Sphingolipids, chromatography

Bartke, N., Fischbeck, A., Humpf, H. U. (2006). Analysis of sphingolipids in potatoes Solanum tuberosum L.) and sweet potatoes Ipomoea batatas (L.) Lam.) by reversed phase high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESl-MS/MS). Mol. Nutr. Food Res., 50,1201-1211. 

Certain classes of lipids are susceptible to degradation under specific conditions. For example, all ester-linked fatty acids in triacylglycerols, phospholipids, and sterol esters are released by mild acid or alkaline treatment, and somewhat harsher hydrolysis conditions release amide-bound fatty acids from sphingolipids. Enzymes that specifically hydrolyze certain lipids are also useful in the determination of lipid structure. Phospholipases A, C, and D (Fig. 10-15) each split particular bonds in phospholipids and yield products with characteristic solubilities and chromatographic behaviors. Phospholipase C, for example, releases a water-soluble phosphoryl alcohol (such as phosphocholine from phosphatidylcholine) and a chloroform-soluble diacylglycerol, each of which can be characterized separately to determine the structure of the intact phospholipid. The combination of specific hydrolysis with characterization of the products by thin-layer, gas-liquid, or high-performance liquid chromatography often allows determination of a lipid structure. 

Our new method of sphingolipid analysis using high performance liquid chromatography allows us to determine not only their quantities but also their homolog compositions in a small amount of tissue. We now feel that less than 1 mg of fresh brain or nerve tissue is sufficient for complete analysis. The application of this new method for analyzing cerebroside and sulfatide-in plaques of brain from a patient with multiple sclerosis has been recently described (15). 

In brief [43, 44], primary neurons were prepared from the cerebellum of 6-day-old mice and the cells were treated with different concentrations of the target compounds for 24 h. Radiolabeled 3-[ 14C] serine or [14C]galactose was added to the culture medium and incorporation into newly synthesized sphingolipids was analyzed after labeling for 24 h. Lipids were extracted, separated by thin-layer chromatography, and visualized with a phosphoimager. Radioactivity found in the selected lipids is expressed in relation to untreated cells. 

Mano, N., Oda, Y. and Yamada, K. (1997) Simultaneous quantitative determination method for sphingolipid metabolites by liquid chromatography/ionspray ionization tandem mass spectrometry. Anal. Biochem., 244 (2), 291-300. 

Bielawski, J., Szulc, Z. M., Hannun, Y. A., Bielawska, A. Simultaneous quantitative analysis of bioactive sphingolipids by high-performance liquid chromatography-tandem mass spectrometry. Methods 39 (2006) 82-91. 

Merrill AH, Jr., Sullards MC, Allegood JC, Kelly S, Wang E. Sphingolipidomics high-throughput, structure-specific, and quantitative analysis of sphingolipids by liquid chromatography tandem mass spectrometry. Methods 36(2005) 207-224. 

On a practical level, the sphingolipid field is caught somewhat awkwardly between old and new nomenclatures due to differences in the information that one gets from traditional analytical methods such as radiolabeling followed by thin-layer chromatography versus more structure-specific methods such as mass spectrometry (MS). 

The analysis and identification of sphingolipids have recently been substantially improved by the use of gas-liquid chromatography, fast-atom bombardment mass spectrometry, nuclear magnetic resonance spectroscopy and selective enzymatic cleavage. Despite these efforts, however, many important members of this class of biomolecules remain relatively inaccessible. Isolation of pure compounds is still difficult because of the diversity and heterogeneity of lipids. Effective synthetic routes to these compounds are, therefore, extremely important to investigate their chemical and biological properties. 

Fang, R, Ho, C.-T., Sang, S., and Rosen, R.T., Determination of sphingolipids in nuts and seeds by a single quadrupole liquid chromatography-mass spectrometry method, J. Food Lipids, 12, 327-343, 2005. 

Schwarz (1960) has discussed infrared absorption analysis of tissue constituents, particularly tissue lipids. He has given a method for examining tissue sections, and has discussed the chromatography of extracted phospholipids, sphingolipids, and fatty acids and neutral lipids. 

In recent years, HPLC coupled with ESI-MS has become a well-established method for the identification and detection of chemical stmctures of lipids, including sphingolipids. Recently, Sugawara and coworkers [44] identified the chemical structures of glucosylceramides from maize, rice, mushroom, and sea cucumber by liquid chromatography-ion trap mass spectrometry with an ESI interface. In the positive full-scan mode, [M-t-HJ" ", [M-l-H—H2O ] , or [M-l-H—162]" (loss of glucose) was used for MS/MS analysis to obtain the product ions, which were used for the identification of the glucocerebrosides. 

Characterizing Sphingolipids by Direct Thin-Layer Chromatography-... 

CHARACTERIZING SPHINGOLIPIDS BY DIRECT THIN-LAYER CHROMATOGRAPHY-MATRIX-ASSISTED LASER DESORPTION IONIZATION COUPLING... 

Seng, J.A., Ellis, S.R., Hughes, J.R., Maccarone, A.T., Truscott, R.J., Blanksby, S.J., and Mitchell, T.W. 2014. Characterisation of sphingolipids in the human lens by thin layer chromatography-desorption electrospray ionisation mass spectrometry, Biochim. Biophys. Acta, 1841 1285-1291. 

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