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Journal of Lipid Research, Vol. 46, 812-816, April 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology

Methods

N-Acyl migration in ceramides

Helena Van Overloop, Gerd Van der Hoeven and Paul P. Van Veldhoven¹

Katholieke Universiteit Leuven, Faculteit Geneeskunde, Departement Moleculaire Celbiologie, Afdeling Farmacologie, Leuven, Belgium

Published, JLR Papers in Press, January 16, 2005. DOI 10.1194/jlr.D400034-JLR200

¹ To whom correspondence should be addressed. e-mail: paul.vanveldhoven{at}med.kuleuven.ac.be

	ABSTRACT

TOP ABSTRACT REFERENCES

 
Upon exposure of truncated ceramides, such as N-acetyl-sphingenine, and long-chain ceramides to moderate acidic conditions, three derivatives are formed. Two of them turned out to be O-acylated sphingenine, 1-O- and 3-O-acyl-sphingenine, and the other was identified as sphingenine. Truncated dihydroceramides (e.g., N-acetyl- and N-hexanoyl-sphinganine) also show this type of rearrangement, which is therefore not related to the presence of the allylic hydroxy group or to the length of the N-acyl chain. Of particular concern is the fact that the O-acylated compounds, which can be considered sphingoid base analogs, can be formed in chloroform or chloroform-methanol mixtures upon storage. For long-term storage, methanol or dichloromethane is the preferred solvent.

A procedure to document the presence/formation of such O-acylated sphingoid bases in ceramide solutions in the picomole range, based on reversed-phase chromatography after derivatization of their amino group with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, is presented.

Supplementary key words O-acylation • N-acylethanolamine • anandamide • hydroxyoxazolidine • ceramide kinase

When analyzing lipid kinase activity in bacterial lysates using labeled truncated ceramide analogs, the presence of unknown labeled compounds was noticed in the organic extracts. With N-[¹⁴C]acetyl-sphingenine, prepared as described previously (1), as substrate, two additional radioactive spots, one with a lower (derivative X) and one with a higher relative mobility (R_f; derivative Y) than the substrate, were observed after TLC in an acidic solvent (Fig. 1A) . These lipids did not comigrate with any known commercially available sphingolipid. As the formation of these derivatives was not dependent on the presence of bacterial proteins (Fig. 1A), the reaction and extraction conditions were investigated. This revealed that the acidification of the assay mixture with HCl, to halt the reaction and to improve the extraction of the ceramide phosphate in the organic phase, was to blame. This was confirmed using unlabeled N-acetyl-sphingenine. After treatment of this lipid with 0.5 N HCl, three extra iodine-reactive spots were formed (Fig. 1B). One derivative comigrated with sphingenine; the others resembled derivatives X and Y with regard to R_f values. All three stained with ninhydrin, suggesting that a migration of the acetyl group from the amino to one of the two adjacent hydroxy groups of the sphingoid base gave rise to the unknown ceramide derivatives X and Y (Fig. 2) . The conversions were concentration dependent and were even noticed when using 0.1 N HCl (Fig. 1B, C; Table 1). Other acids, such as H₂SO₄ and HClO₄, in equivalent normality as 0.5 N HCl, also affected N-acetyl-sphingenine, but 5% acetic acid and 0.5 N H₃PO₄ did not (Fig. 1B, C; data not shown).

View larger version (29K): [in this window] [in a new window]   Fig. 1. TLC analysis of truncated ceramides. A: Autoradiograph of extracts from a ceramide kinase assay with N-[14C]acetyl-sphingenine. The reaction mixture (200 µl, 100 µM substrate, 7,500 dpm/nmol) was stopped with 1.5 ml of chloroform-methanol (1:2, v/v), followed by phase separation by the addition of 0.7 ml of 0.5 N HCl, 2 M NaCl, and 1 ml of chloroform. The organic phase was analyzed by TLC (silica G plates); the solvent was chloroform-acetone-methanol-acetic acid-water (10:4:3:2:1, v/v), and the plate was exposed to Hyperfilm MP (Amersham Bioscience). Lane 1, standard N-[14C]acetyl-sphingenine; lane 2, extract from blank; lane 3, extract from reaction in the presence of bacterial lysate. B, C: N-Acetyl-sphingenine was dissolved at 2 mM in chloroform-methanol (1:1, v/v) containing the indicated amount of acid, kept at room temperature for 3 h, and spotted (40 µl) on silica G plates. After development in the system described in A, lipids were transiently visualized with iodine (B), followed by ninhydrin spray (C). Lane 1, 5% acetic acid; lane 2, 0.1 N HCl; lane 3, 0.5 N HCl; lane 4, 1 N HCl; lane 5, standard N-acetyl-sphingenine. D, E. N-Acetyl-sphingenine was dissolved at 2 mM in the indicated solvents (HPLC grade unless otherwise indicated), kept for 3 h at 60°C, and analyzed as described above (D, iodine; E, ninhydrin). Lane 1, chloroform (Biosolve); lane 2, chloroform-methanol (1:1, v/v); lane 3, methanol (Biosolve); lane 4, dichloromethane (Acros); lane 5, acetone (Fisher Chemicals); lane 6, acetonitrile (Biosolve); lane 7, toluene (Biosolve); lane 8, standard N-acetyl-sphingenine. All panels display TLC plates or corresponding autoradiographs scanned from start to front and converted to black-and-white images.

View larger version (13K): [in this window] [in a new window]   Fig. 2. Rearrangements of ceramide by acyl migration.

View larger version (18K): [in this window] [in a new window]   Fig. 3. HPLC analysis of acyl migration in N-acetyl-sphingenine upon exposure to acid or storage at room temperature. N-Acetyl-sphingenine (Avanti%20Polar%20Lipids">Avanti Polar Lipids; 6 nmol) was dissolved in 100 µl of chloroform-methanol (1:1, v/v; 100 µl) (A) or chloroform-methanol containing 0.5 N HCl (B) and kept at room temperature for 3 h. After the addition of 2 N NH4OH (2-fold molar excess over HCl), methanol and chloroform were added to obtain phase separation (methanol-chloroform-water ratio was 1:1:0.9). An internal standard (700 pmol of 4-hydroxysphinganine; Sigma) was added after the lower phase was dried, and 50 µl of 20 mM triethylamine in methanol, followed by 50 µl of 10 mM 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, prepared as described previously (19), in tetrahydrofuran, was added. The mixture was kept for 45 min at room temperature, and a volume of 20 µl was injected on a Symmetry C18 column (150 x 4.6 mm inner diameter, 5 µm; Waters) via a 717 Plus autosampler (Waters) connected to a 1525 binary HPLC pump (Waters). The column was eluted at a flow rate of 1 ml/min with buffer A (acetonitrile-methanol-water-formic acid-triethylamine, 480:320:188:5:7, v/v) during 25 min, followed by a gradient elution from 100% buffer A to 100% buffer B (acetonitrile-methanol, 60:40, v/v) in 5 min. Elution was monitored by a 2475 multi fluorescence detector (Waters; excitation, 244 nm; emission, 398 nm). C: N-Acetyl-sphingenine was dissolved at 0.06 mM in CHCl3 and stored at room temperature for 1 week. After the addition of 700 pmol of 4-hydroxysphinganine, the sample was dried, treated with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, and analyzed by reversed-phase chromatography as described above.

Acyl migration between adjacent amino and hydroxy groups is a well-known chemical reaction but has been somewhat overlooked in the research on ceramide-mediated biology. NO-Acyl migration has been reported for N-acylated amino alcohols (e.g., N-acetyl-ephedrine) (2) and for peptides containing ß-hydroxy amino acids (serine or threonine) upon exposure to strong acids (3, 4). Linear peptides are converted into O-acyl peptides, and cyclic peptides are isomerized (e.g., cyclosporin A to isocyclosporin A). With regard to lipids, NO-acyl migration was documented long ago in the chemical literature for ethanolamides (5). These fatty amides, now referred to as N-acylethanolamines, are produced from N-acylated phosphatidylethanolamine. Given their endocannabinoid action [e.g., anandamide (N-arachidonoyl-ethanolamine)] (6, 7), N-acylethanolamines are currently actively investigated, and recently, during their analysis in biological systems, acyl migration was (re)noticed (8). Conditions to induce NO migration in anandamide, however, appear more drastic (anhydrous acid, significant heat, sonication) than those for ceramide rearrangements.

Another example of an NO-acyl migration in lipids, explaining the instability of N-acetylated fumonisin B1, was reported by Norred et al. (9). Fumonisin B1 is a mycotoxin that is well known as a potent inhibitor of sphinganine N-acyltransferase (ceramide synthase) (10). Its backbone (2-amino-12,16-dimethyl-3,5,10,14,15-eicosanepentadiol) structurally resembles a sphingoid base, and a free amino group is required to be effective as an inhibitor. Upon storage of N-acetylated fumonisin B1, in solid form but especially in acidic solutions, O-acetylated fumonisin B1 is formed. As both 3-O- and 5-O-acetylated toxins are generated, migration is not limited to vicinal amino/hydroxy groups (9).

With regard to acyl migration in sphingolipids, some notes can be found in older papers dealing with their chemical analysis, normally done by acidic hydrolysis (1–2 N HCl, 75°C, 2–18 h). These drastic procedures result in various by-products of sphingoid bases (11). The isomerization at carbon 3 (threo-sphingoid bases) and the generation of O-methyl-sphingenine have been proposed to rely on the formation of a cyclic intermediate. Upon hydrolysis of lysosphingolipids, lacking the N-acyl chain and hence unable to form an oxazolidine ring, the by-products are not seen (12, 13). As far as we are aware, acyl migration under less drastic conditions has not been documented.

Given the abundant use of N-acetyl-sphingenine as a water-soluble ceramide analog, and of N-acetyl-sphinganine as an inactive analog, as well as various other ceramide analogs (14–18), it seemed of interest to document possible differences in migration between the various ceramides. Moreover, because it cannot be excluded that the O-acetyl derivatives, which can be considered as sphingoid base analogs, possess bioactivity, it appears imperative to verify the stability of these ceramides during storage. To quantify low amounts of derivatives, TLC was not adequate. Therefore, the amino group of the O-acyl sphingoid bases was derivatized by means of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (19), followed by reversed-phase HPLC and postcolumn fluorescence detection, similar to a procedure described for the analysis of sphingoid bases (20). For N-acetyl-sphingenine, after 3 h of exposure to 0.5 N HCl in chloroform-methanol (1:1, v/v) at room temperature, derivative X, derivative Y, and sphingenine were estimated at 12.8, 2.9, and 1.9%, respectively (Table 1; Fig. 3B) . The conversions of N-[¹⁴C]acetyl-sphingenine treated with 0.5 N HCl, based on the radioactivity associated with the derivatized products separated via HPLC or based on direct TLC analysis of the acidic ceramide solution, agreed fairly well (8.2% versus 7.9% for X, 3.7% versus 2.8% for Y). These values indicate that hydrolysis of the O-acyl esters during the extraction/derivatization is negligible and support the validity of the HPLC procedure. The acid-induced rearrangement of ceramides was not limited to N-acetylated compounds. N-Hexanoyl- and N-hexadecanoyl-sphingenine were also converted into ninhydrin-positive products, although the rate of conversion, and certainly further hydrolysis of the O-acyl compounds to sphingenine, appears to diminish with a longer acyl chain (Table 1). Based on data obtained with N-hexanoyl-[4,5-³H]sphinganine (data not shown), the rearrangements are not dependent on the presence of the 4-trans double bond. On the contrary, the double bond suppressed the formation of the 3-O-acyl sphingoid base (derivative Y), based on a comparison between N-acetyl-sphingenine and N-acetyl-sphinganine (Table 1). This is likely attributable to the allylic nature of the 3-hydroxy group.

To quantify conversions during short-term storage, solutions of N-acetyl-sphingenine were kept at room temperature, 4°C, or –20°C for 1 or 4 weeks in chloroform, dichloromethane, or methanol before analysis. This short-term storage did not cause any conversions when the solutions were kept at 4°C or –20°C (data not shown). On the contrary, storage at room temperature for 1 week resulted in the formation of derivative X (1.0%) when chloroform was used as a solvent (Fig. 3C). Derivative Y and sphingenine were not found after 1 week. When methanol or dichloromethane was used as a solvent, no migration was observed.

To simulate long-term storage, ceramide solutions were kept at 60°C for 3 h. When this test was performed in methanol, acetone, dichloromethane, toluene, or acetonitrile, no rearrangements were revealed by TLC. In chloroform and chloroform-methanol mixtures, however, the formation of ninhydrin-positive material was observed (Fig. 1D, E). HPLC analysis of N-acetyl-sphingenine, stored for 3 years at –20°C and dissolved in chloroform-methanol (1:1, v/v), revealed the presence of 0.8% derivative X (data not shown). Likely, in chloroform or chloroform-containing solvents, some acid-catalyzed migration occurs. Aged chloroform is known to become acidic as a result of water- and/or light-catalyzed generation of radicals and HCl.

Summarizing, our data show that ceramides, contrary to other (fatty) amides, are less stable. As a result of the presence of two hydroxy groups, both vicinal to the amide function, two migration routes are possible, presumably via a five-membered ring intermediate (hydroxyoxazolidine), resulting in the formation of 3-O- and 1-O-acyl derivatives (Fig. 2). These rearrangements occur readily upon exposure of ceramides to acid. Also upon storage, especially in chloroform, O-acylated sphingoid bases can be formed. Hence, our observations call for appropriate storage (in methanol or dichloromethane) of ceramides and ceramide-like compounds. At present, it is unknown whether some of the ceramide-mediated cellular effects could have been caused by O-acyl sphingoid bases. The NO-acyl ceramide derivatives are likely to be readily converted by cellular esterases to sphingoid bases, which are known to be cytotoxic and to affect multiple cellular functions (13, 14). If it is stable in the cytosol, however, the O-acyl derivative will probably be lysomotropic and degraded in the lysosomes.

	ACKNOWLEDGMENTS

 
This work was supported by a grant from the Flemish Fonds voor Wetenschappelijk Onderzoek (G.0405.02) and from the Belgian Ministry of Federaal Wetenschapsbeleid Interuniversitaire Attractiepolen (IAP-P5/05). H.V.O. is an aspirant from the Flemish Fonds voor Wetenschappelijk Onderzoek. The technical support of S. Asselberghs is highly appreciated. The authors thank Dr. J. Rozenski (Katholieke Universiteit Leuven, Rega Instituut, Medicinale Chemie) for help with the mass spectral analysis.

Manuscript received November 18, 2004 and in revised form December 30, 2004.

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This Article Abstract Full Text (PDF) All Versions of this Article: D400034-JLR200v1 46/4/812    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Van Overloop, H. Articles by Van Veldhoven, P. P. Search for Related Content PubMed PubMed Citation Articles by Van Overloop, H. Articles by Van Veldhoven, P. P. Social Bookmarking                      What's this?