HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: D200026-JLR200v1 44/1/218    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bodennec, J. Articles by Futerman, A. H. Search for Related Content PubMed PubMed Citation Articles by Bodennec, J. Articles by Futerman, A. H. Social Bookmarking                      What's this?

Journal of Lipid Research, Vol. 44, 218-226, January 2003
Copyright © 2003 by Lipid Research, Inc.

Methods

Aminopropyl solid phase extraction and 2 D TLC of neutral glycosphingolipids and neutral lysoglycosphingolipids

Jacques Bodennec, Dori Pelled and Anthony H. Futerman¹

Department of Biological Chemistry, Weizmann Institute of Science, 76100 Rehovot Israel

Published, JLR Papers in Press, October 1, 2002. DOI 10.1194/jlr.D200026-JLR200

¹ To whom correspondence should be addressed. e-mail: tony.futerman{at}weizmann.ac.il

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Methods for isolation of neutral lysoglycosphingolipids (n-lyso-GSLs) such as glucosylsphingosine and galactosylsphingosine normally involve mild alkaline or acid hydrolysis followed by multiple chromatography steps, yielding relatively low recoveries of n-lyso-GSLs and neutral glycosphingolipids (n-GSLs). We now describe a new technique for isolating these compounds using one chromatography step, resulting in quantitative recovery of n-GSLs and n-lyso-GSLs. Lipids are extracted using a modified Folch procedure in which recovery is optimized by reextracting the Folch upper phase with water-saturated butanol. The extract is applied to an aminopropyl solid phase column from which both n-GSLs and n-lyso-GSLs elute in the same fraction. Separation is achieved using a new two-dimensional thin-layer chromatography procedure. The usefulness of this technique for biological samples was tested by examining Glc[4,5-³H]ceramide and Glc[4,5-³H]sphingosine accumulation in metabolically-labeled neurons treated with an inhibitor of lysosomal glucocerebrosidase. Accurate quantification of both lipids was obtained with Glc[4,5-³H]ceramide and Glc[4,5-³H]sphingosine accumulating at levels of 20 nmol/mg DNA and 40 pmol/mg DNA, respectively.

This simple and rapid technique can therefore be used for the analysis of lyso-GSLs and GSLs in the same tissue, which may permit the determination of their metabolic pathways in normal and in pathological tissues, such as those taken from Gaucher and Krabbe's disease patients.

Abbreviations: CBE, conduritol-B-epoxide; CDH, ceramide dihexoside; CTH, ceramide trihexoside; GalCer, galactosylceramide; GlcCer, glucosylceramide; GlcSph, glucosylsphingosine; GalSph, galactosylsphingosine; GSL, glycosphingolipid; HPLC, high performance liquid chromatography; LacSph, lactosylsphingosine; n-GSL, neutral glycosphingolipid; n-lyso-GSL, neutral lysoglycosphingolipid; SM, sphingomyelin; SPC, sphingosylphosphorylcholine; 2D-TLC, two-dimensional thin-layer chromatography

Supplementary key words glucosylsphingosine • psychosine • Gaucher disease • Krabbe's disease • Niemann-Pick disease • two-dimensional thin-layer chromatography

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Glycosphingolipids (GSLs) are important components of biological membranes where they play both structural and regulatory roles. Their lyso (i.e., N-deacylated) derivatives (lyso-GSLs) have also been implicated in various regulatory processes, although since they are found in small amounts in normal tissues, most attention has focused on attempting to understand their roles upon their accumulation in pathological tissues. For instance, the neutral lyso-GSLs (n-lyso-GSLs), glucosylsphingosine (GlcSph), and galactosylsphingosine (GalSph, psychosine), accumulate in tissues from Gaucher (1) and Krabbe's disease patients (2–5), respectively, and in animal models of these diseases (6–9). GalSph and GlcSph modulate various physiological processes, such as calcium release (10–12) and enzyme activities (13–16), and are also cytotoxic at relatively low concentrations (17–19). It has, however, been difficult to distinguish their functions in vivo from those of their parent neutral glycosphingolipids (n-GSLs) [i.e., galactosylceramide (GalCer) and glucosylceramide (GlcCer)]. Moreover, the biosynthetic pathways leading to n-lyso-GSL accumulation are still poorly understood (5, 20), probably in part due to the lack of simple methods that can be used for metabolically-labeling cultured cells in which n-lyso-GSLs are present in the sub-nmol range and n-GSLs in the sub-µmol range (9, 19, 21–24). Moreover, most isolation and analytical procedures for GSLs involve at least one chemical step, such as mild alkaline or acid hydrolysis, which can inadvertently hydrolyze (25) the parent compounds, yielding artifactually high levels of lyso-GSLs.

The most common method of isolating n-lyso-GSLs is by strong cation exchange chromatography, followed by desalting on a C18 reverse phase column (25), a method recently adapted to commercially available solid phase extraction cartridges (22). These purification methods often result in low (45–60%) recovery of n-lyso-GSLs (8, 22, 25). Moreover, chemical treatments such as alkaline methanolysis interfere with the quantitative recovery of n-lyso-GSLs and can indirectly affect their quantification by high-pressure performance liquid chromatography (HPLC), a problem neglected in the past (26, 27). An additional problem is caused by alkali- and acid-labile cerebroside esters (28–34) and plasmalocerebrosides (35, 36), which are major brain constituents (37, 38). Finally, an alkali-labile (39) 3-O-acetyl-sphingosine series of myelin n-GSLs has been characterized (39), again raising the possibility of artifactual formation of n-lyso-GSLs when mild alkaline hydrolysis is performed.

In the current study, we describe a simple procedure allowing the simultaneous isolation of n-GSLs and n-lyso-GSLs by solid phase extraction using commercially available aminopropyl cartridges, without the need for prior chemical treatment. We also describe a new two-dimensional thin layer chromatography (2D-TLC) procedure to separate n-lyso-GSLs from n-GSLs, and apply this method to quantify levels of Glc[4,5-³H]Sph and Glc[4,5-³H]Cer in a neuronal model of Gaucher disease.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Materials
Galactose oxidase (from Dactylium dendroides, 220 U/mg), 1-ß-D-GlcSph, 1-ß-D-GalSph, sphingomyelin (SM), sphingosylphosphorylcholine (SPC), glucosylceramide (GlcCer), galactosylceramide (GalCer), and calf thymus DNA were from Sigma. D-lactosyl-ß1-1'-D-erythro-sphingosine (LacSph) and lactosylceramide (LacCer) were from Avanti Polar Lipids (Alabaster, AL). Conduritol-B-epoxide (CBE) and a neutral GSL mixture [containing ceramide monohexosides, ceramide dihexosides (CDH), ceramide trihexosides (CTH), and globosides] were from Matreya (Pleasant Gap, PA). Aminopropyl solid phase extraction cartridges (LC-NH2, 100 mg) were from Supelco (Bellefonte, PA). Silica gel 60 TLC plates and sodium tetraborate were from Merck (Darmstadt, Germany). Bisbenzimide dye (Hoescht 33342) was from Molecular Probes (Eugene, OR). L-3-[³H]serine (specific activity of 26 Ci/mmol) and NaB[³]H₄ (specific activity of 11 or 25 Ci/mmol) were from Amersham (Little Chalfont, UK). All solvents were of analytical grade and were purchased from Biolab (Jerusalem, Israel).

Radioactive lipids
[4,5-³H]sphinganine (specific activity of 11 Ci/mmol) was synthesized by reduction of D-erythro-sphingosine with NaB[³]H₄ (11Ci/mmol) (40,41). D-[6-³H]GalSph and D-[6-³H]LacSph were synthesized by oxidation of GalCer and LacCer by galactose oxidase, reduction of the resulting aldehyde by NaB[³]H₄ (25 Ci/mmol) (42), alkaline hydrolysis [100°C, 3 h in butanol-10 N KOH (9:1, v/v)] (43), and purification by Unisil column chromatography and TLC (44) to give a final specific activity of 15 Ci/mmol.

Liquid extraction
D-[6-³H]GalSph and D-[6-³H]LacSph (each 10,000 dpm) were added to a glass tube together with 15 µg each of GlcSph, GalSph, and LacSph, dried under N₂, re-suspended in 4 ml of chloroform-methanol (2:1, v/v) (45), and phase separation was achieved by addition of 1 ml of deionized water. The aqueous and chloroform phases were separately dried under N₂ and spotted on a borate-impregnated TLC plate. Lipids were separated using chloroform-acetone-methanol-acetic acid-water (50:20:10:15:5, v/v/v/v/v) as the first developing solvent, run on two thirds the length of the TLC plate, and after air-drying, chloroform-methanol-water-ammonia (25% v/v) (20:20:2:0.35, v/v/v/v) was used as the second developing solvent, run the whole length of the TLC plate in the same direction as the first solvent. A good resolution between GalSph and GlcSph was obtained by running the plate in the first solvent system, while the greater polarity of the second solvent system gave a good separation from LacSph. Lipids were visualized with iodine, the n-lyso-GSLs recovered by scraping directly into liquid scintillation vials, to which 1 ml of methanol and 5 ml of Optima gold scintillation cocktail (Packard, Downers Groove, IL) were added, and radioactivity determined in a Packard 2100 ß radiospectrometer. The percent recovery of D-[6-³H]GalSph and D-[6-³H]LacSph in the chloroform phase was determined.

Since n-lyso-GSLs were not quantitatively recovered in the chloroform phase, the procedure was modified. First, the aqueous upper phase was re-extracted several times (Fig. 1) by addition of theoretical lower phase [chloroform-methanol-water (86:14:1, v/v/v)]. The lower phases were pooled and dried under N₂, as was the upper phase, and lipids were separated, identified, and quantified as above. Secondly, the upper phase was dried under N₂, resuspended in 2 ml of deionized water to which 2 ml of water-saturated butanol (prepared by adding one volume of butanol to one volume of deionized water, the mixture stirred for 30 min at room temperature, followed by phase separation) was added. After vortexing and phase separation, the butanol upper phase was pooled with the chloroform lower phase from the initial partitioning step. The effect of varying the number of water-saturated butanol extraction steps on the recovery of individual n-lyso-GSLs was determined (Fig. 1C), as was the efficiency of the water-saturated butanol extraction using varying amounts of n-lyso- GSLs by adding increasing amounts of GalSph and LacSph to glass tubes containing 10,000 dpm each of D-[6-³H]GalSph and D-[6-³H]LacSph.

View larger version (64K): [in this window] [in a new window]   Fig. 1. Recovery of galactosylsphingosine (GalSph) and lactosylsphingosine (LacSph) by liquid extraction. A: GalSph (squares) and LacSph (circles) recovery in the chloroform lower phase after multiple Folch partitions of the aqueous upper phase was compared with (C) extraction of the Folch upper phase by water saturated-butanol. The data are means ± SD for five different experiments. Representative TLC plates of the partitioned lipids are shown in B and D. Lipids were separated on a borated TLC plate using the following developing solvents: chloroform-acetone-methanol-acetic acid-water (50:20:10:15:5, v/v/v/v/v) run on two thirds the length of the plate, followed by chloroform-methanol-deionized water-ammonia (25% v/v) (20:20:2:0.35, v/v/v/v) run on the whole length of the plate. Lipids were visualized using the orcinol reagent. B: authentic standards are shown in lane 1, the lipids recovered in the lower phase after four extractions of the Folch upper phase in lane 2, and the LacSph lost to the upper phase in lane 3. D: Lane 1 shows lipid standards, lane 3 shows the quantitative recovery of the lipids in the pooled butanol-extracted upper phase combined with the initial chloroform phase, and lane 2 the absence of any lyso-glycosphingolipids (GSLs) in the aqueous phase after butanol extraction.

The efficiency of the solid phase extraction procedure was determined using D-[6-³H]GalSph and D-[6-³H]LacSph (10,000 dpm each) together with varying amounts of GlcSph, GalSph, and LacSph. The radioactivity remaining on the silica-based matrix after elution was also determined. The extent of cross-contamination of D-[6-³H]GalSph and D-[6-³H]LacSph in the other fractions was calculated after resolving the different fractions eluted from the column by TLC and the radioactivity in each individual n-lyso-GSL spot for each fraction counted. The percentage of cross-contamination was calculated as: percentage contamination in fraction X = (dpm in fraction X/dpm in all the fractions) x 100.

2D-TLC
Fraction 3 from the aminopropyl column was re-suspended in chloroform-methanol (2:1, v/v) and spotted 2 cm from the edge of a 10 x 10 cm TLC plate (Fig. 2) . The TLC plate was developed in the first direction using tetrahydrofuran-acetone-methanol-deionized water (25:10:20:3, v/v/v/v) run two thirds the length of the TLC plate, which was then air-dried and developed in the same direction with the same solvent system, but run until 0.5 cm from the edge of the plate. After air drying, the plate was briefly dipped (10 s) in a solution of 1% sodium tetraborate in methanol. After air drying, the area of the plate that was not dipped in borate was sprayed with borate, and after air-drying again, the TLC plate was developed in the second direction with chloroform-acetone-methanol-acetic acid-deionized water (50:20:10:15:5, v/v/v/v/v). Migration was stopped when the solvent front reached two thirds of the length of the plate. After air-drying, the plate was developed in chloroform-methanol-deionized water-25% ammonia (20:20:2:0.35, v/v/v/v), which was allowed to run the full-length of the TLC plate. Lipids were detected using either the orcinol reagent or iodine.

View larger version (55K): [in this window] [in a new window]   Fig. 2. 2D-TLC of neutral glycosphingolipids (n-GSLs) and their lyso-derivatives. An example of the 2D-TLC separation of GSLs and their lyso-derivatives is shown. Arrow 1 shows the first developing solvent used [tetrahydrofuran-acetone methanol-deionized water (25: 10:20:3, v/v/v/v)], which was run two thirds the length of the TLC plate. The same solvent system was used again but run until it was 0.5 cm from the front of the plate (arrow 2). The dashed line indicates the extent to which the TLC plate was dipped in the methanol/1% sodium tetraborate, and the rest of the plate (non-borated zone) was sprayed with borate. Arrow 3 indicates the direction of separation and extent of migration of the third solvent system [chloroform-acetone-methanol-acetic acid-deionized water (50:20:10:15:5, v/v/v/v/v)], and arrow 4 the final solvent system [chloroform-methanol-deionized water-25% ammonia (20:20:2:0.35, v/v/v/v)], which was allowed to migrate the full extent of the TLC plate. GSLs were visualized by orcinol. The position of migration of [4,5-3H]sphinganine, which can leak to some extent into fraction 3 of the aminopropyl column (46), is indicated.

At the end of the chase, neurons were washed with ice-cold PBS and scraped with a rubber policeman into 3 ml of ice-cold methanol, and sonicated. An aliquot was removed for DNA quantification using bisbenzimide (51). ³H-labeled GSLs were extracted by the addition of chloroform to the methanol extract to give a final ratio of 2:1 (v/v) (45), followed by addition of 2.25 ml deionized water. The aqueous and chloroform phases were dried under N₂, and the aqueous phase re-suspended in 3 ml of deionized water and 3 ml of water-saturated butanol. The butanol upper phase was pooled with the initial chloroform phase and dried under N₂. Lipids were re-suspended in 200 µl chloroform-methanol (23:1, v/v) and loaded onto an aminopropyl column, eluted as described above, and recovered. Fraction 3 containing n-GSLs and n-lyso-GSLs was separated by 2D-TLC. The extent of recovery of n-lyso-GSLs from the neuronal tissue was estimated by adding D-[6-³H]GalSph and D-[6-³H]LacSph to a neuronal methanol-extract that had not been metabolically labeled, and subsequently processed identically as for the samples labeled with [4,5-³H]sphinganine.

The possible contamination of the n-GSLs isolated from biological samples with glycerolipids was analyzed by incubating 6-day- old hippocampal neurons with L-3-[³H]serine (10 µCi per ml of medium). Lipids were extracted as above, loaded onto an aminopropyl cartridge, and fraction 3 collected. GSL standards were added to fraction 3, half of which was resolved by 2D-TLC without further treatment, and half of which was subjected to mild alkaline hydrolysis (46) by drying the samples under N₂, re-suspending in 2 ml chloroform-0.6 N NaOH in methanol (1:1, v/v) for 1 h at room temperature, and neutralizing with 10 N HCl. Deionized water (0.8 ml) was added to the mixture and the resulting upper and lower phases dried. The upper phase was extracted twice with water-saturated butanol and the butanol phases pooled with the initial chloroform lower phase. After 2D-TLC separation, lipids were detected with iodine, and radioactivity determined for each n-lyso-GSL and n-GSL. The distribution of ³H-radioactivity within the individual lipids was compared between samples that had or had not been subjected to mild alkaline hydrolysis.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Preliminary experiments demonstrated that only 64% and 42% respectively of GalSph and LacSph were recovered in the chloroform phase of a Folch extract, with the remainder lost to the aqueous phase (Fig. 1A). Re-extraction of the aqueous phase with theoretical lower phase improved the recovery, but even after four reextractions, 15% of LacSph was lost (Fig. 1A, and Fig. 1B, lane 3), and this was not improved by using KCl, NaCl, or CaCl₂ instead of deionized water to achieve phase separation. In contrast, recovery was drastically improved by re-extracting the Folch upper phase with water-saturated butanol, with one extraction yielding quantitative recovery of both GalSph and LacSph (Fig. 1C, D). The extraction efficiency using water-saturated butanol remained constant irrespective of the initial amount of GalSph and LacSph used, with 97–98% recovery of GalSph and 93–97% recovery of LacSph using starting amounts between 0.1 ng and 1 µg (not shown). Thus, this method is appropriate for n-lyso-GSL extraction in the mass range in which they are found in cultured cells (23, 24).

When applied to aminopropyl solid phase extraction cartridges, n-lyso-GSLs were recovered in one fraction, namely fraction 3 (Fig. 3A) , the same fraction as that in which the parent n-GSLs are eluted (46), with only a small amount of GalSph and LacSph lost to fraction 4. The n-lyso- GSLs were well resolved from SPC and SM, which elute in fraction 4 (Fig. 3B), in which the other neutral phospholipids also elute (46). Free long-chain bases, such as sphingosine, dihydrosphingosine and phytosphingosine, are recovered in fraction 2 (46) with a small amount of the free long chain bases found in fraction 3. However, the long chain bases did not interfere with the subsequent analysis of n-lyso-GSLs since they were well-separated by 2D-TLC (Fig. 2). The extent of recovery of n-lyso-GSLs in fraction 3 was essentially constant irrespective of the amount added to the column, with >90% recovered when applying between 10 ng to 50 µg of either GalSph or LacSph and <5% remaining on the cartridge. Precise quantification of GlcSph recovery could not be obtained due to the lack of radioactive GlcSph, but since the recovery of GlcSph in fraction 3 was always complete (Fig. 3A), we assume that GlcSph recovery is at least similar to that obtained for GalSph and LacSph. Together, these results show that solid phase extraction of n-lyso-GSLs is a highly efficient process for their quantitative recovery in the same fraction as the parent n-GSLs.

View larger version (89K): [in this window] [in a new window]   Fig. 3. Fractionation of neutral lyso-glycosphingolipids (n-lyso-GSLs) by aminopropyl solid phase extraction. A: Fifteen micrograms each of LacSph, GalSph, and glucosylsphingosine (GlcSph) were separated on a pre-equilibrated aminopropyl cartridge, eluted, and separated on a borated TLC plate with the developing solvents used in Fig. 1B and 1D, and lipids visualized using the orcinol reagent. The arrows in lane 4 show the small amounts of LacSph and GalSph that are lost to fraction 4 (F4); note that GlcSph was never observed in this fraction. B. Ten micrograms each of sphingomyelin (SM) and sphingosylphosphorylcholine (SPC) were separated on a pre-equilibrated aminopropyl cartridge, eluted, and fractions 1 to 5 (F1 to F5) separated by TLC using chloroform-methanol-25% ammonia (65:35:7.5, v/v/v) as the developing solvent. Lipids were visualized using cupric sulfate and charring the plate at 180°C for 5 min, and identified using authentic standards (Std).

View larger version (11K): [in this window] [in a new window]   Fig. 4. Analysis of n-lyso-GSLs and n-GSLs in cultured hippocampal neurons. Hippocampal neurons were incubated with or without conduritol-B-epoxide (CBE), metabolically labeled with [4,5-3H]sphinganine, the lipids extracted, fractionated on an aminopropyl column, fraction 3 resolved by 2D-TLC, and Glc[4,5-3H]Sph, Glc[4,5-3H]Cer, and [4,5-3H]ceramide dihexoside (CDH) analyzed. Statistical differences (Student's t-test) between control and CBE-treated neurons are indicated. Results are means ± SD from four independent cultures.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

View larger version (24K): [in this window] [in a new window]   Fig. 5. Schematic diagram of methods used for n-GSL and n-lyso-GSL isolation and analysis. See text for further details.

A further advantage of the method described herein is the use of only one column for n-GSL and n-lyso-GSL separation. This is in contrast to other published methods that use a combination of cation-exchange and reverse phase and silica gel chromatography (Fig. 5) (22), affecting the quantitative recovery of both n-GSLs and n-lyso-GSLs, and resulting in the loss of other lipid classes. Using an aminopropyl solid phase column, all cellular lipids are recovered, and can be stored for further analysis; aminopropyl columns are preferable to silicic acid columns as they are slightly less polar (56), resulting in recovery of n-lyso-GSLs and n-GSLs in the same fraction. Use of different aminopropyl solid phase column sizes, from between 100–500 mg, renders the method useful for separation of different amounts of cellular lipids, ranging between 4–20 mg (46). Of the most importance, n-GSLs and n-lyso-GSLs are recovered in one fraction without significant contamination of other major cellular lipids, although some minor lipids, such as dihydrosphingosine (46), may co-elute with them. However, dihydrosphingosine is well separated from n-GSLs and n-lyso-GSLs using a 2D-TLC system (Fig. 2) in which the first developing solvent was run to two thirds of the length of the TLC plate, which also resulted in optimal separation of GlcSph and GalSph. Glyceroglycolipids, such as galactosylglycerolipid, may also co-elute in fraction 3 of the aminopropyl column, and since these lipids occur at low levels in bacteria and plants (57) but also in some mammalian tissues such as testis and myelin (58, 59), appropriate caution must be taken to assess n-lyso- GSL and n-GSL purity after TLC analysis. One way to avoid potential contamination with glyceroglycolipids is to subject fraction 3 to mild alkaline hydrolysis, although this itself must be performed with caution to avoid artifactual formation of n-lyso-GSLs.

The last advantage of our method is the use of a novel 2D-TLC procedure to completely separate n-GSLs and n-lyso-GSLs from each other and from dihydrosphingosine, and to separate GalSph from GlcSph. The latter has proven difficult by HPLC after derivatization of the free amino group with o-phthalaldehyde (60), although some success has been reported by derivatization with 4-fluoro-7-nitrobenzofurazan (7, 61). To date, no procedure has been described for the separation of these compounds on the same TLC plate, and the solid phase extraction procedure could also be used as a first step to obtain pure n-GSLs and n-lyso-GSLs from biological tissues prior to their subsequent quantification by other analytical methods. The high recovery of n-lyso-GSLs and n-GSLs, and their purification within the same fraction, together with their separation on the same TLC plate, renders this method particularly suitable for metabolic studies. Since this method can also be applied to small amounts of metabolically-labeled tissues, and since the metabolic products and/or precursors (such as ceramide and free sphingoid bases) are recovered in separate fractions (46, 62), we anticipate that it could be used to resolve the metabolic pathways by which n-lyso-GSLs are formed. Moreover, since a similar method has also been applied to recover GSLs from various tissue sources, such as melanoma tumors (46), fish gills (63), and rat mitochondria (64), we also anticipate that it will be useful to determine their levels of accumulation in pathological tissues obtained from patients with lysosomal storage diseases, such a Gaucher, Krabbe's, and Niemann-Pick disease.

	ACKNOWLEDGMENTS

 
This work was supported by the Children's Gaucher Research Fund. Jacques Bodennec is supported by a Koshland Scholar award.

Manuscript received July 21, 2002 and in revised form September 18, 2002.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

1. Nilsson, O., and L. Svennerholm. 1982. Accumulation of glucosylceramide and glucosylsphingosine (psychosine) in cerebrum and cerebellum in infantile and juvenile Gaucher disease. J. Neurochem. 39: 709–718.[CrossRef][Medline]

2. Miyatake, T., and K. Suzuki. 1972. Globoid cell leukodystrophy: additional deficiency of psychosine galactosidase. Biochem. Biophys. Res. Commun. 48: 539–543.[CrossRef][Medline]

3. Svennerholm, L., M. T. Vanier, and J. E. Mansson. 1980. Krabbe disease: a galactosylsphingosine (psychosine) lipidosis. J. Lipid Res. 21: 53–64.[Abstract]

4. Kobayashi, T., H. Shinoda, I. Goto, T. Yamanaka, and Y. Suzuki. 1987. Globoid cell leukodystrophy is a generalized galactosylsphingosine (psychosine) storage disease. Biochem. Biophys. Res. Commun. 144: 41–46.[CrossRef][Medline]

5. Suzuki, K. 1998. Twenty five years of the "psychosine hypothesis": a personal perspective of its history and present status. Neurochem. Res. 23: 251–259.[CrossRef][Medline]

6. Igisu, H., and K. Suzuki. 1984. Progressive accumulation of toxic metabolite in a genetic leukodystrophy. Science. 224: 753–755.[Abstract/Free Full Text]

7. Orvisky, E., E. Sidransky, C. E. McKinney, M. E. Lamarca, R. Samimi, D. Krasnewich, B. M. Martin, and E. I. Ginns. 2000. Glucoylsphingosine accumulation in mice and patients with type 2 Gaucher disease begins early in gestation. Pediatr. Res. 48: 233–237.[Medline]

8. Shinoda, H., T. Kobayashi, M. Katayama, I. Goto, and H. Nagara. 1987. Accumulation of galactosylsphingosine (psychosine) in the twitcher mouse: determination by HPLC. J. Neurochem. 49: 92–99.[CrossRef][Medline]

9. Luzi, P., M. A. Rafi, M. Zaka, M. Curtis, M. T. Vanier, and D. A. Wenger. 2001. Generation of a mouse with low galactocerebrosidase activity by gene targeting: a new model of globoid cell leukodystrophy (Krabbe disease). Mol. Genet. Metab. 73: 211–223.[CrossRef][Medline]

10. Okajima, F., and Y. Kondo. 1995. Pertussis toxin inhibits phospholipase C activation and Ca2+ mobilization by sphingosylphosphorylcholine and galactosylsphingosine in HL60 leukemia cells. Implications of GTP-binding protein-coupled receptors for lysosphingolipids. J. Biol. Chem. 270: 26332–26340.[Abstract/Free Full Text]

11. Liu, R., M. C. Farach-Carson, and N. J. Karin. 1995. Effects of sphingosine derivatives on MC3T3–E1 pre-osteoblasts: psychosine elicits release of calcium from intracellular stores. Biochem. Biophys. Res. Commun. 214: 676–684.[CrossRef][Medline]

12. Himmel, H. M., D. Meyer zu Heringdorf, B. Windorfer, C. J. van Koppen, U. Ravens, K. H. and Jakobs. 1998. Guanine nucleotide-sensitive inhibition of L-type Ca2+ current by lysosphingolipids in RINm5F insulinoma cells. Mol. Pharmacol. 53: 862–869.[Abstract/Free Full Text]

13. Hannun, Y. A., and R. M. Bell. 1987. Lysosphingolipids inhibit protein kinase C: implications for the sphingolipidoses. Science. 235: 670–674.[Abstract/Free Full Text]

14. Igisu, H., N. Hamasaki, A. Ito, and W. Ou. 1988. Inhibition of cytochrome c oxidase and hemolysis caused by lysosphingolipids. Lipids. 23: 345–348.[Medline]

15. Sohal, P. S., and R. B. Cornell. 1990. Sphingosine inhibits the activity of rat liver CTP:phosphocholine cytidylyltransferase. J. Biol. Chem. 265: 11746–11750.[Abstract/Free Full Text]

16. Ariga, T., W. D. Jarvis, and R. K. Yu. 1998. Role of sphingolipid-mediated cell death in neurodegenerative diseases. J. Lipid Res. 39: 1–16.[Abstract/Free Full Text]

17. Cho, K. H., M. W. Kim, and S. U. Kim. 1997. Tissue culture model of Krabbe's disease: psychosine cytotoxicity in rat oligodendrocyte culture. Dev. Neurosci. 19: 321–327.[Medline]

18. Tanaka, K., and H. D. Webster. 1993. Effects of psychosine (galactosylsphingosine) on the survival and the fine structure of cultured Schwann cells. J. Neuropathol. Exp. Neurol. 52: 490–498.[Medline]

19. Tohyama, J., J. Matsuda, and K. Suzuki. 2001. Psychosine is as potent an inducer of cell death as C6-ceramide in cultured fibroblasts and in MOCH-1 cells. Neurochem. Res. 26: 667–671.[CrossRef][Medline]

20. Kozutsumi, Y., T. Kanazawa, Y. Sun, T. Yamaji, H. Yamamoto, and H. Takematsu. 2002. Sphingolipids involved in the induction of multinuclear cell formation. Biochim. Biophys. Acta. 1582: 138–143.[Medline]

21. Atsumi, S., C. Nosaka, I. Hironobu, and K. Umezawa. 1993. Accumulation of tissue glucosylsphingosine in Gaucher-like mouse induced by the glucosyceramidase inhibitor cyclophellitol. Arch. Biochem. Biophys. 304: 302–304.[CrossRef][Medline]

22. Whitfield, P. D., P. C. Sharp, R. Taylor, and P. Meikle. 2001. Quantification of galactosylsphingosine in the twitcher mouse using electrospray ionization-tandem mass spectrometry. J. Lipid Res. 42: 2092–2095.[Abstract/Free Full Text]

23. Yamaguchi, Y., N. Sasagasako, I. Goto, and T. Kobayashi. 1994. The synthetic pathway for glucosylsphingosine in cultured fibroblasts. J. Biochem. (Tokyo). 116: 704–710.[Abstract/Free Full Text]

24. Sasagasako, N., T. Kobayashi, Y. Yamaguchi, N. Shinnoh, and I. Goto. 1994. Glucosylceramide and glucosylsphingosine metabolism in cultured fibroblasts deficient in acid beta-glucosidase activity. J. Biochem. (Tokyo). 115: 113–119.[Abstract/Free Full Text]

25. Igisu, H., and K. Suzuki. 1984. Analysis of galactosylsphingosine (psychosine) in the brain. J. Lipid Res. 25: 1000–1006.[Abstract]

26. Kwon, O-S., G. D. Newport, and W. Slikker. 1998. Quantitative analysis of free sphingoid bases in the brain and spinal cord tissues by high-performance liquid chromatography with a fluorescence detection. J. Chromatogr. B. 720: 9–14.[CrossRef]

27. Dasgupta, S., and E. L. Hogan. 2001. Chromatographic resolution and quantitative assay of CNS tissue sphingoids and sphingolipids. J. Lipid Res. 42: 301–308.[Abstract/Free Full Text]

28. Norton, W. T., and M. Brotz. 1963. New galactolipids of brain: a monoalkyl-monoacyl-glycerylgalactoside and cerebroside fatty acid esters. Biochem. Biophys. Res. Commun. 12: 198–203.[CrossRef][Medline]

29. Klenk, E., and M. Doss. 1966. About the occurrence of ester-cerebrosides in brain. Hoppe Seylers Z. Physiol. Chem. 346: 296–298.

30. Klenk, E., and J. P. Lohr. 1967. On the ester cerebroside of brain. Hoppe Seylers Z. Physiol. Chem. 348: 1712–1714.[Medline]

31. Tamai, Y., T. Taketomi, and T. Yamakawa. 1967. New glycolipids in bovine brain. Jpn. J. Exp. Med. 37: 79–81.[Medline]

32. Tamai, Y. 1968. Further study on fast running glycolipid in brain. Jpn. J. Exp. Med. 38: 65–73.[Medline]

33. Kishimoto, Y., M. Wajda, and N. S. Radin. 1968. 6-acyl-cerebroside of pig brain. J. Lipid Res. 9: 27–33.[Abstract]

34. Tamai, Y., K. Nakamura, K. Takayama-Abe, T. Kasama, and H. Kobatake. 1993. Less polar glycolipids in Alaskan pollack brain: isolation and characterization of acyl galactosyl ceramide and acyl glucosyl ceramide. J. Lipid Res. 34: 601–608.[Abstract]

35. Levery, S. B., E. D. Nudelman, and S. Hakomori. 1992. Novel modification of glycosphingolipids by long-chain cyclic acetals: isolation and characterization of plasmalocerebroside from human brain. Biochemistry. 31: 5335–5340.[CrossRef][Medline]

36. Yachida, Y., M. Kashiwagi, T. Mikami, K. Tsuchihashi, T. Daino, T. Akino, and S. Gasa. 1998. Stereochemical structures of synthesized and natural plasmalogalactosylceramides from equine brain. J. Lipid Res. 39: 1039–1045.[Abstract/Free Full Text]

37. Nudelman, E. D., S. B. Levery, Y. Igarashi, and S. Hakomori. 1992. Plasmalopsychosine, a novel plasmal (fatty aldehyde) conjugate of psychosine with cyclic acetal linkage. Isolation and characterization from human brain white matter. J. Biol. Chem. 267: 11007–11016.[Abstract/Free Full Text]

38. Hikita, T., K. Tadano-Aritomi, N. Iida-Tanaka, J. K. Anand, I. Ishizuka, and S. Hakomori. 2001. A novel plasmal conjugate to glycerol and psychosine ("Glyceroplasmalopsychosine"). Isolation and characterization from bovine brain white matter. J. Biol. Chem. 276: 23084–23091.[Abstract/Free Full Text]

39. Dasgupta, S., S. B. Levery, and E. L. Hogan. 2002. 3-O-acetyl-sphingosine-series myelin glycolipids: characterization of novel 3-O-acetyl-sphingosine galactosylceramide. J. Lipid Res. 43: 751–761.[Abstract/Free Full Text]

40. Hirschberg, K., J. Rodger, and A. H. Futerman. 1993. The long-chain sphingoid base of sphingolipids is acylated at the cytosolic surface of the endoplasmic reticulum in rat liver. Biochem. J. 290: 751–757.

41. Schwarzmann, G. 1978. A simple and novel method for tritium labeling of gangliosides and other sphingolipids. Biochim. Biophys. Acta. 529: 106–114.[Medline]

42. Radin, N. S., and G. P. Evangelatos. 1981. The use of galactose oxidase in lipid labeling. J. Lipid Res. 22: 536–541.[Abstract]

43. Taketomi, T., and T. Yamakawa. 1963. Immunochemical studies of lipids. I. Preparation and immunological properties of synthetic psychosine-protein antigens. J. Biochem. (Tokyo). 54: 444–451.[Free Full Text]

44. Miyatake, T., and K. Suzuki. 1972. Galactosylsphingosine galactosyl hydrolase. Partial purification and properties of the enzyme in rat brain. J. Biol. Chem. 247: 5398–5403.[Abstract/Free Full Text]

45. Folch, J., M. Lees, and G. H. S. Stanley. 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226: 497–509.[Free Full Text]

46. Bodennec, J., O. Koul, I. Aguado, G. Brichon, G. Zwingelstein, and J. Portoukalian. 2000. A procedure for fractionation of sphingolipid classes by solid-phase extraction on aminopropyl cartridges. J. Lipid Res. 41: 1524–1531.[Abstract/Free Full Text]

47. Schwarz, A., E. Rapaport, K. Hirschberg, and A. H. Futerman. 1995. A regulatory role for sphingolipids in neuronal growth - Inhibition of sphingolipid synthesis and degradation have opposite effects on axonal branching. J. Biol. Chem. 270: 10990–10998.[Abstract/Free Full Text]

48. Hirschberg, K., R. Zisling, G. van Echten-Deckert, and A. H. Futerman. 1996. Ganglioside synthesis during the development of neuronal polarity. Major changes occur during axonogenesis and axon elongation, but not during dendrite growth or synaptogenesis. J. Biol. Chem. 271: 14876–14882.[Abstract/Free Full Text]

49. Brewer, G. J., J. R. Torricelli, E. K. Evege, and P. J. Price. 1993. Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination. J. Neurosci. Res. 35: 567–576.[CrossRef][Medline]

50. Goslin, K., H. Asmussen, and G. Banker. 1998. Rat hippocampal neurons in low-density culture. In Culturing Nerve Cells. G. Banker and K. Goslin, eds. MIT press, Cambridge, MA. 339–370.

51. Labarca, C., and K. Paigen. 1980. A simple, rapid, and sensitive DNA assay procedure. Anal. Biochem. 102: 344–352.[CrossRef][Medline]

52. Legler, G., and E. Bieberich. 1988. Active site directed inhibition of a cytosolic beta-glucosidase from calf liver by bromoconduritol B epoxide and bromoconduritol F. Arch. Biochem. Biophys. 260: 437–442.[CrossRef][Medline]

53. Korkotian, E., A. Schwarz, D. Pelled, G. Schwarzmann, M. Segal, and A. H. Futerman. 1999. Elevation of intracellular glucosylceramide levels results in an increase in endoplasmic reticulum density and in functional calcium stores in cultured neurons. J. Biol. Chem. 274: 21673–21678.[Abstract/Free Full Text]

54. Kralik, S. F., X. Du, C. Patel, and J. P. Walsh. 2001. A method for quantitative extraction of sphingosine 1-phosphate into organic solvent. Anal. Biochem. 294: 190–193.[CrossRef][Medline]

55. Bligh, E. G., and W. J. Dyer. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37: 911–917.

56. Ruiz-Gutierrez, V., and M. C. Perez-Camino. 2000. Update on solid-phase extraction for the analysis of lipid classes and related compounds. J. Chromatogr. A. 885: 321–341.[CrossRef][Medline]

57. Pahlson, P., S. L. Spitalnik, P. F. Spitalnik, J. Fantini, O. Rakotonirainy, S. Ghardashkhani, J. Lindberg, P. Konradsson, and G. Larson. 2001. Characterization of galactosyl glycerolipids in the HT29 human colon carcinoma cell line. Arch. Biochem. Biophys. 396: 187–198.[CrossRef][Medline]

58. Inoue, T., D. S. Deshmukh, and R. A. Pieringer. 1971. The association of the galactosyl diglycerides of brain with myelination. I. Changes in the concentration of monogalactosyl diglyceride in the somal and myelin fractions of brain of rats during development. J. Biol. Chem. 246: 5688–5694.[Abstract/Free Full Text]

59. Sastry, P. S. 1974. Glycosyl glycerides. Adv. Lipid Res. 12: 251–310.[Medline]

60. Merrill, A. H., E. Wang, R. E. Mullins, W. C. Jamison, S. Nimkar, and D. C. Liotta. 1988. Quantitation of free sphingosine in liver by high-performance liquid chromatography. Anal. Biochem. 171: 373–381.[CrossRef][Medline]

61. Nozawa, M., T. Iwamoto, T. Tokoro, and Y. Eto. 1992. Novel procedure for measuring psychosine derivatives by an HPLC method. J. Neurochem. 59: 607–609.[CrossRef][Medline]

62. Bodennec, J., G. Brichon, G. Zwingelstein, and P. Portoukalian. 2000. Purification of sphingolipid classes by solid phase extraction with aminopropyl and weak cation exchanger cartridges. Methods Enzymol. 312: 101–114.[Medline]

63. Bodennec, J., G. Brichon, J. Portoukalian, and G. Zwingelstein. 1999. Improved methods for the simultaneous separation of free diacylglycerol species from ceramides containing phytosphingosine and sphingosine bases with non-hydroxy and alpha-hydroxy fatty acids. J. Liquid Chrom. & Rel. Technol. 22: 1493–1502.[CrossRef]

64. Ardail, D. 2001. Occurrence of ceramides and neutral glycolipids with unusual long-chain base composition in purified rat liver mitochondria. FEBS Lett. 488: 160–164.[CrossRef][Medline]

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				E. Lloyd-Evans, D. Pelled, C. Riebeling, J. Bodennec, A. de-Morgan, H. Waller, R. Schiffmann, and A. H. Futerman Glucosylceramide and Glucosylsphingosine Modulate Calcium Mobilization from Brain Microsomes via Different Mechanisms J. Biol. Chem., June 20, 2003; 278(26): 23594 - 23599. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: D200026-JLR200v1 44/1/218    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bodennec, J. Articles by Futerman, A. H. Search for Related Content PubMed PubMed Citation Articles by Bodennec, J. Articles by Futerman, A. H. Social Bookmarking                      What's this?