Hiroya Hidaka *1), Masaki Takiwaki 2), Mine Yamashita 2), Shinya Otsuki 1), Kenji Kawasaki 3), Mitsutoshi Sugano 3) and Takayuki Honda 4)

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1 Mild acid hydrolysis of sphingolipids yields lysosphingolipids: a matrix-assisted laser desorption and ionization time-of-flight mass spectrometry study Hiroya Hidaka *1), Masaki Takiwaki 2), Mine Yamashita 2), Shinya Otsuki 1), Kenji Kawasaki 3), Mitsutoshi Sugano 3) and Takayuki Honda 4) The mechanisms underlying the generation of lysosphingolipids remain unclear. The present study investigated whether sphingolipids can be de-n-acylated under relatively mild acidic conditions to produce lysosphingolipids. Sphingolipids (ceramide, sphingomyelin, cerebroside, lactosylceramide, trihexosylceramide, globoside and Forssman glycolipid) were dissolved in chloroform/methanol (2:1 v/v) and then treated with 0.5N HCl at room temperature. The hydrolysis products were analyzed using thin layer chromatography and matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry. We found that for all sphingolipid species tested, the relatively mild acid treatment resulted in the production of a de-n-acylated sphingolipid. A time-course study showed that approximately 15% of the sphingomyelin sample was hydrolyzed in the first 24 h of treatment, and that hydrolysis continued at a slower rate for 3 weeks thereafter. It appeared that sphingolipid deacylation was induced by cyclic elimination of a fatty acid due to N-acyl to O-acyl conversion. The present findings indicate that lysosphingolipids can be generated from sphingolipids under relatively mild acidic conditions. Sphingomyelin, Lysosphingomyelin, Ceramide, Glycosphingolipid, Mass analysis

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3 Journal of Analytical Bio-Science Vol. 35, No 3 (2012) 2.4. Mass spectrometry Mass spectrometer measurements were performed as previously described using a MALDI-TOF mass spectrometer (Voyager Elite XL, PerSeptive Biosystems, Framingham, MA)19). Extracted lipids were mixed with an equal quantity of 2,5-DHB, and 1μl of that mixture was applied to the spectrometer metal sample plate. The sample was allowed to dry, after which routine MALDI-TOF mass spectrometry analysis in positive ion mode was performed Thin layer chromatography Samples (1μl of 1 mg/ml in C/M, 2:1 v/v) were applied to TLC silica gel plates and the plates were placed in a chamber with a solvent mixture of chloroform, methanol and aqueous ammonia (65: 35: 8, v/v/v). Lipids were visualized by spraying the plate with a solution of primuline reagent and then exposing the plate to UV light (366 nm). 0.5N HCl for 3 weeks at room temperature, and then analyzed using TLC. Untreated sphingomyelin (1 mg/ml in C/M, 2:1 v/v) appeared as two adjacent spots following TLC (Fig. 1). Treatment with 0.2 and 0.5N HCl resulted in the generation of lysosphingomyelin, whereas treatment with 0.02 or 0.1N HCl did not. MALDI-TOF mass spectrometry analysis of untreated sphingomyelin revealed a number of ion peaks (Fig. 2-A). The peaks at m/z 669.8, 703.6, 725.6, 781.8, and represented various sphingomyelin molecular species containing different fatty acids. An ion peak at m/z represented the sodium ion of 2,5-DHB ([M+Na]+). Treatment of sphingomyelin with 0.5N HCl for 3 weeks at room temperature resulted in a significant reduction in the intensity of the peaks between m/z and 837.7, and the appearance of new peaks at m/z and (Fig. 2-B). The latter peaks were identified as protonated and sodium-positive sphingosylphosphorylcholins ([M+H]+ and [M+Na]+), respectively (Table 1). 3. Results A time-course study was performed to examine 3.1. Degradation of sphingomyelin Sphingomyelin was treated with 0.02, 0.1 or Fig. 1 The generation of lysosphingomyelin following relatively mild acid hydrolysis of sphingomyelin. Untreated sphingomyelin or sphingomyelin treated for 3 weeks with various concentrations of HCl was subjected to TLC analysis as described in the Methods (developing solvent: chloroform, methanol and aqueous ammonia at 65:35:8 v/v/v ratio). Lane 1; untreated sphingomyelin, Lanes 25; sphingomyelin treated with 0.02, 0.1, 0.2 and 0.5N HCl, respectively. Fig The effect of mild acid hydrolysis on sphingomyelin MALDI-TOF mass spectra. Sphingomyelin was treated without (A) or with (B) 0.5N HCl for 3 weeks at room temperature, and then analyzed using positive ion mode MALDI-TOF mass spectrometry.

4 Journal of Analytical Bio-Science Table 1 Identification of MALDI-TOF mass spectrometry molecular ion peaks formed following treatment of sphin-golipids with 0.5N HCl at room temperature. m/z: mass/charge, LCB: long chain base = sphingosine bases, FA: fatty acid, M: identified compound (sphingolipids). Fig. 3 Time course of sphingomyelin hydrolysis. Pure sphingomyelin (1 mg/ml in C/M, 2:1 v/v) was treated with 0.5N HCl at room temperature. Samples were taken at the times indicated and analyzed in positive ion mode using MALDITOF MS. : Sphingomyelin (total height of peaks at m/z 703 and 725), : lysosphin gomyelin (total height of peaks at m/z 466 and 488). the rate of lysosphingomyelin production during the 3week treatment. We found that approximately 15% of the sphingomyelin sample was hydrolyzed in the first 24 h, after which degradation occurred at a slower rate over the 3-week period (Fig. 3). Fig. 4 Mild acidic hydrolysis of ceramide. Ceramide (Cer) was treated without (A) or with (B) 0.5N HCl at room temperature for 1 week, and the products analyzed in positive ion mode using MALDI-TOF mass spectrometry Degradation of ceramide We examined the effect of mild acid hydrolysis on 244

5 Journal of Analytical Bio-Science Vol. 35, No 3 (2012) Fig. 5 ceramides. MALDI-TOF mass spectrometry analysis of untreated ceramides with various fatty acids revealed ion peaks at m/z 508.7, 520.7, 536.8, 560.7, 564.8, 592.8, and (Fig. 4-A). The ion peak at m/z represented the sodium ion of 2,5DHB ([M+Na]+). Following treatment with 0.5N HCl for one week at room temperature, additional ion peaks were detected at m/z 300.4, and (Fig. 4-B). Those peaks corresponded to protonated sphingosine ([M+H]+), dehydroxy sphingosine ([MOH]+), and two protonated molecules of dehydrated sphingosine ([M-2H2O+H]+) (Table1). In addition, there was a decrease in the intensity of peaks in the Mild acidic hydrolysis of glycosphingolipids. Cerebroside (CMH; Gal-Cer) (panel A), lactosylceramide (CDH; Gal-Glu-Cer) (panel B) or trihexosyl ceramide (CTH; Gal-Gal-Glu-Cer) (panel C) was treated without (a) or with (b) 0.5N HCl at room temperature for one week, and the products analyzed in positive ion mode using MALDI-TOF mass spectrometry. region from m/z to (i.e., ceramide) Degradation of glycosphingolipids The effect of a 1-week treatment with 0.5N HCl at room temperature on three glycosphingolipids was investigated. MALDI-TOF mass spectrometry analysis of untreated cerebroside (CMH) revealed mass ion peaks at m/z 738.6, 750.7, 766.7, 792.8, 806.7, 832.8, 834.8, 848.8, and (Fig. 5-A-a). After treatment, two new ion peaks were detected at m/z and (Fig. 5-A-b), which were identified as individual molecular species of protonated and sodium-positive lysocerebroside (Table 1). 245

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