Sulfatide Species Research Articles

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.

Distribution of C16:0, C18:0, C24:1, and C24:0 sulfatides in central nervous system lipid rafts by quantitative ultra-high-pressure liquid chromatography tandem mass spectrometry

Sulfated galactosylceramides (sulfatides) are glycosphingolipids associated with cholesterol- and sphingolipid-enriched membrane microdomains (lipid rafts) and are highly expressed in brain tissue. Although it is known that sulfatide species show heterogeneity in their fatty acid acyl group composition throughout brain development, their lipid raft distribution and biological relevance is poorly understood. We validated a fast and sensitive ultra-high-pressure liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) method to measure developmentally regulated sulfatide species (C16:0, C18:0, C24:1, and C24:0) in central nervous system (CNS) lipid rafts isolated without using detergent. Our UHPLC–MS/MS assay showed good accuracy and precision with a linear range of 5 to 1000nM for C18:0 and C24:1 sulfatides and 10 to 1000nM for C16:0 and C24:0 sulfatides. We applied this quantitative analysis to detergent-free lipid rafts isolated from wild-type mice and arylsulfatase A-deficient (ASA knockout) mice that accumulate sulfatides. All four sulfatide species were more abundant in raft membranes than in non-raft membranes, with a significant increase in lipid rafts isolated from ASA knockout mice. This is the first description of an analytical method to study these sulfatide species in raft and non-raft membranes and has the potential to be applied to preparations from other tissues.

Ultra-performance liquid chromatography/tandem mass spectrometry for determination of sulfatides in dried blood spots from patients with metachromatic leukodystrophy

Metachromatic leukodystrophy (MLD) is a genetic autosomal recessive disease caused by a deficiency in arylsulfatase A. Accumulated sulfatides can be detected in the urine and detection of sulfatiduria is a useful test for diagnosis and monitoring. To our knowledge, no studies have explored the accumulation of sulfatides in dried blood spots (DBSs). We developed an ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) method for measuring sulfatides in DBSs from patients with MLD. DBSs were eluted with internal standard. After mixing and centrifugation, the organic layer was transferred to a 96-well microplate and dried, then resuspended in methanol/propanol solution. Samples were analyzed on an UPLC system. Total running time was 4 min. Quantification was achieved by multiple reaction monitoring using a tandem mass spectrometer. We evaluated the precision, linearity, and ion suppression of the method and analyzed sulfatide concentrations in DBS specimens from MLD patients (n = 9), pseudodeficiency (PD) patient (n = 1), obligate heterozygotes (OH) (n = 2) and normal controls (n = 124). In negative-ion mode, sulfatides species subjected to collision-induced dissociation readily fragment to produce an intense ion at m/z 96.8 (HSO4(-)). The precisions of low and high concentration controls ranged from 5.4 to 19.9%. The sulfatides produced linear responses. Molecular species of sulfatides were barely detected in DBSs from normal individuals and the PD-OH group [mean (range), 0.07 (<0.05-0.34) and 0.13 (<0.05-0.22) µg/mL, respectively]. In contrast, the DBSs from MLD patients showed a marked increase in several molecular species of sulfatide [mean (range), 2.02 (1.18-3.89) µg/mL]. Simultaneous detection for sulfatides using UPLC/MS/MS can be successfully applied to DBS analysis. This method provides a fast and effective screening and monitoring tool for the diagnosis and treatment of MLD.

Newborn screening of mucopolysaccharidosis II (Hunter syndrome) and VI (Maroteaux-Lamy syndrome) using flow injection tandem mass spectrometry and newly developed reagents

sulfatides can be monitored both in positive and negative ion mode although with different sensitivity. Fragment ions in both ion modes are independent of the sulfatide acyl chain length and thus suitable for sulfatide profiling using selected reaction monitoring (SRM). We collected urine and blood samples from confirmed MLD patients and analyzed them in parallel. Our results show clearly that accumulation of sulfatide species in dried urine spots allows for the detection of MLD. Dried blood spots are probably not suitable for newborn screening or diagnosis since sulfatide levels were barely detectable and elevated in only a subset of MLD samples.

Alterations in Mouse Brain Lipidome after Disruption of CST Gene: A Lipidomics Study

To investigate the effects of a critical enzyme, cerebroside sulfotransferase (CST), involving sulfatide biosynthesis on lipid (particularly sphingolipid) homeostasis, herein, we determined the lipidomes of brain cortex and spinal cord from CST null and heterozygous (CST(-/-) and CST(+/-), respectively) mice in comparison to their wild-type littermates by multi-dimensional mass spectrometry-based shotgun lipidomics. As anticipated, we demonstrated the absence of sulfatide in the tissues from CST(-/-) mice and found that significant reduction of sulfatide mass levels was also present, but in an age-dependent manner, in CST(+/-) mice. Unexpectedly, we revealed that the profiles of sulfatide species in CST(+/-) mice were significantly different from that of littermate controls with an increase in the composition of species containing saturated and hydroxylated fatty acyl chains. Contrary to the changes of sulfatide levels, shotgun lipidomics analysis did not detect significant changes of the mass levels of other lipid classes examined. Taken together, shotgun lipidomics analysis demonstrated anticipated sulfatide mass deficiency in CST defect mouse brain and revealed novel brain lipidome homeostasis in these mice. These results might provide new insights into the role of CST in myelin function.

Correction of Brain Oligodendrocytes by AAVrh.10 Intracerebral Gene Therapy in Metachromatic Leukodystrophy Mice

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder characterized by accumulation of sulfatides in glial cells and neurons, the result of an inherited deficiency of arylsulfatase A (ARSA; EC 3.1.6.8) and myelin degeneration in the central and peripheral nervous systems. No effective treatment is currently available for the most frequent late infantile (LI) form of MLD, which results in rapid neurological degradation and early death after the onset of clinical manifestations. To potentially arrest or reverse disease progression, ARSA enzyme must be rapidly delivered to brain oligodendrocytes of patients with LI MLD. We previously showed that brain gene therapy with adeno-associated virus serotype 5 (AAV5) driving the expression of human ARSA cDNA under the control of the murine phosphoglycerate kinase (PGK) promoter alleviated most long-term disease manifestations in MLD mice. Herein, we evaluated the short-term effects of AAVrh.10 driving the expression of human ARSA cDNA under the control of the cytomegalovirus/β-actin hybrid (CAG/cu) promoter in 8-month-old MLD mice that already show marked sulfatide accumulation and brain pathology. Within 2 months, and in contrast to results with the AAV5-PGK-ARSA vector, a single intrastriatal injection of AAVrh.10cuARSA resulted in correction of brain sulfatide storage, accumulation of specific sulfatide species in oligodendrocytes, and associated brain pathology in the injected hemisphere. Better potency of the AAVrh.10cuARSA vector was mediated by higher neuronal and oligodendrocyte transduction, axonal transport of the AAVrh.10 vector and ARSA enzyme, as well as higher CAG/cu promoter driven expression of ARSA enzyme. These results strongly support the use of AAVrh.10cuARSA vector for intracerebral gene therapy in rapidly progressing early-onset forms of MLD.

The Detection of Glycosphingolipids in Brain Tissue Sections by Imaging Mass Spectrometry Using Gold Nanoparticles

Glycosphingolipids (GSLs) are amphiphilic molecules consisting of a hydrophilic carbohydrate chain and a hydrophobic ceramide moiety. They appear to be involved primarily in biological processes such as cell proliferation, differentiation, and signaling. To investigate the mechanism of brain function in more detail, a more highly sensitive method that would reveal the GSL distribution in the brain is required. In this report, we describe a simple and efficient method for mapping the distribution and localization of GSLs present in mouse brain sections using nanoparticle-assisted laser desorption/ionization imaging mass spectrometry (IMS). We have developed and tested gold nanoparticles (AuNPs) as a new matrix to maximize the detection of GSLs. A matrix of AuNPs modified with alkylamine was used to detect various GSLs, such as minor molecular species of sulfatides and gangliosides, in mouse brain sections; these GSLs were hardly detected using 2,5-dihydroxybenzoic acid (DHB), which is the conventional matrix for GSLs. We achieved approximately 20 times more sensitive detection of GSLs using AuNPs compared to a DHB matrix. We believe that our new approach using AuNPs in IMS could lead to a new strategy for analyzing basic biological mechanisms and several diseases through the distribution of minor GSLs.

Selective desorption/ionization of sulfatides by MALDI-MS facilitated using 9-aminoacridine as matrix

Recently, we used the favorable properties of 9-aminoacridine (9-AA) as matrix for the quantitative analysis of acidic metabolites and glycerophospholipids from extracts of biological materials [Sun, G., Yang, K., Zhao, Z., Guan, S., Han, X., and Gross, R.W. (2007) A shotgun metabolomics approach for rapid analysis of negatively-charged water-soluble cellular metabolites from mouse heart tissue. Anal. Chem. 79: 6629-6640; Sun, G., Yang, K., Zhao, Z., Guan, S., Han, X., and Gross, R.W. (2008) Matrix-assisted laser desorption/ionization-time of flight mass spectrometric analysis of cellular glycerophospholipids enabled by multiplexed solvent dependent analyte-matrix interactions. Anal. Chem. 80: 7576-7585.] by MALDI-MS. Herein, we extend this discovery and identified the selective desorption/ionization of sulfatides over other examined anionic lipids present in lipid extracts of biological samples by MALDI-MS using 9-AA as matrix. Through this approach, a high throughput method for the quantitative analysis of low to very low abundance sulfatide molecular species directly from crude lipid extracts has been developed. This method possessed a linear dynamic range of over 1,000-fold, a detection limit at the high attomole level, and a reproducibility of approximately 10% deviation. Many potential factors that might affect the quantitation of sulfatide species employing the method were examined and their effects were found to be negligible within experimental error. Collectively, these results demonstrate a powerful high throughput method for the measurement of sulfatides directly from extracts of biological samples, facilitating the study of sulfatide metabolism, trafficking, and homeostasis in health and disease.

Sulfatide with different fatty acids has unique distributions in cerebellum as imaged by Time-Of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS)

In order to elucidate the biological role of sulfatide it is important to define the cellular and subcellular distribution of its various molecular species (e.g. fatty acid chain length and hydroxylation). We determined sulfatide species distribution in the rat cerebellum using time-of-flight secondary ion mass spectrometry (TOF-SIMS). TOF-SIMS detects ions up to m/ z 10000 and enables simultaneous imaging of the lateral distribution of substances on an exposed surface, in this case a section through cerebellum. In addition to TOF-SIMS we analyzed sulfatide distribution in rat cerebellum using a sulfatide monoclonal antibody and confocal laser scanning microscopy. In the white matter, TOF-SIMS showed a uniform distribution of sulfatide with short chain fatty acids and a patchy distribution of sulfatide with C24 fatty acids. These patches had a low cholesterol signal. The granular layer showed a more uniform distribution of the sulfatide species, with the highest signal of C24. The molecular layer and Purkinje cells were devoid of sulfatide signals. Immunofluorescence showed the highest intensity in the white matter, lower intensity in the granular layer and absence of fluorescence in the molecular layer and Purkinje cells. The results are discussed in relation to previously published data and possible functional roles.

Establishment of a quantitative, qualitative, and high-throughput analysis of sulfatides from small amounts of sera by matrix-assisted laser desorption ionization–time of flight mass spectrometry

Based on our previous measurements of sulfatides, we further developed a quantitative, qualitative, and high-throughput analytical method for serum sulfatides as forms of lysosulfatides by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Using 0.1 N NaOH in 90% MeOH for saponification instead of absolute MeOH, as previously used, we succeeded in eliminating the formation of lysosulfatide artifacts, facilitating much more sensitive detection. The use of MonoTip C18 allowed quantitation of serum sulfatides from 100 50-μl serum specimens within 1 working day. Purification of lysosulfatides with MonoTip C18 also gave rise to clear MALDI-TOF MS spectra, allowing overall analysis of sphingoid molecular species of sulfatides in serum. The composition was as follows: d18:1 (61.3 ± 2.8%), d18:2 (13.3 ± 1.7%), t18:0 (11.8 ± 1.5%), d18:0 (7.6 ± 0.8%), d20:0 (3.0 ± 1.2%), t20:0 (2.3 ± 0.8%), and d20:1 (1.6 ± 0.5%). This is also the first detailed report on sphingoid molecular species of sulfatides in human serum. We believe that this method is suitable for daily clinical analysis of sulfatides in various clinical samples such as blood, urine, cerebrospinal fluid, and specimens from biopsies.

Rapid demonstration of diversity of sulfatide molecular species from biological materials by MALDI-TOF MS

By combining the partition method for enrichment of sulfatides without any chromatographic procedures and the preparation method of lysosulfatides, we succeeded in analyzing these sulfated glycosphingolipids from biological materials by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) to reduce the complexity of mass fragmentation patterns within a day. We found that sulfated GalCer (HSO3-3Gal beta 1Cer) (SM4s [galactosylsulfatide]) was composed of different species. While composition of SM4s specifically depended on source materials, it always contained hydroxy fatty acids of various degrees. In addition to the common sphingoid 4-sphingenine (d18:1), uncommon/unusual sphingoids phytosphingosine (4-hydroxysphinganine) (t18:0), eicosasphinganine (d20:0), 4-eicosasphingenine (d20:1), and sphingadienine (d18:2) were easily detected. Finally, in addition to SM4s, sulfatide sulfated LacCer (HSO3-3Gal beta 4Glc beta 1Cer) (SM3 [sulfated lactosylceramide]) and sulfated Gg3Cer (GalNAc beta 4(HSO3-3)Gal beta 4Glc beta 1Cer) (SM2 [sulfated gangliotriaosylceramide]) were clearly detected in renal tubule cells. The major SM4s was composed of ceramides possessing d18:1 with C22 hydroxy fatty acids (C22:0 h), C23:0 h, and C24:0 h, whereas the major SM3/SM2 were composed of ceramides possessing t18:0 with C22 normal fatty acids (C22:0), C23:0, C24:0. Namely, in these two series of sulfatides, either fatty acids or sphingoids were hydroxylated, and chain lengths of these components were exactly the same, consequently resulting in a similar polarity of ceramide moieties in these sulfatide species. These results demonstrated diversities of sulfatide molecular species, not only with respect to sugar moieties but also to ceramide moieties, which are probably important for specific effective functions in particular microenvironments such as lipid membrane microdomains.

Substantial sulfatide deficiency and ceramide elevation in very early Alzheimer's disease: potential role in disease pathogenesis.

In addition to pathology in the gray matter, there are also abnormalities in the white matter in Alzheimer's disease (AD). Sulfatide species are a class of myelin-specific sphingolipids and are involved in certain diseases of the central nervous system. To assess whether sulfatide content in gray and white matter in human subjects is associated with both the presence of Alzheimer's disease (AD) pathology as well as the stage of dementia, we analyzed the sulfatide content of brain tissue lipid extracts by electrospray ionization mass spectrometry from 22 subjects whose cognitive status at time of death varied from no dementia to very severe dementia. All subjects with dementia had AD pathology. The results demonstrate that: (i) sulfatides were depleted up to 93% in gray matter and up to 58% in white matter from all examined brain regions from AD subjects with very mild dementia, whereas all other major classes of lipid (except plasmalogen) in these subjects were not altered in comparison to those from age-matched subjects with no dementia; (ii) there was no apparent deficiency in the biosynthesis of sulfatides in very mild AD subjects as characterized by the examination of galactocerebroside sulfotransferase activities in post-mortem brain tissues; (iii) the content of ceramides (a class of potential degradation products of sulfatides) was elevated more than three-fold in white matter and peaked at the stage of very mild dementia. The findings demonstrate that a marked decrease in sulfatides is associated with AD pathology even in subjects with very mild dementia and that these changes may be linked with early events in the pathological process of AD.

Analysis of sulfatide from rat cerebellum and multiple sclerosis white matter by negative ion electrospray mass spectrometry

The accumulation of sulfatide (sulfatogalactosyl cerebroside) and changes in the sulfatide species present have been examined in the cerebellum of day 6–32 aged rats and in multiple sclerosis (MS) tissue samples. Negative ion electrospray mass spectrometry with daughter and parent ion analyses were used to distinguish the fatty acyl character in the amide linkage of sulfatide; measurement was done by selected ion and multiple reaction monitoring of individually identified sulfatide molecules. Sulfatide accumulation in rat cerebellum shows that 18:0- and hydroxylated 18:0-sulfatide are the first sulfatide molecules detectable. Very long fatty acyl chain sulfatide molecules (>20:0) are present at day 7 and the ratio of non-hydroxylated compared to hydroxylated sulfatide rises as the amount of non-hydroxylated sulfatide increases. 24:1-sulfatide accumulates at a ratio of about 3:1 over 24:0-sulfatide during active myelination. Analyses of the sulfatide in human tissue have shown differences between MS plaque tissues, normal appearing adjacent white matter and control tissues. The findings show that total sulfatide is reduced by 60% in the plaque matter and decreased 25% in adjacent normal appearing white matter. There are significant increases ( P=0.05) in the amount of hydroxylation of sulfatide, demonstrated by an increase in the percentage of hydroxylated h24:0-sulfatide (hydroxy-lignoceroyl sulfatide).

Electrospray ionization tandem mass spectrometric analysis of sulfatide.: Determination of fragmentation patterns and characterization of molecular species expressed in brain and in pancreatic islets

The sphingolipid sulfatide is a component of myelin and some non-neuronal cells. Antibodies to sulfatide occur in some patients with autoimmune neuropathies and in patients with insulin-dependent diabetes mellitus (IDDM) caused by immunologic destruction of insulin-secreting pancreatic islet β-cells. Distinct sulfatide molecular species may differ in immunogenicity, and facile means to identify sulfatide species in islets and other tissues obtainable in only small amounts could be useful. Electrospray ionization mass spectrometry (ESI/MS) permits structural determination of small quantities of phospholipids and is applied here to sulfatide analysis. We find that sulfatide standards are readily analyzed by negative ion ESI/MS, and tandem mass spectra of individual species exhibit some ions common to all species and other ions that reflect distinct fatty acid substituents in different sulfatide molecules. A signature ion cluster resulting from cleavage directed by the α-hydroxy group of sulfatide species with a hydroxylated fatty acid substituent identifies such species. Sulfatide profiles in tissue lipid extracts can be obtained by ESI/MS/MS scanning for common sulfatide ions and for ions reflecting fatty acid substituents. Islets are demonstrated to contain sulfatide and to exhibit a profile of species different from that of brain.

The lipid composition of the electric organ of the ray, Torpedo marmorata, with specific reference to sulfatides and Na+-K+-ATPase.

The lipids from the electric organ of the ray, Torpedo marmorata, have been isolated and characterized. The major lipids were cholesterol, choline phospholipids, ethanolamine phospholipids, and sphingomyelins. The major fatty acids of ethanolamine phospholipids were 18:1, 18:0, 22:6, and 20:4. More than 50% of the acids in choline phospholipids were 16:0. The sphingomyelins consisted of five major ceramide species, all with sphingosine and the fatty acids 14:0, 15:0, 16:0, 22:1, and 24:1. The fatty acid 15:0 was mostly branched (n-2), a fatty acid earlier identified in sphingomyelins of the rectal gland of spiny dogfish. All long-chain bases were dihydroxy bases with a small percentage of branched chains. Sulfatides (cerebroside sulfate) made up the largest glycolipid fraction. The polar moiety wase galactose-3-sulfate. The fatty acids were normal and 2-hydroxy; the homologue 24:1 was the most abundant in both types of fatty acids. Most fatty acids were higher homologues of mono-unsaturated acids, but normal 18:0 fatty acid was also found. The long-chain bases were both dihydroxy and trihydroxy, with very small amounts of branched chains. The two major ceramide species of sulfatides were sphingosine combined with normal and hydroxy 24:1 fatty acids, respectively. Smaller amounts of trihydroxy base (18:0) were found linked to hydroxy 24:1 fatty acid, but not to its normal homologue. The cerebrosides contained the two major species mentioned above but lacked the trihydroxy base-hydroxy fatty acid species. The ratio of the activity of Na+-K+-dependent ATPase (EC 3.6.1.3) and the concentration of sulfatides was similar to ratios found for other tissues with normal and increased Na+ and K+ transporting capacity. The significance of this finding is discussed.