SE197:/S02

Sample Set Information

ID	SE197
Title	Separation and quantification of 2-acyl-1-lysophospholipids and 1-acyl-2-lysophospholipids in biological samples by LC-MS/MS
Description	Lysophospholipids (LysoGPs) serve as lipid mediators and precursors for synthesis of diacyl phospholipids(GPs ). LysoGPs detected in cells have various acyl chains attached at either the sn-1 or sn-2 position of the glycerol backbone. In general, acyl chains at the sn-2 position of 2-acyl-1-LysoGPs readily move to the sn-1 position, generating 1-acyl-2-lyso isomers by a nonenzymatic reaction called intra-molecular acyl migration, which has hampered the detection of 2-acyl-1-LysoGPs in biological samples. In this study, we developed a simple and versatile method to separate and quantify 2-acyl-1- and 1-acyl-2-LysoGPs. The main point of the method was to extract LysoGPs at pH 4 and 4°C, conditions that were found to completely eliminate the intra-molecular acyl migration. Under the present conditions, the relative amounts of 2-acyl-1-LysoGPs and 1-acyl-2-LysoGPs did not change at least for 1 week. Further, in LysoGPs extracted from cells and tissues under the present conditions, most of the saturated fatty acids (16:0 and 18:0) were found in the sn-1 position of LysoGPs, while most of the PUFAs (18:2, 20:4, 22:6) were found in the sn-2 position. Thus the method can be used to elucidate the in vivo role of 2-acyl-1-LysoGPs.
Authors	Okudaira, M., A. Inoue, A. Shuto, K. Nakanaga, K. Kano, K. Makide, D. Saigusa, Y. Tomioka, and J. Aoki.
Reference	J. Lipid Res. 2014. 55: 2178–2192
Comment

The original paper is available at PubMed.

The original paper is available at The Journal of Lipid Research site.

Sample Information

ID	S02
Title	Mouse tissues
Organism - Scientific Name
Organism - ID	NCBI taxonomy 10090
Compound - ID
Compound - Source
Preparation	C57BL/6 mice were purchased from CLEA Japan and maintained according to the Guidelines for Animal Experimentation of Tohoku University. Mouse tissues (approximately 100 mg) were placed in 1.5 ml siliconized sample tubes. Then, nine volumes of acidic methanol (pH 4.0), 1 μM (final concentration) of 17:0-LPA, and 10 μM (final concentration) of 17:0-LPC were added to the tube. The obtained mixture was homogenized for 10 min by Micro Smash MS-100R (TOMY) (3,000 rpm at 4°C). After an initial centrifugation at 1,000 g for 10 min at 4°C, the supernatants were further centrifuged at 21,500 g for 10 min at 4°C. The resulting supernatant was passed through a filter (0.2 μm pore size, 4 mm inner diameter; YMC), and 10 μl of the filtrate was subjected to LC-MS/MS. To obtain a serum sample, a blood sample was collected with a noncoated capillary (910-01-75; Hirschmann Laborgerate), incubated at 37°C for 1 h, allowed to stand at 4°C for 12 h, and centrifuged at 1,500 g for 10 min at 25°C. Plasma was collected with a heparinized capillary (1-040-7500-HC; Drummond), followed by addition of 1 mM of EDTA (final concentration), and centrifuged at 1,500 g for 10 min at 25°C. The plasma and serum samples (10 μl each) were placed in 1.5 ml siliconized sample tubes, deproteinized by mixing with 90 μl acidic methanol (pH 4.0), homogenized for 3 min in an ultrasonic bath, and centrifuged at 21,500 g for 10 min at 4°C (ice-cold water). The supernatants were filtered and 10 μl of the filtrate was subjected to the LC-MS/MS.
Sample Preparation Details ID
Comment