-ESI-MS/MS-- Lyso-SM3, lyso-SM2a, and lyso-SB1a as well as lyso-GM3 and lyso-GM2 were obtained by treatment of the purified compounds with SCDase according to Ref. 26. The crude products were purified by silica gel column flash chromatography using an appropriate mixture of C/M/W as running solvent system. (Prior to activation 2-hydroxy fatty acids were esterified with acetic anhydride as follows: 2-hydroxy-myristinic acid was dissolved in 200 µl of anhydrous and alcohol-free chloroform + 50 µl of acetic anhydride + 50 µl of 0.1% N,N-dimethylaminopyridine in chloroform. The reaction mixture was incubated for 60 min at 37 °C. Then 500 µl of toluol were added and the sample dried under a gentle stream of nitrogen at 37 °C. The sample was dissolved again in 200 µl of toluol and dried again as before.)

Fatty acids (65 µmol) were dissolved in 4 ml of dry tetrahydrofuran under nitrogen gas and activated with 0.82 equivalents of dicyclohexylcarbodiimide and 0.93 equivalents of N-hydroxysuccinimide. Reaction took place overnight at room temperature.

For condensation, about 100 nmol of lyso-GSL was dissolved in 2 ml of dry N,N-dimethylformamide and 4 µl of triethylamine. Then 2 ml of the activated fatty acid were added. The reaction mixture was incubated at room temperature for 2-5 days and monitored by TLC. Upon the long incubation some GSL were also acylated at hydroxyl groups. This resulted in a smear on TLC, running faster than the GSL standard. The reaction mixtures were, therefore, treated with 0.1 M methanolic KOH for 2 h at room temperature. This mild base treatment was also necessary to remove the acetate ester from the 2-hydroxy group of SM4s (18:1,h14:0). The crude products were purified by silica gel column flash chromatography.

Quantification of the Synthesized GSL MS Standards-- Aliquots of the synthesized GSL MS standards in the range of 0.3-1.0 nmol were spotted on TLC using a Linomat IV from CAMAG (Muttenz, Switzerland). On adjacent lanes corresponding GSL TLC standards were spotted in different concentrations to obtain calibration curves. After development in chloroform, methanol, 0.2% aqueous CaCl₂ (60/35/8) GSL bands were developed with orcinol/sulfuric acid spray reagent at 110 °C for 20 min or with 10% CuSO₄ in 8% H₃PO₄ at 150 °C for 20 min. The amount of the GSL compounds was determined by densitometric scanning of each lane at a wave length of 440 nm (Shimadzu CS-9301 TLC scanner). For each GSL the C14, C19, and C27 fatty acid containing compounds were mixed in an equimolar ratio resulting in the ready-to-use MS standard of known concentration.

Extraction of GSLS from Murine Kidney for Mass Spectrometric Analysis-- Kidneys wet weight was determined, and the kidney homogenized on ice in 5 ml of distilled water with a Ultra Turrax T25 basic (6 × 30 s of homogenizing at 24,000 rpm with pauses of 30 s in between).

GSL MS standards were transferred to glass tubes and the solvent was evaporated with a gentle nitrogen stream. An aliquot of the aqueous kidney homogenate, equal to 20 mg of organ wet weight, was added, and the sample was sonicated for 5 min. Thereafter, the sample was lyophilized and extracted 2 times with 2 ml of acetone. The residual pellet then was extracted twice with 1.5 ml C/M/W (10/10/1) and with 2 ml of C/M/W (30/60/8) for GSL. Neutral and acidic GSL of the combined C/M/W extracts were separated on DEAE A-25 columns (bed volume: 300 µl). The flow-through and wash yielded fraction 1, containing the neutral GSL. Acidic GSL, collected as fraction 2, were eluted with 4 ml of 500 mM methanolic potassium acetate. Solvent was evaporated and acid GSL (fraction 2) desalted with RP-18 (200 mg RP-18 material per column) column chromatography. Fraction 1 was dissolved in 100 µl of 5 mM methanolic ammonium acetate, and fraction 2 in 100 µl of methanol. If necessary, samples were further diluted for nano-ESI-MS/MS.

Extraction of GSLS from Human Liver for Mass Spectrometric Analysis-- Human liver GSLs were extracted in analogy to the mouse kidney protocol. Since the Tay-Sachs liver was stored frozen for more than 25 years it lost barely any weight by freeze drying. Therefore GSL concentrations were calculated per mg dry weight. Extraction without MS standards was carried out with 150 mg dry weight, introducing MS standards with 20 mg.

Determination and Characterization of Sulfatides and GSLS by Nano-ESI-MS/MS-- All analyses were performed with a triple quadrupole instrument (VG micromass (Cheshire, UK) model Quattro II) equipped with a nano-electrospray source operating at an estimated flow rate of 20-50 nl/min. Usually, 10 µl of a samples, dissolved in methanol or methanolic ammonium acetate (5 mM), was filled into a gold-sputtered capillary. The capillary was positioned at a distance of 1-3 mm in front of the cone. The source temperature was set to 30 °C and the spray was started by applying 800-1200 V to the capillary. For each spectrum 20-50 scans of 15-30 s duration were averaged. All tandem MS experiments were performed with argon as collision gas at a nominal pressure of 2.2-2.7 × 10³ mbar. The parameters for the cone voltage and the collision energy of the different scan-modes are listed in Table I.

Evaluation of the Nano-ESI-MS/MS Data and Quantification of Lipids-- Quantitative spectra were measured with an average mass resolution of 1200 (ion mass/full width half-maximum). Peak height values of the first mono-isotopic peak of each compound were taken for evaluation. From the peak intensities of the corresponding internal standard lipids a linear trend was calculated. The obtained calibration curve represented the intensity of the internal standard molar amount at a given m/z value. In addition, a linear trend for n+2 molecular isotopic signal intensities (molecules containing either two ¹³C-atoms or one ³⁴S-atom, and, thereby, shifted by m/z 2 upwards) was calculated from the internal standards. If necessary, signal intensities were corrected first from influence of n+2 signal overlap. This overlap appears if lipids, that contain one additional double bond, are present. Then their n+2 signal overlaps with the main signal of the lipid without this double bond. From the corrected intensity ratio (sample lipid/internal standard trend) and the amount of internal standard added the quantity of the individual molecular species (e.g. SM4s (18:1, 16:0) or SM4s (18:1, 24:1) etc.) was calculated. From the sum of individual molecular species then the amount of a lipid (SM4s) resulted. Endogenous SB1a, GM3, and GM2 were correlated to the sole corresponding standard.

Extraction of GSLS from Murine Kidney for TLC Analysis-- For TLC analysis, 50 mg of kidney wet weight were extracted as above using the appropriate volumes. The neutral and acidic GSL fractions were each taken up in 100 µl of C/M/W (10/10/1). Aliquots according to the Figure legends were spotted on TLC plates with a Linomat IV from CAMAG (Muttenz, CH). A pre-run was performed with chloroform/alcohol (C/A) (1/1). Then the plates were dried and GSL were separated with the running solvent chloroform, methanol, 0.2% aqueous CaCl₂ (60/35/8), if not otherwise noted. Bands were detected with orcinol/sulfuric acid spray reagent at 110 °C for 10-20 min.

Carbohydrate Constituent Analysis-- Carbohydrate constituents were released by acid hydrolysis after hydrofuran treatment, converted into their corresponding alditol acetates and analyzed by capillary GC/MS as detailed elsewhere (27).

Carbohydrate Permethylation Analysis-- For determination of linkage positions of monosaccharide constituents, glycolipids were permethylated and hydrolyzed (28). Partially methylated alditol acetates obtained after sodium borohydride reduction and peracetylation were analyzed by GC/MS using the instrumentation and microtechniques described previously (29, 30).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Synthesis of Internal Standards for Nano-ESI-MS/MS-- For quantification of GSLs by nano-ESI-MS/MS an appropriate internal standard must be applied (24). Therefore, sulfatides and gangliosides with unusual fatty acid composition (myristic acid (14:0), 2-hydroxymyristic acid (h14:0), nonadecanoic acid (19:0), and heptacosanoic acid (27:0)) were synthesized from the corresponding lyso compounds. The latter were prepared enzymatically from the corresponding sulfatides, SM4s, SM3, SM2a, and SB1a, and gangliosides, GM3 and GM2 using SCDase (for SM2a, see Fig. 2A). Coupling the lyso-GSL to a fatty acid as described under "Experimental Procedures," we produced SM4s (18:1,14:0), SM4s (18:1,19:0), SM4s (18:1,27:0), SM3 (18:1,14:0), SM3 (18:1,19:0), SM3 (18:1,27:0), SM2a (h18:0,14:0), SM2a (h18:0,19:0), SM2a (h18:0,27:0), SB1a (h18:0,19:0), and recently GM3 (18:1,19:0), GM2 (18:1,14:0), and SM4s (18:1,h14:0). The sphingoid of human kidney SM3 consisted solely of C18-sphingosine (data not shown), whereas rat kidney SM2a and SB1a contained a mixture of C18-sphingosine and C18-phytosphingosine as verified by nano-ESI-MS/MS (Fig. 2B).

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Generation of lyso-SM2a by enzymatic digestion of rat kidney SM2a. SM2a was purified from rat kidney and hydrolyzed for 24 h with the enzyme sphingolipid ceramide N-deacylase (SCDase) from Pseudomonas sp. as described under "Experimental Procedures." A, reaction products of the aqueous phase were taken up in C/M/W (10/10/1, v/v), separated on TLC with running solvent C/M/0.2% aqueous CaCl2 (45/45/10) and stained for sugars with orcinol/sulfuric acid. Lane 1, purified SM2a from rat kidney; lane 2, SCDase digest of SM2a. Whereas GSL stained purple, taurodesoxycholate (TDC), used in the assay, turned light blue after several hours at room temperature, and no remaining SM2a staining could be observed in the digest. Densitometric quantification of the product bands revealed a ratio of 57: 100 for sphingosyl-lyso-SM2a to phytosphingosyl-lysoSM2a. B, nano-ESI-MS/MS precursor ion m/z 97-spectra of rat kidney SM2a (i) and its products of SCDase digestion (spectrum ii) corresponding to lanes 1 and 2 of A), respectively. m/z 97 represents the fragment [HSO4] produced in the collision chamber. By this scan only compounds bearing a sulfate group (m/z 97) are detected and plotted in the spectrum. Therefore no TDC m/z 1019.5, carrying a sulfono- (giving rise to [·SO3] with m/z 80) but not a sulfate group, is detected. A ratio of 59:100 for lyso-SM2a (18:1) to lyso-SM2a (h18:0) was determined.

Densitometric quantification of the product bands of lyso-SM2a from the orcinol/sulfuric acid-stained TLC (Fig. 2A) revealed a ratio of 57:100 for sphingosyl-lyso-SM2a (18:1) to phytosphingosyl-lyso-SM2a (h18:0). This was in good agreement with the ratio of the corresponding peaks in the nano-ESI-MS/MS spectrum (Fig. 2B) showing a sphingoid ratio of 59 (18:1):100 (h18:0).

Each standard solution was quantified by densitometric scanning of the orcinol/sulfuric acid- or CuSO₄/phosphoric acid-stained TLC band. On TLC, standards with C14, C19, or C27 fatty acid migrated sequentially faster as compared with one another (data not shown). For mass spectrometric quantification of each sulfatide, the three respective fatty acid derivative standards were mixed in an equimolar ratio. Since the concentration of the different sulfated GSLs, i.e. SM4s, SM3, SM2a, and SB1a, was not identical in murine kidney (Fig. 3, top, lane 3), different amounts of corresponding MS standards were added to the kidney samples. For most samples, 432 pmol of SM4s, 156.6 pmol of SM3, 102.6 pmol of SM2a, and 152.2 pmol of SB1a-MS standards were added (Fig. 4). Correlating the endogenous sulfatide signals to those of the corresponding standards levels of kidney sulfatides were quantified as described under "Experimental Procedures."

View larger version (113K): [in this window] [in a new window]   Fig. 3.   TLC of acidic GSL from kidneys of mutant mice. Acidic GSL were extracted from kidney homogenate, lipids corresponding to 4 mg of kidney wet weight separated on TLC and stained with orcinol/sulfuric acid as described: top, lanes 1 and 9, and B, lanes 1 and 10: ganglioside standards, from top to bottom: GM3, GM2, GM1, GD1a, GD1b, and GT1b; A, lanes 2 and 8; and B, lanes 2, 5, and 9: sulfated GSL standards, from top to bottom: SM4s, SM3, SM2a, SB2, and SB1a. Top, lane 3, wild type; 4, Gm2a/; 5, Hexa/; 6, Hexb/; 7, Hexa+/ and Hexb/. Bottom, lane 3, ASA/; 4, ASA+/+; 6, GD3S/ and GalNAcT+/+; 7, GM3 only GD3S/ and GalNAcT/; 8, GD3S/ and GalNAcT+/. TLC, top, shows the strong storage of SM2a in Hexa/ (lane 5) and Hexa+/ and Hexb/ kidney (lane 7) and minor storage of SM2a in Hexb/ (lane 6) and Gm2a/ kidney (lane 4, faint band), whereas no SM2a could be detected in wild type kidney (lane 3). The acidic GSL pattern of Hexa/ and Hexb/ kidney is identical to that of Hexa/ kidney (data not shown). TLC, bottom, shows (i) strong storage of SM4s, SM3, and SB1a in ASA/ kidney (lane 2), with SM4s and SM3 not separated and (ii) complete lack of SB1a with corresponding accumulation of SM3 in kidney of GalNAcT/ mice (lane 7).

View larger version (11K): [in this window] [in a new window]   Fig. 4.   Nano-ESI-MS/MS spectrum of a MS standard mixture for sulfatide determination. Synthetic sulfated GSL standards were mixed as follows: C14-, C19-, and C27-SM4s: 4.8 pmol/µl each; C14-, C19-, and C27-SM3: 1.74 pmol/µl each; C14-, C19-, and C27-SM2a, 1.14 pmol/µl each; and C19-SB1a, 1.69 pmol/µl. The mixture was scanned by nano-ESI-MS/MS in negative mode using a precursor ion scan with m/z 97 (corresponding to [HSO4]) specific for sulfated compounds (31, 32, 40, 53, 54). Aliquots of this mixture later were added to the kidney GSL samples for quantification.

At Higher Collision Energies 2-Hydroxy Fatty Acid-containing Sulfatides Are Measured in the Precursor Ion Mode (m/z 97) with the Same Abundance as Sulfatides Containing Non-hydroxy Fatty Acids-- Since sulfatides SM4s with a 2-hydroxy fatty acid give rise to additional product ions (due to a break between the carboxyl-carbon- and the -carbon-atom of the fatty acid), this might affect the relative abundances of the common fragments (e.g. [HSO₄] used for quantification) (31, 32). These additional fragments could be detected for SM4s (2hFA) at collision energies of 50-60 eV with not more than 7% of the intensity of fragment m/z 97 ([HSO₄]). At collision energies of 90-115 eV that were used to quantify SM4s in the precursor ion mode, these fragments were not detectable or had an abundance smaller than 0.2%. For SM3 and SM2a additional fragments due to a 2-hydroxy fatty acid could also be detected at collision energies of 65-70 eV with up to 7% abundance relative to m/z 97. But none of these fragment appeared at collision energies of 90-115 eV, which were relevant for quantification.

For SM4s a 2-hydroxy fatty acid containing standard SM4s (18:1,h14:0) was synthesized and mixed in an equimolar ratio with SM4s (18:1,14:0), SM4s (18:1,19:0), and SM4s (18:1,27:0). From the peak intensities of the three non-hydroxy fatty acid containing SM4s a linear trend was calculated. At low collision energy (60 eV) the measured intensity of the 2-hydroxy standard SM4s (18:1,h14:0) reached 90% of the linear trend whereas at 90 eV it differed no more than 2% from the linear trend.

Linearity of the Mass Spectrometric Method in Comparison to TLC Densitometry-- To test the linearity of the mass spectrometric method, a constant amount of SM4s standard (272 pmol) was mixed in several samples with different amounts of bovine brain sulfatide (8.5 to 17.7 nmol). The values obtained by mass spectrometry as plotted against the amounts used showed that linearity was achieved from 35 to 8830 pmol (Fig. 5). The average concentration evaluated from the 9 data points in this range differed by 1.7% from the theoretical value with a standard deviation of 8%. Since bovine brain sulfatide is a mixture of sulfatides with different ceramide compositions, values obtained for some representative individual sulfatides were also plotted in this diagram to demonstrate linearity for the individual species. The results indicate that individual signal intensities down to 2.5% and up to 1000% of the standard signal intensities were in the range of linearity.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Quantification of SM4s by nano-ESI-MS/MS. Bovine brain sulfatide (SM4s) from 8.5 pmol to 17.7 nmol was mixed in 220 µl of methanol with SM4s-MS standard, containing C14-, C19-, and C27-SM4s, each in an amount of 272 pmol. Samples were measured with a precursor ion scan specific for sulfated lipids as described under "Experimental Procedures." Besides the total sulfatide, the amount of different ceramide compositional SM4s species are plotted. The number indicates the m/z value of the single sulfatide with: m/z 806, SM4s(18:1,h18:0); m/z 862, SM4s(18:1,22:0); m/z 888: SM4s(18:1,24:1); m/z 890, SM4s(18:1,24:0); m/z 904: SM4s(18:1,h24:1); m/z 916, SM4s(18:1,26:1). Data points for 806, 862, 904, and 916 are more or less overlapping.

Kidney of Wild Type Mice Contain the Sulfated GSLS SM4S, SM3, and SB1A-- Acidic GSLs were isolated and separated on TLC as described. Staining with orcinol/sulfuric acid revealed GSLs with migration rates comparable to SM4s, SM3 and SB1a and GM3 (Fig. 3A, lane 3). Except for the compound migrating with GM3, the TLC bands also stained with azur A indicating that they are sulfated glycolipids (data not shown). Quantification by nano-ESI-MS/MS revealed SM4s, SM3, and SB1a to make up 83, 10, and 7% of the sulfated GSLs of mouse whole kidney in close agreement with an earlier report by Tadano-Aritomi and co-workers (8) (Fig. 6A; Table II). As compared with their data, however, the present analyses showed an ~1.5 times higher concentration of sulfated GSLs. SM2a and SB2, that are present in rat kidney, could not be detected in the kidney of wild type mice. Considering the limiting background noise of the mass spectra obtained, the concentration of SM2a was calculated to be less than 1.4 pmol/mg wet weight corresponding to less than 0.3% of total sulfated GSL.

View larger version (35K): [in this window] [in a new window]   Fig. 6.   Comparison of kidney sulfatides in wild type, Hexa/ and "GM3-only" mice by nano-ESI-MS/MS. Kidney homogenate was mixed with MS standards for sulfatides and extracted for acidic GSL as described. Sulfatides were detected by nano-ESI-MS/MS in negative mode using the precursor ion scan m/z 97. Spectrum A, wild type; B, Hexa/; and C, GM3-only (GD3S/ and GalNAcT/) mice. Signals correspond to 20 mg wet weight kidney, and 432 pmol of SM4s, 156.6 pmol of SM3, 102.6 pmol of SM2a, and 152.1 pmol of SB1a internal standards, each. Signals for internal standards are pointed out in spectrum A). Mass per charge (m/z) range for mouse kidney endogenous sulfatide signals: SB1a, 692 (18:1,16:0) to 756 (18:1,h24:0); SM4s, 776 (18:1,16:1) to 944 (18:1,28:1); SM3 938 (18:1,16:1) to 1080 (18:1,26:0); and SM2a, 1143 (18:1,16:0) to 1283 (18:1,26:0). Signal m/z 713 (18:1,19:0) is due to SB1a internal standard (see Fig. 4). Clearly, accumulating SM2a is detected in high amounts in B, whereas in C, no kidney SB1a could be detected (*).

View this table: [in this window] [in a new window]   Table II SM2 accumulation in kidney of -hexosaminidase or GM2 activator protein-deficient mice as determined by nano-ESI-MS/MS The standard deviation of the method is smaller than 8%.

With regard to mouse kidney sulfatide ceramide composition, C18-sphingosine was the most prominent sphingoid with less than 6% of additional C18-phytosphingosine and 60-70% fatty acids of C22- and C24-aliphatic chain length. In addition, fatty acids of C-16, C-18, C-20, C21, C-23, C26, and C28 chain length were also detected. More than 75% of the fatty acids were saturated and the amount of 2-hydroxylated fatty acids, ~60% of the total, was twice as high for SM4s than for SM3 and SB1a with ~30%. 2-Hydroxylation was identified by both, molecular mass in mass spectrometry, as well as, the additional fragments m/z 522, 540, and 568, that appeared in the corresponding product ion spectra of SM4s (data not shown). These fragments have been reported to be characteristic for sulfatide with 2-hydroxy fatty acids (31, 32).

Gg₃Cer, Gb₄Cer, and IV⁶GlcNAC-Gb₄Cer Accumulate in Hexa/ and HexB/ Kidney-- Neutral and acidic GSLs of double mutant Hexa/ and Hexb/ mice were isolated. As compared with the wild type mouse, TLC of the neutral GSLs revealed two double and one single bands that stained intensely with orcinol/sulfuric acid indicating the accumulation of three glycolipid components (Fig. 7, lane 1). The upper double band had a TLC migration rate corresponding to Gg₃Cer, and the lower with Gb₄Cer. Both GSLs are known to accumulate in these mice. The lower single TLC band, designated compound X, showed a migration between Forssman glycolipid and Gg₄Cer.

View larger version (62K): [in this window] [in a new window]   Fig. 7.   Neutral GSL storage compounds in kidney of Hexb/, Hexa+/ and Hexb/, and Hexa/ and Hexb/ mice. Neutral GSL corresponding to 5 mg of kidney wet weight were separated on TLC with the running solvent chloroform, methanol, 0.2% CaCl2 (60/35/8) and stained with orcinol/sulfuric acid as described. Lanes 1, Hexa/ and Hexb/; 2 and 8, neutral GSL standard, from top to bottom: GalCer (triple band), LacCer (double band), Gg3Cer and Forssman lipid (double band, mixture from sheep erythrocytes and chicken heart); lane 3, wild type; 4, Hexa/; 5, neutral GSL standard, from top to bottom: GlcCer (double band), LacCer (strong double band), Gb3Cer (double band), Gb4Cer (strong band), Lc4Cer, nLc4Cer and Gg4Cer (strong band); 6, Hexb/; 7, Hexa+/ and Hexb/. Whereas there is no significant difference between Hexa/ (lane 4) and wild type mice (lane 3), Hexa/ and Hexb/ (lane 1), Hexb/ (lane 6), as well as, Hexa+/ and Hexb/ kidney (lane 7) accumulate Gg3Cer and Gb4Cer. In addition, only Hexa/ and Hexb/ mice (lane 1) accumulate a third neutral GSL (compound X), running between Gg4Cer and Forssman glycolipid. Fuc-LacNAc-Gb5Cer: Gal-1,4 (Fuc-1,3-)-GlcNAc1,6(Gal-1,3-)-GalNAc1,3-Gb3Cer.

Investigating the neutral GSL fraction in nano-ESI-MS/MS with a precursor ion scan of m/z 264 significantly increased signals for neutral GSL with the sequence Cer-Hex-Hex-HexNAc (as in Gg₃Cer) and Cer-Hex-Hex-Hex-HexNAc (as for Gb₄Cer) were detected, as compared with wild type kidney. m/z +264 represents the protonated and dehydrated C18-sphingosine base, which is obtained as a characteristic fragment of neutral GSLs under these conditions. The ascribed sequence was confirmed from the collision induced fragments obtained from these molecules (data not shown). Scanning for higher neutral GSLs in nano-ESI-MS/MS, we also used a precursor ion scan of m/z +204. m/z +204 represents a protonated and dehydrated HexNAc residue which should be present at the terminus in all storage compounds of this mutant mouse. By both of these scans, signals for a GSL containing 3 Hex and 2 HexNAc residues could be identified that were not present in wild type kidney. Thus, the third accumulating GSL, compound X, contained five sugar residues.

Comparing the collision induced fragments of the protonated storage compound X with that of protonated Forssman glycolipid by nano-ESI-MS/MS indicated that the characteristic fragments were identical (Fig. 8A). From these data the structure for compound X could be assigned as HexNAc-HexNAc-Hex-Hex-Hex-Cer.

View larger version (39K): [in this window] [in a new window]   Fig. 8.   Fragmentation patterns of compound X from Hexa/ and Hexb/ and of Forssman glycolipid from sheep erythrocytes by nano-ESI-MS/MS-product ion mode. A, fragments in positive mode: (i) compound X (18:1,24:0) with m/z 1542.9 and (ii) Forssman glycolipid (GalNAc-1,3-Gb4Cer (18:1,24:0)) with m/z 1542.9. Comparing the fragments of both compounds demonstrates, that the sugar increments are ordered in the same sequence: Cer-Hex-Hex-Hex-HexNAc-HexNAc. B, fragments in negative mode (i) compound X (18:1,24:0) with m/z 1540.9 and (ii) Forssman glycolipid (18:1,24:0) with m/z 1540.9. Fragment m/z 322 is generated only by compound X (i) and represents the terminal HexNAc with a fragment of the sub-terminal HexNAc generated by a ring cleavage between C2-C3 and ring O-C1. On the other hand, fragment m/z 154 can only be generated by Forssman glycolipid (ii), indicating that both compounds are not identical. C, structures and fragmentation schemes of compound X and Forssman glycolipid.

Since in Forssman glycolipid the terminal HexNAc residue is -glycosidically linked and not a substrate for -hexosaminidase, it is assumed not to accumulate in the GM2 gangliosidosis mice. In addition, the TLC band of compound X did not co-migrate with the Forssman lipid standard. Compound X was further analyzed by nano-ESI-MS/MS. Comparing the collision induced fragments of the deprotonated compound X and Forssman glycolipid in the negative product ion mode of nano-ESI-MS/MS, distinct differences were observed. First, the storage compound did not yield a fragment of m/z 154 that appeared in Forssman lipid standard from sheep erythrocytes (Fig. 8B, ii) or from chicken heart (data not shown). Second, a fragment with m/z 322, not present in Forssman lipid, appeared with compound X (Fig. 8B, i). This is a terminal fragment produced by ring cleavage between C2-C3 and C5-oxygen ring of the subterminal HexNAc residue. To ensure that this fragment was not due to impurities, the neutral GSL fraction was scanned for compound X using this fragment in a nano-ESI-MS/MS precursor ion mode. The storage compound with the same ceramide pattern (C18-sphingosine combined with 16:0, 22:0, 24:1, and 24:0 fatty acids) as described before with a precursor ion scan of m/z 220, representing the deprotonated terminal HexNAc-residue was again detected (Fig. 8A, iii and iv). Since Forssman glycolipid from chicken heart had a distinctly different ceramide composition, including ceramide (C18-sph,18:0) and (C18-sph,20:0) (Fig. 9A, ii), it was admixed with the GSL fraction containing compound X. Both compounds could be detected when scanning the mixed sample either in nano-ESI-MS/MS total negative ion mode (Fig. 9B, i), or with collision induced fragments (m/z 405, Fig. 9B, ii, or m/z 220, Fig. 9B, iii) that appear in the product ion scans of both compounds. In contrast, by scanning with the compound X collision-induced fragment the m/z 322 only compound X could be detected; no signals for Forssman glycolipid appeared (Fig. 9B, iv). Therefore, compound X with the structure HexNAc-HexNAc-Hex-Hex-Hex-Cer, must be different from Forssman glycolipid. For a further investigation of the nature of compound X, the glycolipid was isolated by preparative TLC. Subsequent carbohydrate constituent analysis by GC/MS revealed the monosaccharides Gal, Glc, GlcNAc, and GalNAc in the ratio (2.0:1.25:1.0:0.9). And additional permethylation analysis identified 4-substituted Glc, 4-substituted Gal, 3-substituted Gal, 6-substituted GalNAc, and terminal GlcNAc (data not shown).

View larger version (20K): [in this window] [in a new window]   Fig. 9.   Mass spectrometric differences between Forssman glycolipids and compound X from Hexa/ and Hexb/ kidney. A, compositions of Forssman glycolipid from chicken heart or from sheep erythrocytes, and from compound X, respectively, as measured by precursor ion scanning. According to the fragmentation patterns of Forssman glycolipid and compound X in Fig. 7B, the dominant fragment m/z 405 was taken to measure Forssman glycolipid from sheep erythrocytes (i) and from chicken heart (ii), and the dominant fragments m/z 220 (iii) and 322 (iv) were taken to scan for compound X in the neutral GSL fraction of Hexa/ and Hexb/ kidney. m/z 220 represents [HexNAc  H+], m/z 322 a terminal fragment derived by ring cleavage through the subterminal HexNAc (see text) and m/z 405 represents [HexNAc2  H3O+]. Compound X contains mainly the fatty acids: 16:0, 22:0, 24:1, and 24:0. Forssman glycolipid from chicken heart contains mainly the fatty acids: 16:0, 18:0, 20:0, and 22:0, whereas that of sheep erythrocytes contains mainly 24:1 and 24:0 fatty acids. Forssman glycolipid was not detectable by the precursor ion scan m/z 322. B, nano-ESI-MS/MS-spectra of a mixture of chicken heart Forssman glycolipid (A, ii) with the neutral GSL fraction from Hexa/ and Hexb/ kidney (A, iii or iv). (i) total negative ion spectrum; (ii) precursor ion scan m/z 405; (iii) precursor ion scan m/z 220; and (iv) precursor ion scan m/z 322. Whereas both compounds, Forssman glycolipid and compound X, are detected with the precursor ions of m/z 220 (iii) and 405 (ii) (although with different sensitivities), the precursor ion m/z 322 (iv) is specific for compound X; no Forssman glycolipid (lack of m/z 1457 and 1485) is detected and the intensity pattern in (iv) returns to that of the pure neutral GSL fraction as shown (A, iv).

It is known that in mouse kidney a characteristic globo-/neolacto-series glycolipid occurs, Gal1-4(Fuc1-3)GlcNAc1-6(Gal1-3)Gb₄Cer, which could be detected by nano-ESI-MS/MS in wild type and in mutant kidney samples (data not shown). Therefore, it appears highly likely that compound X is an accumulated degradation product of this glycolipid with its remnant N-acetylhexosamine-terminal core structure IV⁶-GlcNAc-Gb₄Cer.

Besides C18-sphingosine and non-hydroxylated fatty acids of C16 up to C24 aliphatic chain length, hydroxy fatty acids, as well as, phytosphingosine were determined in Gb₄Cer and Gg₃Cer. This explains the appearance of TLC double bands for both of these glycolipids (data not shown).

SM2A Accumulates in HexA/ and HexB/ Mouse Kidney-- Separation of the acid GSL fraction on TLC and staining with orcinol/sulfuric acid revealed a new prominent band running at the level of SM2a that does not appear in wild type kidney. No significant increase of a band at the level of GM2 was observed.

Whereas quantification of SM4s, SM3, or SB1a by nano-ESI-MS/MS showed no significant changes in concentrations, a large amount of SM2a (239 pmol/mg wet weight) was identified in kidney from a 9-week-old mutant mouse. This corresponds to an SM2a increase of at least 172-fold as compared with kidney from a wild type mouse (Table II and Fig. 10). No significant changes in the ceramide compositions of SM4s, SM3, and SB1a, or accumulated SM2a compared with wild type were detected.

View larger version (22K): [in this window] [in a new window]   Fig. 10.   Accumulation of SM2a in kidney of -hexosaminidase or GM2-activator protein-deficient mice. Values obtained by quantitative nano-ESI-MS/MS as described under "Experimental Procedures" are plotted in a logarithmic scale. For absolute values see Table II. For wild type, SM2a detection limit (1.39 pmol/mg wet weight) was set to 100% since no SM2a could be detected in wild type. Age of mice in weeks is indicated on the y axis.

SM2A but Not Neutral GSLS Accumulate in Hexa/ Kidney-- TLC analysis of the neutral GSLs of Hexa/ mice kidney showed no significant differences as compared with wild type (data not shown). In contrast, the acidic GSL component profile was characterized by a prominent band running at the level of SM2a that was not present in lipids of wild type kidney (Fig. 3, top, lane 5). However, no significant increase of a TLC band at the level of GM2 could be observed. Similar to TLC analysis, quantification of the sulfated GSL by nano-ESI-MS/MS revealed no significant changes in SM4s, SM3, or SB1a concentrations (Fig. 6B). However, in the case of kidney from 19- and 20-week-old Hexa/ mice, large amounts (248 ± 18 pmol/mg wet weight) of SM2a were detected that corresponded to an average increase of at least 180-fold as compared with the wild type (Table II and Fig. 10). No significant changes in the ceramide compositions of sulfated GSL compared with wild type were detected The SM2a pattern was similar to that of Hexa/ and Hexb/ mice.

Accumulation of Gg₃Cer and Gb₄Cer but Not IV⁶GlcNAC-Gb₄Cer in HexB/ Kidney-- TLC analysis of the neutral GSLs of kidney from Hexb/ mice showed storage of Gg₃Cer, Gb₄Cer, but no accumulation of GlcNAc1-6Gb₄Cer. There was no significant difference in Gg₃Cer and Gb₄Cer TLC-band intensities between Hexb/ and Hexa/ and Hexb/ double mutant kidney (Fig. 7).

SM2A Accumulates in Hexb/ Kidney-- In the case of the Hexb/ mouse mutant, TLC of the kidney acidic GSLs showed the appearance of a faint band migrating identically to the SM2a standard (Fig. 3, top, lane 6). By nano-ESI-MS/MS, 24.7 ± 0.05 pmol of SM2a per mg wet weight was quantified in a 13- and 18-week-old mutant kidney corresponding to an average increase of at least 18-fold as compared with the wild type (Table II, Fig. 10). No significant changes in SM4s, SM3, or SB1a-concentrations were found. No significant differences in the ceramide compositions of sulfated GSL compared with wild type were detected. The SM2a pattern was similar to that of Hexa/ and Hexb/ mice.

SM2A Accumulates in Gm2a/ Kidney-- TLC analysis of the neutral GSL fraction from Gm2a/ kidney showed no significant differences as compared with wild type kidney (data not shown). A faint TLC band of the acidic GSLs, not seen in the wild type, co-migrated with SM2a (Fig. 3, top, lane 4). As determined by nano-ESI-MS/MS, kidney from 23-week-old mutant mice contained 7.1 ± 1.8 pmol of SM2a per mg wet weight corresponding to an average increase of at least 5-fold over wild type (Table II and Fig. 10).

No significant changes in the ceramide compositions of sulfated GSL compared with wild type were detected. The SM2a pattern was similar to that of double mutant Hexa/ and Hexb/ mice.

All Sulfatides Accumulate in Arylsulfatase A-deficient Kidney-- The neutral GSLs of ASA/ mouse kidney were not different from the wild type (data not shown). In contrast, TLC of the mutant mouse kidney acidic GSL fraction showed strong increases in bands co-migrating with SM4s, SM3, and SB1a. All were stained by orcinol/sulfuric acid spray reagent (Fig. 3, bottom, lane 3) and with azur A (data not shown).

In a 11-week-old ASA/ kidney, 11-, 4.4-, and 15-fold accumulation of SM4s, SM3, and SB1a, respectively, was quantified by nano-ESI-MS/MS. Analysis of a kidney from a 1-year-old ASA/ mouse demonstrated a further increase in the accumulation of SM4s, SM3, and SB1a to about 80-, 40-, and 60-fold, respectively. However, no further increase in the accumulation of these GSLs was seen in a 2-year-old ASA/ kidney. Sulfatide concentrations were very similar to those of the 1-year-old ASA/ kidney (Table III and Fig. 11). No significant changes in the ceramide constituent compositions of SM4s, SM3, and SB1a compared with wild type were detected.

View larger version (15K): [in this window] [in a new window]   Fig. 11.   Accumulation of sulfatides in kidney of arylsulfatase A-deficient mice. Values obtained by quantitative nano-ESI-MS/MS as described under "Experimental Procedures" are plotted in a logarithmic scale. For absolute values see Table III. Age of mice in weeks is indicated on the y axis.

Mice Deficient in -GalNac Transferase Lack SB1A in the Kidney-- No significant differences, as compared with wild type, were observed by TLC for the neutral kidney GSLs of GD3S/ and GalNAcT+/+, GD3S/ and GalNAcT+/, and GD3S/ and GalNAcT/ mutants (data not shown). With regard to the acidic GSLs, the TLC profile of the GD3S and GalNAcT/ kidney was characterized by the disappearance of a band co-migrating with the SB1a standard (Fig. 3, bottom, lane 7). Quantification of the sulfated GSL in these mutants revealed a 20% decrease of SB1a with a corresponding increase in SM3 in GD3S/ and GalNAcT+/ kidney as compared with GD3S/ and GalNAcT+/+ mutant. Kidneys from GD3S/ and GalNAcT/ mutant mice showed an increase in SM3 with SB1a being undetectable (Table IV).

View this table: [in this window] [in a new window]   Table IV Lack of SB1a in kidney of -GalNAc transferase-deficient mice as determined by nano-ESI-MS/MS The standard deviation of the method is smaller than 8%.

SM2A Accumulates in Addition to GM2 in a Tay-Sachs Patient's Liver-- Acidic GSL were extracted with and without internal MS standards from a Tay-Sachs patient's and a control human liver. In both livers comparable amounts (±10%) of GM3 were detected in good agreement with the data published by Nilsson and Svennerholm (33). In addition, significant amounts of SM2a and GM2 could only be detected in the Tay-Sachs liver (Table V). The ceramide composition of all three GSLs, GM3, GM2, and SM2a, was different from each other, whereby that of GM3 and GM2 were comparable to the values reported earlier (33) (Figs. 12 and 13). With 57% GM2 containing stearic acid (GM2(18:1,18:0)) was the main GM2 species. In contrast to this, with 31% SM2a containing a C24:1-fatty acid (SM2a(18:1,24:1)) was the main SM2a species.

View larger version (20K): [in this window] [in a new window]   Fig. 12.   Composition of accumulated SM2a in a human Tay-Sachs patient's liver as determined by ESI-MS/MS-precursor ion m/z 97 mode.

View larger version (11K): [in this window]