Glycobiology Advance Access originally published online on May 2, 2006
Glycobiology 2006 16(8):719-728; doi:10.1093/glycob/cwj122
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Rapid demonstration of diversity of sulfatide molecular species from biological materials by MALDI-TOF MS
2 Division of Molecular Pathology, Aichi Cancer Center Research Institute, 1-1, Kanokoden Chikusa-ku, Nagoya, Aichi, 464-8681, Japan; 3 Department of Histopathology and 4 Department of Metabolic Regulation, Institute on Aging and Adaptation, Shinshu University Graduate School of Medicine, 3-1-1, Asahi, Matsumoto, Nagano, 390-8621, Japan
1 To whom correspondence should be addressed; e-mail: mkyogashi{at}aichi-cc.jp
Received on January 28, 2006; revised on April 24, 2006; accepted on April 25, 2006
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Abstract |
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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 ß 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 ß 4Glc ß 1Cer) (SM3 [sulfated lactosylceramide]) and sulfated Gg3Cer (GalNAc ß 4(HSO3-3)Gal ß 4Glc ß 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 (C22:0h), C23:0h, and C24:0h, 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.
Key words: biological materials / lysosulfatides / MALDI-TOF MS / sphingoid / sulfatides
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementReferences |
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Sulfoglycolipids known as sulfatides have important physiological and pathological roles (Ishizuka, 1997
). In the nervous system, sulfatide sulfated GalCer (HSO3-3Gal ß 1Cer) (SM4s [sulfated galactosylceramide]) is well known as a critical component of myelin. Abnormal accumulation of SM4s induces demyelination in patients with metachromatic leukodystrophy (Gieselmann et al., 2003), and recently, significant depletion of SM4s in patients with Alzheimer’s disease has been reported (Han et al., 2002). Sulfatides are also known as adhesive molecules that bind P- (Aruffo et al., 1991) and L-selectins (Suzuki et al., 1993), and many proteins in the extracellular matrix such as laminin (Roberts, Rao, et al., 1985), thrombospondin-1 (Roberts, Haverstick, et al., 1985), and von Willebrand factor (Roberts et al., 1986), suggesting their potential roles in invasion and metastasis in cancer cells. Indeed, various cancer types not only express simple sulfatides such as SM4s (Yoda et al., 1979; Kiguchi et al., 1992) and sulfated LacCer (HSO3-3Gal ß 4Glc ß 1Cer) (SM3 [sulfated lactosylceramide]) (Sakakibara et al., 1989; Kuboshiro et al., 1992; Wu et al., 2004) in high amounts but also express complicated sulfatides (Hiraiwa et al., 1988, 1990). In the cardiovascular system, SM4s is the major glycosphingolipid of serum lipoproteins (Hara and Taketomi, 1987) and highly accumulates in the walls of atheromatous arteries (Hara and Taketomi, 1991). It can exhibit thrombogenic and paradoxically anti-thrombogenic activities under certain conditions (Kyogashima et al., 1998; Kyogashima, 2004). With regard to the immune system, SM4s was reported to bind to serious pathogens like HIV (Harouse et al., 1995) or Helicobacter pyroli (Saitoh et al., 1991) and was found to be presented on CD1a as a self-antigen (Zajonc et al., 2003). Furthermore, SM4s was reported to play roles in the processing and secretion of insulin from pancreatic islets (Blomqvist et al., 2003). Thus, sulfatides are probably involved in many diseases, and rapid, accurate, high-throughput applicable analyses to determine sulfatide species from small amounts of biological samples are desired. Immunological approaches such as enzyme-linked immunosorbent assay/radioimmunoassay (ELISA/RIA) have been previously used, but specificities of antibodies against sulfatides, especially against SM4s, are sometimes questionable, because some IgM antibodies nonspecifically bind to SM4s (Roberts, 1987). Furthermore, antibodies cannot characterize different ceramide species of sulfatides. In colon cancers, for example, a limited number of SM4s species have been suggested to correlate with lymph node metastasis (Morichika et al., 1996). In multiple sclerosis, increase of a particular species possessing C24:0h fatty acid was reported, while total amount of SM4s in myelin decreased (Marbois et al., 2000). In diabetic mice, it was reported that SM4s possessing 16:0 nonhydroxy fatty acid (C16:0) was specifically absent (Blomqvist et al., 2003). Thus, it is critical to characterize species of sulfatides with regard to ceramide moieties and their carbohydrate moieties.
Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a powerful tool that has been used to characterize glycosphingolipids including sulfatide (Harvey, 1995; Taketomi and Sugiyama, 2000). Furthermore, this technique provides very short analytical time, and many samples can be analyzed using sample plates with 100 loading positions resembling microtiter plates. These conditions are very critical for high-throughput analysis, especially for daily analysis of clinical samples. We previously introduced simple and rapid preparations of both sulfatide SM4s (Hara and Radin, 1979) and lysosulfatide SM4s (Sugiyama et al., 1999). Here, using MALDI-TOF MS, we further developed methods for the analysis of sulfatides not only SM4s but also SM3 and sulfated Gg3Cer (GalNAc ß 4(HSO3-3)Gal ß 4Glc ß 1Cer) (SM2) from small amounts of biological materials by combining with these two indispensable preparative methods and revealed distinct profiles for sulfatide species.
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Results |
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SM4s and lysoSM4s from spinal cord, lung, colon, and stomach in rats
Peaks for sulfatide SM4s from spinal cord, stomach, lung, and colon from rats were adequately detected, although those from the latter two are not shown, indicating that our preparative partition method by using a mixture of hexane, 2-propanol, water, and alkali resulted in successful SM4s analysis by MALDI-TOF MS (Figure 1).
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Peaks at 888.6, 890.6, 904.6, and 906.6 were detected as major peaks, in addition to peaks at 862.6 and 878.6 in spinal cord and lung samples, and exhibited similar mass patterns. Small peaks at 806.5, 822.5, 834.6, and 918.6 were also detected from spinal cord samples. In the colon, characteristic peaks at 868.6 and 924.6 were detected with additional peaks at 862.6, 888.6, 890.6, 904.6, and 906.6. A very peculiar pattern was observed for stomach samples. Indeed, although the commonly found peak at 906.6 was prominent, peaks at 888.6 and 890.6, both being major in other organs, were absent or scarce. In addition, the peak at 878.6 was higher, and peaks at 778.5 and 794.5 were specific to this organ. Figure 2 illustrates representatively chemical preparation of lysoSM4s from the colon and the stomach. For all organs, peak at 540.3 was detected as a major component possessing sphingoid of 4-sphingenine (d18:1), which is a sole component of sphingoid of SM4s from the lung. In the spinal cord, lysoSM4s possessing sphinganine (d18:0) was expected because of a slightly larger area of the 542.3 peak compared to that of the theoretically calculated heteroisotopic peak at 542.3, solely derived from lysoSM4s possessing d18:1 in the spinal cord (data not shown). Thus, sphingoid composition in lysoSM4s of the spinal cord was calculated to be 93% of d18:1, and 7% of d18:0, on the basis of peak area data. Additional peaks at 558.3 and 570.3 were detected in the colon sample and were assigned to derive from lysoSM4s possessing phytosphingosine (4-hydroxysphinganine) (t18:0) and eicosasphinganine (d20:0), respectively. A small but definite peak at 538.3 was detected in the stomach sample and was assigned to be from sphingadienine (d18:2), which has been recently reported in mouse brain (Colsch et al., 2004) (Figure 2).
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Together with the results of lysoSM4s, we assigned peaks of intact SM4s to most likely correspond to individual SM4s species by using data of percentages of relative peak areas (Table I). Species possessing d18:2, d18:0, and d20:0 were omitted because of their limited occurrence and lower incidence (Figure 2). Peaks at 888.6, 890.6, 904.6, and 906.6 corresponded to SM4s of d18:1-C24:1, d18:1-C24:0, d18:1-C24:1h, and d18:1-C24:0h, respectively, and these were major species in spinal cord, lung, and colon. The colon contained characteristic species of t18:0-C22:0h and t18:0-C24:0h. The stomach contained SM4s with hydroxy fatty acids such as d18:1-C16:0h, d18:1-C22:0h, d18:1-C23:0h, and d18:1-C24:0h with very little of d18:1-C24:0 and undetectable amounts of d18:1-C24:1, showing a very unique pattern.
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SM4s, SM3, and SM2, and their lyso-forms from renal tubule cells in rats
Next, we applied the method for 10–40 mg of wet weight tissue from rat renal tubule cells, for preparation of sulfatide fractions using the partition method, that were further analyzed by thin-layer chromatography (TLC) (Figure 3). It should be noted that this single partition method could effectively eliminate cholesterol, sphingomyelin, and alkali labile glycerophospholipids together with large amounts of fatty acids derived from alkali-treated glycerophospholipids, which obstructively migrated in the solvent front. From the sulfatide fraction obtained, we detected peaks corresponding to sulfatides SM4s, SM3, and SM2 (sulfated gangliotriaosylceramides) (Figure 4), indicating that the partition procedure, originally introduced for the preparation of SM4s from large amounts of brain samples, was applicable not only for SM4s but also for complicated sulfatides such as SM3 and SM2.
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In all types of sulfatides, three prominent peaks with a constant single mass interval of 14.0 were detected. These were 878.6, 892.6, and 906.6 in SM4s; 1042.7, 1056.7, and 1070.7 in SM3; and 1245.8, 1259.8, and 1273.8 in SM2. Thus, differences between the three peaks in each group were because of a single methylene group -CH2-. In addition, small peaks at 850.6, 864.6, and 924.6 in SM4s; at 996.6, 1014.6, 1024.6, 1052.7, and 1084.7 in SM3; and at 1199.7, 1217.7, 1227.7, 1255.8, 1271.8, and 1287.8 in SM2 were also detected. If SM4s (HSO3-3Gal ß 1Cer) and SM3 (HSO3-3Gal ß 4Glc ß 1Cer) possessed the same ceramide, difference in molecular mass between these two species should be a single unit of Hex minus H2O, 162.1. When peaks at 878.6 in SM4s and at 1042.7 in SM3 were comparatively examined (Figure 4A and B), difference in molecular mass between them was 164.1, suggesting that their ceramides were not identical and that species at 878.6 might contain an additional double bond in its ceramide compared to species at 1042.7. The same relationships were observed in other groups at 892.6/1056.7 and 906.6/1070.7. On the contrary, if SM3 and SM2 (GalNAc ß 4(HSO3-3)Gal ß 4Glc ß 1Cer) possessed identical ceramides, difference in molecular mass between these two species should be a single unit of HexNAc minus H2O, 203.1. When peaks at 1042.7 in SM3 and at 1245.8 in SM2 were comparatively examined (Figure 4B and C), difference in molecular mass between them was 203.1, indicating that these two peaks were from identical ceramides. The same relationships were observed in other groups at 1056.7/1259.8 and 1070.7/1273.8. Thus, the three major peaks in the three types of sulfatides suggested that SM4s possessed unique ceramides, whereas SM3 and SM2 possessed common ceramides.
Analysis of lyso-forms revealed further information (Figure 5). In SM4s, the major lysoSM4s peak was at 540.3, indicating the presence of a sphingoid of d18:1, and minor peaks at 538.3, 558.3, 568.3, and 570.3 were assigned to lysoSM4s of d18:2, t18:0, 4-eicosasphingenine (d20:1), and d20:0. Results from Figures 4A and 5A revealed that in SM4s, major peaks at 878.6, 892.6, and 906.6 were assigned to d18:1-C22:0h, d18:1-C23:0h, d18:1-C24:0h, respectively, and minor peaks at 850.6, 864.6, and 924.6 were assigned to d18:1-C20:0h, d18:1-C21:0h, and t18:0-C24:0h, respectively (Table II). In comparison to other organs, SM4s in renal tubule cells mostly resembled the one from the stomach with regard to scarcity of species possessing d18:1-C24:1 and d18:1-C24:0. In lyso-forms of SM3 and SM2 (Figure 5B and C), three peaks with similar patterns at 702.3, 720.3, and 732.4 in SM3 and at 905.4, 923.4, and 935.5 in SM2 were found, suggesting that lysoSM3 and lysoSM2 mainly possessed sphingoids of t18:0 and specific amounts of d18:1 and d20:0. Molecular mass differences between peaks at 702.3 and 905.4, 720.3 and 923.4, and 732.4 and 935.5 were all calculated to equal to a single number of 203.1 corresponding to a single unit of HexNAc minus H2O. No peaks corresponding to N-deacetyl-lysoSM2 of d18:1 ([M-H]–, 863.4), t18:0 ([M-H]–, 881.4), and d20:0 ([M-H]–, 893.5) were detected (data not shown). Although previously reported methods for chemical conversion of glycosphingolipids to lyso-forms showed induction of N-deacetylation in HexNAc to various degrees (Suzuki et al., 1984; Taketomi et al., 1996), the present deacylation procedure might protect from this unfavorable reaction. Similar results were observed in the preparation of lysoganglioside GM1 (Sonnino et al., 1992). Results from Figure 4B and C and Figure 5B and C characterized peaks in SM3 and SM2 and are summarized in Table II. These showed that peaks for SM3 and SM2 isomers could be derived from different types of fatty acids, that t18:0 was the major sphingoid, and d18:1 and d20:0 were the minor ones in SM3/SM2. Consequently, major ceramides in SM3 and SM2 were mainly composed of t18:0 and nonhydroxy fatty acids with acyl chains of C22, C23, and C24. This composition contrasted with results on ceramides in SM4s, composed of d18:1 and hydroxy fatty acids with acyl chains of C22, C23, and C24 (Table II).
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementReferences |
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We successfully reported a procedure for sulfatide analysis from biological materials, using MALDI-TOF MS within a day which was based on two simple and rapid methods. The first method is the partition method for enrichment of sulfatides without any chromatographic procedures (Hara and Radin, 1979
). Without enrichment of sulfatide fractions, it is difficult to analyze sulfatides in total lipids using TLC or MALDI-TOF MS because of very large amounts of nonsulfatide lipids as shown in Figure 3. The second method is the preparation method of lysosulfatides (Sugiyama et al., 1999) which was useful but not appropriate to analyze underivatized forms of sulfatides from biological materials. Therefore, the combination of these two methods with modification for micro-scale applicable devices was indispensable for the determination of ceramide molecular species in sulfatides using MALDI-TOF MS to reduce the complexity of mass fragmentation patterns and to further easily detect uncommon/unusual sphingoids. In addition, for the desalting process, we employed C18 tips that were easily attached to Gilson pipettes, instead of C18 cartridges, which contributed to minimize the dead space to avoid loss of samples, especially in the case of very small amounts of biological materials such as renal tubule cells.
We examined sulfatides in rats from several organs, including spinal cord, lung, colon, and stomach, and small amounts of renal tubule cells. Results for SM4s are summarized as follows. Firstly, the compositions of SM4s molecular species from different sources were different, although some similarities were found. Secondly, as expected, ceramides commonly possessed major sphingoids of d18:1, and some less common or unusual sphingoids included d18:0, t18:0, d20:1, d20:0, and d18:2 that occurred organ specifically. Thirdly, ceramides from different sources always contained hydroxy fatty acids of various degrees, and this was particularly observed in the stomach and the renal tubule cells. These results seem to agree with very early (Morell and Radin, 1969) and recent (Schaeren-Wiemers et al., 1995) observations that ceramide galactosyltransferase in endoplasmic reticulum, responsible for the synthesis of GalCer (Schulte and Stoffel, 1993), which is a direct precursor of SM4s, preferred ceramides possessing hydroxy fatty acids as substrates than those possessing nonhydroxy fatty acids. Recently, Fewou et al. 2005) directly demonstrated the importance of both hydroxylated GalCer and SM4s for the stability of myelin using transgenic mice overexpressing ceramide galactosyltransferase. In addition, they also suggested that SM4s possessing hydroxy fatty acids might be more potent inhibitors of oligodendrocyte differentiation than those possessing nonhydroxy fatty acids. Importance of SM4s with hydroxy fatty acids outside the nervous system should further be examined.
On the other contrary, it was very interesting that, in the renal tubule cells, the major species of SM3/SM2 commonly possessed C22, 23, and 24 nonhydroxy fatty acids and sphingoid t18:0, containing an additional –OH group compared to d18:1, whereas major species of SM4s possessed C22, 23, and 24 hydroxy fatty acids and sphingoid d18:1. Thus, different series of sulfatides contain different but, incidentally, similar hydroxyceramides with respect to same lengths of both fatty acyl chains and sphingoid chains (Figure 6). These observations suggested that for the major species of different series of sulfatides in rat renal tubule cells, these types of hydroxyceramides might be necessary for previously reported functions of ion transport (Ishizuka, 1997) and L-selectin ligand (Ogawa et al., 2004). The hydroxylation of ceramides in glycosphingolipids was reported to significantly influence the cell membrane with regard to strengthening lateral interactions of neighboring proteins and lipids, consequently greatly affecting membrane lipid microdomains (Graf et al., 2002). Recently, hydroxylases concerned with phytosphingosine (Mizutani et al., 2004; Omae et al., 2004) and hydroxy fatty acids (Alderson et al., 2004; Eckhardt et al., 2005) of sphingolipids were successively reported in mice and humans.
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Different but similar hydroxyceramides between SM4s and SM3/SM2 might be reflected by differences of synthetic pathways. Unlike SM4s, SM3/SM2 are derived from GlcCer synthesized by ceramide glucosyltransferase (Hirabayashi and Ichikawa, 2002) catalyzing reactions at the outer surface of the Golgi (Marks et al., 1999). Although preferred characteristics were not reported for this enzyme, a preference might exist for the presence of phytoceramides in the microenvironment surrounding renal tubule cells. Alternatively, cerebroside sulfotransferase responsible for the synthesis of not only SM4s but also SM3 and SM2 (Honke, 2002) might prefer precursor glycosphingolipids possessing hydroxyceramides, especially phytoceramides in cases of SM3 and SM2. Although details are unclear, our analytical results suggested the occurrence of a sophisticated preference system for the glycosylation of ceramides that may be important for appropriate functions in their microenvironments such as lipid membrane microdomain (lipid raft) (Brown and London, 2000). Indeed, recently, occurrence of SM4s was reported in lipid raft of the basolateral membrane of rainbow trout gill epithelium (Lingwood et al., 2005).
We detected significant amounts of sphingoid d20:0 in SM4s, SM3, and SM2. Previously, Tadano and Ishizuka (1982) reported that in addition to d18:1 and t18:0, significant amounts of unknown sphingoid was present in rat SM2, which probably corresponded to our findings of d20:0. Usually, sphingoids of d20:1 and d20:0 are characteristic components of gangliosides in the nervous system, with d20:0 still being considered as a minor component compared to that of d20:1 (Ando and Yu, 1984), although their roles are still unknown.
Recently, t18:0 has been reported to induce apoptosis in cancer cells through caspases, but roles of the involved caspases are still controversial (Park et al., 2003; Nagahara et al., 2005). However, in these previous studies, exogenous t18:0 has been used in assay systems, and nothing was reported for roles of the endogenous form. Sphingosine, ceramide, and their metabolites have been well studied to play crucial roles in cell signaling, proliferation, and death (Ogretmen and Hannun, 2004). However, these studies were mainly based on ubiquitous phosphosphingolipid of sphingomyelin which possessed the common sphingoid d18:1 and nonhydroxy fatty acids (Cuvillier, 2002). The hydroxylation of ceramides occurs in limited types of glycosphingolipids including sulfatides, and this may be related to appropriate the expressions of sugar chains which probably relate to cell signaling, proliferation, and death in specific microenvironments. Actually, we previously reported the importance of hydroxyceramide affecting the expression of glycosphingolipid tumor antigen. Namely, the murine lymphoma cells exhibited higher antigenicity of asialo GM2 possessing ceramide with C16 hydroxy fatty acid, whereas the cells exhibited less antigenicity of asialo GM2 possessing ceramide with C16 nonhydroxy fatty acid and additionally contained ganglioside GM1b with nonhydroxy fatty acid (Kannagi et al., 1983). We believe our approach can also contribute to resolve these issues.
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Materials and methods |
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Standards of sulfatide SM4s and ganglioside GM1, both from bovine brain and matrix compound
-cyano-4-hydroxycinnamic acid (-CHCA) were obtained from Sigma (St. Louis, MO). Standards of GalCer, lysoSM4s, and hydrogenated N-acetyl-lysoSM4s were prepared from pig spinal cord in our laboratory (Sugiyama et al., 1999). LacCer, Gb3Cer, and Gb4Cer from human erythrocytes were prepared in our laboratory. High performance TCL (HPTLC) plates (silica gel 60) were from Merck (Darmstadt, Germany). Sep-Pak C18 cartridges and Monotips C18 were from Waters (Milford, MA) and GL Sciences (Tokyo, Japan), respectively. F344 rats (12 weeks old) were obtained from Charles River Laboratories (Wilmington, MA). Rats were sacrificed following ether anesthesia, and organs including spinal cord, lungs, stomach, colon, and kidneys were collected. Renal tubule cell fractions were prepared from rat kidneys according to the graded sieving method using sieves of 125, 105, and 53 µM, as previously described (Sato et al., 1997).
Preparation of sulfatide fractions
Sulfatides were prepared as previously described (Hara and Radin, 1979), with some modifications for micro-scale analysis. Total lipids were extracted from approximately <0.5–1.0 g of wet weight rat organs, using 10 volumes of 20:10:1 (v/v/v) mixture, and subsequently a 10:20:1 (v/v/v) mixture of chloroform/methanol/water. The lipid extract was completely evaporated and re-dissolved in 10 ml 60:40:3 mixture of hexane/2-propanol/water to which 200 µL of 5 N NaOH in methanol was added with vigorous shaking for 1 min. After standing for 30 min, the upper layer was carefully removed, and the pellet-like lower layer containing sulfatides was neutralized with acetic acid, and desalted using a Sep-Pak C18 cartridge. For renal tubule cell analysis, 10–40 mg of wet weight tissues was directly homogenized with 1.2 ml of 3:2 (v/v) hexane/2-propanol mixture and subsequently with the same volume of 60:40:3 (v/v/v) hexane/2-propanol/water with sonication (Hara and Radin, 1978). After evaporating organic solvents using a centrifuge evaporator (CVE-2000, Tokyo Rikakikai, Tokyo, Japan), we re-dissolved total lipids in 1 ml 60:40:3 (v/v/v) hexane/2-propanol/water to which we added 30 µL of 5 N NaOH in methanol with vigorous shaking. After centrifugation, the upper layer was carefully removed, and the pellet-like lower layer was neutralized with acetic acid, evaporated, and then desalted using Monotip C18 (GL Sciences). The fraction thus obtained was used for further analysis.
Micro-scale preparation of lysosulfatides
Lysosulfatides were prepared from sulfatides as previously described (Sugiyama et al., 1999), with some modifications for micro-scale analysis. A fraction containing 
10–100 pmol of sulfatides was dissolved in 300 µL of 0.1 N NaOH in methanol in a Pyrex glass tube (13 x 100 mm), tightly screw-capped with a heat-resistant cap. The tube was heated at 150°C for 10 min in an oven. After immediate cooling, 20 µL of 3 N HCl was gradually added to adjust pH to below 4.0, and then 520 µL of hexane was added, followed by vigorous shaking. After centrifugation and removal of the hexane layer containing fatty acids, the lower layer containing lysosulfatides was evaporated using a centrifuge evaporator and desalted by Monotip C18.
MALDI-TOF MS
A saturated solution of -CHCA in a 1:1 mixture of acetonitrile/water containing 0.1% trifluoroacetic acid was used as the matrix solution. One microliter of matrix solution and 1 µL of sulfatide fraction (20–200 pmol of sulfatides) or lysosulfatides (5–50 pmol) in 60:40:5 (v/v/v) mixture of hexane/2-propanol/water were mixed and loaded onto a sample plate with 100 loading positions for crystallization. The sample plate was inserted into the Voyager Elite XL (6.5 m flight length in the reflector mode) Biospectrometry Workstation (PerSeptive Biosystems, Framingham, MA). A nitrogen laser (337 nm) was used for ionization. Negative ion mode detection was employed for this study. A two-point external calibration was performed in all experiments by appropriately choosing two of the following monoisotopic molecular ions: lysoSM4s possessing sphingoid d18:1 ([M-H]–, 540.2843), its hydrogenated form possessing sphingoid d18:0 ([M-H]–, 584.3105), SM4s from bovine brain possessing ceramide of d18:1-C24:1 ([M-H]–, 888.6235) and ganglioside GM1 possessing ceramide of d18:1-C18:0 ([M-H]–, 1544.8689).
TLC
Lipids were separated by TLC in a solvent system consisting of chloroform/methanol/water (65:25:4). Lipids were visualized by spraying with cupric-phosphoric acid charring reagent.
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Conflict of interest statement |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementReferences |
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None declared.
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Acknowledgements |
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In this article, sulfatides referred to glycolipids containing sulfate ester. Subclasses of sulfatides were abbreviated according to Ishizuka (1997)
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Abbreviations |
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In this article, sulfatides referred to glycolipids containing sulfate ester. Subclasses of sulfatides were abbreviated according to Ishizuka (1997).;
-CHCA, -cyano-4-hydroxycinnamic acid; C16:0, 16:0 nonhydroxy fatty acid; C16:0h, C16:0 hydroxy fatty acid; d18:0, sphinganine; d18:1, 4-sphingenine; d18:1-C16:0, ceramide possessing d18:1 with C16:0; d18:2, sphingadienine; d20:0, eicosasphinganine; d20:1, 4-eicosasphingenine; HPTLC, high performance TLC; MALDI-TOF MS, matrix-assisted laser desorption and ionization time-of-flight mass spectrometry; SM2, sulfated Gg3Cer (GalNAc ß 4(HSO3-3) Gal ß 4Glc ß 1Cer); SM3, sulfated LacCer (HSO3-3Gal ß 4Glc ß 1Cer); SM4s, sulfated GalCer (HSO3-3Gal ß 1Cer); t18:0, phytosphingosine (4-hydroxysphinganine); TLC, thin-layer chromatography or chromatogram |
References |
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