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 0.1N 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-mul 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.     

Determination of urinary sulfatides and other lipids by combination of reversed-phase and thin-layer chromatographies

A fast and simple method for determination of sulfatides in the urine of patients with metachromatic leukodystrophy (MLD, arylsulfatase A deficiency) has been developed. The procedure consists of two steps: extraction of total urinary lipids by reversed-phase chromatography and their HPTLC separation. Two types of sorbents based on different matrixes were compared, of which the hydroxyethyl methacrylate C-18 type sorbent was found to be superior. Twenty-milliliter aliquots of urine are sufficient for the analysis. The technique is appropriate for simultaneous qualitative identification and semiquantitative densitometric determination and is suitable for routine work. The amount of sulfatides is expressed in relation to sphingomyelin, which copurifies with sulfatides and better reflects the level of membrane lipids in urine than commonly used parameters (creatinine, urine volume, etc.). The ranges were found to be 0.15-0.68 nmol sulfatide/nmol sphingomyelin for control individuals and 3.5-27.2 nmol sulfatide/nmol sphingomyelin for MLD patients. The excretion of sulfatides is pathonognomic for true MLD (due to the accumulation in kidney) and therefore its analysis is important for evaluation of suspected MLD cases including clinically and enzymatically atypical cases. The method is also useful as a complementary analysis for other lipidoses with high excretion of sphingolipids in urine (e.g., Fabry disease).     

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.     

Sulfogalactosylceramides in motor and psycho-cognitive adult metachromatic leukodystrophy: relations between clinical, biochemical analysis and molecular aspects

Metachromatic leukodystrophy (MLD) is a human autosomal recessive lysosomal neurodegenerative disorder that results from the accumulation of sulfatides in the central and peripheral nervous system. It is due to the enzyme deficiency of the sulfatide sulfatase i.e. arylsulfatase A (ASA). During adolescence and/or adulthood, there are 2 clinical presentations. It may be that of a degenerative disease of the central nervous system with mainly spastic manifestations or a spino-cerebellar ataxia, or that of a psychosis. As several lines of evidence indicate that the psychotic form of MLD could be a model of psychosis, we decided to do a pluridisciplinary study on 11 psycho-cognitive cases involving mental and psychiatric testing, in comparison with 5 adult motor cases, a biochemical study with enzyme assays and quantitative mass spectrometry of urinary sulfatides, so as to determine whether there were biochemical particularities related to the psychotic forms. For quantitative mass spectrometry (MS), a non physiological sulfatide with C17:0 fatty acid was synthesized. The major sulfatide isoforms were present in the 2 clinical forms with the following fatty acids and sphingoid bases: C22:1/d18:1, and /or C22:0/d18:2 (m/z 862.5), C22:0 (OH)/d18:1 (m/z 878,5), C24:0/d18:1 and / or C24:0/C23:1(OH)/d18:2 (m/z 890,3), C24:0 (OH)/d18:1(m/z 906.5). We had shown previously that there were different ASA mutations in the psychiatric adult form (heterozygous I179S) versus the adult motor form (homozygous P426L). We show here that there were no relations with the level of ASA and with the mass spectrometric study of the sulfatide isoforms which were identical in the 2 clinical forms.     

Renal sulfatides: sphingoid base-dependent localization and region-specific compensation of CerS2-dysfunction

Mammalian kidneys are rich in sulfatides. Papillary sulfatides, especially, contribute to renal adaptation to chronic metabolic acidosis. Due to differences in their cer­amide (Cer) anchors, the structural diversity of renal sulfatides is large. However, the underling biological function of this complexity is not understood. As a compound’s function and its tissue location are intimately connected, we analyzed individual renal sulfatide distributions of control and Cer synthase 2 (CerS)2-deficient mice by imaging MS (IMS) and by LC-MS² (in controls for the cortex, medulla, and papillae separately). To explain locally different structures, we compared our lipid data with regional mRNA levels of corresponding anabolic enzymes. The combination of IMS and in source decay-LC-MS² analyses revealed exclusive expression of C20-sphingosine-containing sulfatides within the renal papillae, whereas conventional C18-sphingosine-containing compounds were predominant in the medulla, and sulfatides with a C18-phytosphingosine were restricted to special cortical structures. CerS2 deletion resulted in bulk loss of sulfatides with C23/C24-acyl chains, but did not lead to decreased urinary pH, as previously observed in sulfatide-depleted kidneys. The reasons may be the almost unchanged C22-sulfatide levels and constant total renal sulfatide levels due to compensation with C16- to C20-acyl chain-containing compounds. Intriguingly, CerS2-deficient kidneys were completely depleted of phytosphingosine-containing cortical sulfatides without any compensation.     

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.     

Acute kidney injury induced by protein-overload nephropathy down-regulates gene expression of hepatic cerebroside sulfotransferase in mice, resulting in reduction of liver and serum sulfatides

Sulfatides, possible antithrombotic factors belonging to sphingoglycolipids, are widely distributed in mammalian tissues and serum. We recently found that the level of serum sulfatides was significantly lower in hemodialysis patients than that in normal subjects, and that the serum level closely correlated to the incidence of cardiovascular disease. These findings suggest a relationship between the level of serum sulfatides and kidney function; however, the molecular mechanism underlying this relationship remains unclear. In the present study, the influence of kidney dysfunction on the metabolism of sulfatides was examined using an established murine model of acute kidney injury, protein-overload nephropathy in mice. Protein-overload treatment caused severe proximal tubular injuries within 4 days, and this treatment obviously decreased both serum and hepatic sulfatide levels. The sphingoid composition of serum sulfatides was very similar to that of hepatic ones at each time point, suggesting that the serum sulfatide level is dependent on the hepatic secretory ability of sulfatides. The treatment also decreased hepatic expression of cerebroside sulfotransferase (CST), a key enzyme in sulfatide metabolism, while it scarcely influenced the expression of the other sulfatide-metabolizing enzymes, including arylsulfatase A, ceramide galactosyltransferase, and galactosylceramidase. Pro-inflammatory responses were not detected in the liver of these mice; however, potential oxidative stress was increased. These results suggest that down-regulation of hepatic CST expression, probably affected by oxidative stress from kidney injury, causes reduction in liver and serum sulfatide levels. This novel mechanism, indicating the crosstalk between kidney injury and specific liver function, may prove useful for helping to understand the situation where human hemodialysis patients have low levels of serum sulfatides.     

Apolipoprotein E genotype and LRP1 polymorphisms in patients with different clinical types of metachromatic leukodystrophy

Metachromatic leukodystrophy (MLD) is a severe, neurodegenerative, metabolic disease which is caused by deficient activity of arylsulfatase A (ARSA). Sulfatides and other substrates of ARSA are stored in central and peripheral nervous systems, and in some other organs. Accumulated sulfatides are especially toxic to oligodendrocytes and Schwann cells leading to progressive demyelination. The kind of apolipoprotein E (apoE) isoform is of essential significance for the modulation of sulfatide quantity in the brain as apoE4 contains more sulfatides than apoE3. Taking into consideration the fact that apoE4 leads to the loss of sulfatides in CSF of Alzheimer's disease patients, we examined if apoE isoforms display any impact on clinical outcome in patients with different forms of MLD in whom sulfatides accumulate. The significant association of age at the onset of MLD symptoms with APOE ε2/ε3/ε4 and LRP1 c.766C>T polymorphisms was shown in multivariate stepwise regression analysis, in which other factors known to affect age at onset of the disease, i.e. clinical type of MLD, family connection of the patient and sex were also analyzed. As expected, the clinical type of MLD explained about 80% of the variance of the dependent variable. The impact of both polymorphisms on age of onset of the disease was considerably lower: 2.0% in the case of APOE polymorphism and 1.0% in the case of LRP1 polymorphism. Thus, the clinical outcome in MLD patients is related principally to the genotype of the ARSA gene, while the polymorphisms in the APOE and LRP1 genes are only slightly modifying factors.     

Multi-dimensional mass spectrometry-based shotgun lipidomics and the altered lipids at the mild cognitive impairment stage of Alzheimer's disease

Multi-dimensional mass spectrometry-based shotgun lipidomics (MDMS-SL) is a well-developed technology for global lipid analysis, which identifies and quantifies individual lipid molecular species directly from lipid extracts of biological samples. By using this technology, we have revealed three marked changes of lipids in brain samples of subjects with mild cognitive impairment of Alzheimer's disease including sulfatides, ceramides, and plasmalogens. Further studies using MDMS-SL lead us to the identification of the potential biochemical mechanisms responsible for the altered lipids at the disease state, which are thoroughly discussed in this minireview. Specifically, in studies to identify the causes responsible for sulfatide depletion at the mild cognitive impairment stage of Alzheimer's disease, we have found that apolipoprotein E is associated with sulfatide transport and mediates sulfatide homeostasis in the nervous system through lipoprotein metabolism pathways and that alterations in apolipoprotein E-mediated sulfatide trafficking can lead to sulfatide depletion in the brain. Collectively, the results obtained from lipidomic analyses of brain samples provide important insights into the biochemical mechanisms underlying the pathogenesis of Alzheimer's disease.     

The Pathogenic Implication of Abnormal Interaction Between Apolipoprotein E Isoforms, Amyloid-beta Peptides, and Sulfatides in Alzheimer’s Disease

Alzheimer’s disease (AD) is the most common cause of dementia in the aging population. Prior work has shown that the ε4 allele of apolipoprotein E (apoE4) is a major risk factor for “sporadic” AD, which accounts for >99% of AD cases without a defined underlying mechanism. Recently, we have demonstrated that sulfatides are substantially and specifically depleted at the very early stage of AD. To identify the mechanism(s) of sulfatide loss concurrent with AD onset, we have found that: (1) sulfatides are specifically associated with apoE-associated particles in cerebrospinal fluid (CSF); (2) apoE modulates cellular sulfatide levels; and (3) the modulation of sulfatide content is apoE isoform dependent. These findings not only lead to identification of the potential mechanisms underlying sulfatide depletion at the earliest stages of AD but also serve as mechanistic links to explain the genetic association of apoE4 with AD. Moreover, our recent studies further demonstrated that (1) apoE mediates sulfatide depletion in amyloid-β precursor protein transgenic mice; (2) sulfatides enhance amyloid β (Aβ) peptides binding to apoE-associated particles; (3) Aβ42 content notably correlates with sulfatide content in CSF; (4) sulfatides markedly enhance the uptake of Aβ peptides; and (5) abnormal sulfatide-facilitated Aβ uptake results in the accumulation of Aβ in lysosomes. Collectively, our studies clearly provide a link between apoE, Aβ, and sulfatides in AD and establish a foundation for the development of effective therapeutic interventions for AD.     

Endosomes and lysosomes play distinct roles in sulfatide-induced neuroblastoma apoptosis: potential mechanisms contributing to abnormal sulfatide metabolism in related neuronal diseases

Alterations in sulfatide metabolism, trafficking and homoeostasis are present at the earliest clinically recognizable stages of Alzheimer's disease and are associated with metachromatic leukodystrophy. However, the role of sulfatide in these disease states remains unknown. In the present study, we investigated the sequelae of NB (neuroblastoma) cells upon sulfatide supplementation and the biochemical mechanisms contributing to the sulfatide-induced changes. By using shotgun lipidomics, we showed dramatic accumulations of sulfatide, ceramide and sphingosine in NB cells in a time- and dose-dependent manner. Further studies utilizing subcellular fractionation and shotgun lipidomics analyses demonstrated that most of the increased ceramide content was generated in the endosomal compartment, whereas sulfatides predominantly accumulated in lysosomes. In addition, we determined that the sulfatide-mediated increase in endosomal ceramide content mainly resulted from beta-galactosidase activity, which directly hydrolyses sulfatide to ceramide without a prior desulfation step. Substantial cell apoptosis occurred in parallel with the accumulation of sulfatides and ceramides, as revealed by mitochondrial membrane depolarization, by phosphatidylserine translocation and by the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) assay. These findings were also demonstrated with primary neuron cultures. Collectively, our results demonstrate that abnormal sulfatide metabolism can induce cell apoptosis due to endosome-mediated ceramide generation and the accumulation of cytotoxic levels of sulfatides in lysosomes.     

Brain Galactolipid Content in a Patient with Pseudo-Arylsulfatase A Deficiency and Coincidental Diffuse Disseminated Sclerosis, and in Patients with Metachromatic, Adreno-, and Other Leukodystrophies

A 4-year old boy died of diffuse disseminated sclerosis (DDS) of the brain and was found to have also pseudoarylsulfatase A deficiency (PASAD) with about 20% residual arylsulfatase A (ASA) and cerebroside sulfatase (CS) activity. The reexamination of lipids did not show any sulfatide accumulation in the patient's organ extracts. Although the residual CS activity in the patient's extracts was clearly demonstrable only after partial purification, it was concluded that this activity protects organ tissues from sulfatide accumulation in PASAD, since in sulfatide lipidosis (metachromatic leukodystrophy, MLD) no residual CS activity was detectable. The study of residual ASA activity in the patient's fibroblasts by gel electrofocusing resulted in an almost normal enzyme microheterogeneity. However, the detailed study of the brain galactolipids in the patient revealed an elevated ratio of sulfatide/galactocerebroside content, despite the decrease of both lipids. In tissues of other patients with severe demyelinating diseases different from DDS and MLD, this galactolipid ratio was also found to be increased, especially in three patients with adrenoleukodystrophy. A general mechanism of this anomaly in severe demyelination is considered.     

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).     

Roles of galactose and sulfate residues in sulfatides for their antagonistic functions in the blood coagulation system

We previously reported that the sulfatide (galactosylceramide I³-sulfate) may have contradictory functions, namely both coagulant and anticoagulant roles in vivo: sulfatide induced giant thrombi formation when injected into rats with vein ligation, whereas no thrombi were formed when sulfatide was injected into rats without vein ligation. Rather it prolonged bleeding time. To investigate the structural features of sulfatide for both functions, a synthetic sulfatide (galactosylceramide I⁶-sulfate) which does not occur naturally, cholesterol 3-sulfate and ganglioside GM4 were examined together with naturally occurring sulfatide. Both sulfatides and cholesterol 3-sulfate induced giant thrombi in the rats with vein ligation within ten minutes of injection, although cholesterol 3-sulfate exhibited weaker coagulant activity than the sulfatides. On the contrary, both sulfatides significantly prolonged bleeding time but cholesterol 3-sulfate barely prolonged it when injected without vein ligation. GM4 exhibited neither coagulant nor anticoagulant activity. These results suggested that sulfate moiety in the sulfatides is essential for coagulant activity and that galactose residue enhances the activity, whereas both galactose and sulfate residues seem to be important for anticoagulant activity. This is because the sulfatides possess both residues but GM4 possesses galactose without sulfate and cholesterol 3-sulfate possesses sulfate without galactose. We previously reported that the possible mechanism of anticoagulation by sulfatide was due to its binding to fibrinogen, thereby inhibiting the conversion to fibrin. In this paper we reveal that both sulfatides inhibited thrombin activity independent of heparin cofactor II, thus providing evidence of another anticoagulation mechanism for the sulfatides.     

Deletion of fatty acid amide hydrolase reduces lyso-sulfatide levels but exacerbates metachromatic leukodystrophy in mice

An inherited deficiency of arylsulfatase A (ASA) causes the lysosomal storage disease metachromatic leukodystrophy (MLD) characterized by massive intralysosomal storage of the acidic glycosphingolipid sulfatide and progressive demyelination. Lyso-sulfatide, which differs from sulfatide by the lack of the N-linked fatty acid, also accumulates in MLD and is considered a key driver of pathology although its concentrations are far below sulfatide levels. However, the metabolic origin of lyso-sulfatide is unknown. We show here that ASA-deficient murine macrophages and microglial cells express an endo-N-deacylase that cleaves the N-linked fatty acid from sulfatide. An ASA-deficient astrocytoma cell line devoid of this activity was used to identify the enzyme by overexpressing 13 deacylases with potentially matching substrate specificities. Hydrolysis of sulfatide was detected only in cells overexpressing the enzyme fatty acid amide hydrolase (FAAH). A cell-free assay with recombinant FAAH confirmed the novel role of this enzyme in sulfatide hydrolysis. Consistent with the in vitro data, deletion of FAAH lowered lyso-sulfatide levels in a mouse model of MLD. Regardless of the established cytotoxicity of lyso-sulfatide and the anti-inflammatory effects of FAAH inhibition seen in mouse models of several neurological diseases, genetic inactivation of FAAH did not mitigate, but rather exacerbated the disease phenotype of MLD mice. This unexpected finding was reflected by worsening of rotarod performance, increase of anxiety-related exploratory activity, aggravation of peripheral neuropathy, and reduced life expectancy. Thus, we conclude that FAAH has a protective function in MLD and may represent a novel therapeutic target for treatment of this fatal condition.     

Age-dependent PPARα activation induces hepatic sulfatide accumulation in transgenic mice carrying the hepatitis C virus core gene

Sulfatides, a type of glycosphingolipid, are associated with carcinogenesis. Peroxisome proliferator-activated receptor α (PPARα) is involved in the regulation of sulfatide metabolism as well as in cancer development. We previously reported that transgenic (Tg) mice expressing hepatitis C virus core protein (HCVcp) exhibited age-dependent PPARα activation and carcinogenesis in liver. However, the metabolism of sulfatides in hepatocellular carcinoma is unknown. To examine the relationship between sulfatide metabolism, carcinogenesis, HCVcp, and PPARα, age-dependent changes of these factors were examined in HCVcpTg, PPARα inhibitor-treated HCVcpTg, and non-Tg mice. The sulfatide content in liver, the hepatic expression of two key enzymes catalyzing the initial and last reactions in sulfatide synthesis, the hepatic expression of known sulfatide-transferring protein, oxidative stress, and hepatic PPARα expression and its activation were age-dependently increased in HCVcpTg mice. The increased synthesis and accumulation of sulfatides and PPARα activation were significantly enhanced in liver cancer lesions. These changes were attenuated by PPARα inhibitor treatment and not observed in non-Tg mice. These results suggest that HCVcp-induced age-dependent PPARα activation increases synthesis of sulfatides and the resulting sulfatide accumulation affects HCV-related liver cancer. The monitoring of hepatic sulfatide content and the modulation of sulfatide generation by intervention using a PPARα inhibitor might be useful for the prediction and prevention of HCV-related hepatocarcinogenesis, respectively.     

A quantitative analysis of serum sulfatide by matrix-assisted laser desorption ionization time-of-flight mass spectrometry with delayed ion extraction.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry with delayed ion extraction (DE MALDI-TOF MS) was applied for the first time for the quantitation of sulfatide content in serum at the picomole level. The total lipids extracted by n-hexane:isopropanol (3:2, v/v) from 100 microliter of serum were saponified to convert sulfatide to its lyso form, and then the lysosulfatide was directly determined using DE MALDI-TOF MS in the presence of other degraded lipids. Hydrogenated N-acetyl lysosulfatide was used as an internal standard. The relative peak height of sulfatide was calculated and plotted versus its contents. This plot showed linearity between 2 pmol and 1 nmol of sulfatide (regression coefficient r > 0.95). Sulfatide contents of normal human sera and rabbit serum were quantitated by this method. The results corresponded well to the reported data determined by gas-liquid chromatography. This new approach was found to be sensitive, convenient, and reliable. It is expected to be applied to quantitate sulfatide from other small amounts of body fluids or tissues and to clinical examination. It is also expected to be applicable to quantitate other glycosphingolipids.     

Structure and composition of sulfatides isolated from livers of patients with metachromatic leukodystrophy: galactosyl sulfatide and lactosyl sulfatide

The livers of four patients with metachromatic leukodystrophy contained galactosyl sulfatide and lactosyl sulfatide, whereas these substances were undetectable in normal human liver. On the basis of methanolysis and permethylation studies, both sulfatides were shown to be substituted with sulfate at the C-3 position of the galactose moiety. Examination of the fatty acid compositions of these sulfatides showed that C(22:0) and higher 2-hydroxy and nonhydroxy fatty acids predominated in both. Both sulfatides contained the same long-chain bases, predominantly sphingosine, dihydrosphingosine, and phytosphingosine. Using as criteria the proportion of lactosyl sulfatide to galactosyl sulfatide, and the fatty acid and long-chain base compositions, the liver sulfatides from subjects with metachromatic leukodystrophy closely resemble those in the kidney and differ from those in brain and peripheral nerve.     

Sulfatides facilitate apolipoprotein E-mediated amyloid-beta peptide clearance through an endocytotic pathway

Amyloid-β (Aβ) accumulation and fibril formation are key pathologic characteristics of Alzheimer's disease (AD). We have previously found that sulfatide depletion occurs at the earliest stages of AD. To further identify the role of sulfatides in the pathogenesis of AD as well as the interactions between apolipoprotein E (apoE), sulfatides, and Aβ peptides, we examined alterations in the clearance of apoE-mediated Aβ peptides after sulfatide supplementation to cell culture systems. We demonstrated that sulfatides markedly facilitate apoE-mediated clearance of Aβ peptides endogenously generated from H4-APPwt cells through an endocytotic pathway. Moreover, we found that the uptake of Aβ42 mediated by sulfatides was selective in comparison to that of Aβ40. We excluded the possibility that the supplementation of sulfatides and/or apoE altered the production of Aβ peptides from H4-APPwt cells through determination of the clearance of Aβ peptides from conditioned H4-APPwt cell media by neuroblastoma cells which do not appreciably generate Aβ peptides. Finally, we demonstrated that the sulfate galactose moiety of sulfatides is essential for the sulfatide-facilitated clearance of Aβ peptides. Collectively, the current study provides insight into a molecular mechanism leading to Aβ clearance/deposition, highlights the significance of sulfatide deficiency at the earliest clinically recognizable stage of AD, and identifies a potential new direction for therapeutic intervention for the disease.