A high-performance liquid chromatographic method to measure sphingosine 1-phosphate and related compounds from sphingosine kinase assays and other biological samples.

Published

Journal Article

Sphingosine 1-phosphate is an intermediate of sphingosine catabolism as well as a potent signaling compound. Conditions were established for the extraction and analysis of sphingosine 1-phosphate and other sphingoid base 1-phosphates from in vitro sphingosine kinase assays and other biological samples. The sphingoid base 1-phosphates were extracted in high yield (85%) using small C-18 reverse-phase columns (LiChroprep RP-18). After the extracts were treated with 0.1 N KOH to remove glycerolipids, the sphingoid base 1-phosphates were converted to fluorescent o-phthalaldehyde derivatives that were separated by HPLC using C-18 columns with a mobile phase of methanol:10 mM potassium phosphate (pH 7.2):1 M tetrabutylammonium dihydrogen phosphate (in water) (83:16:1, v/v/v). The o-phthalaldehyde derivative of sphingosine 1-phosphate was reasonably stable (t(1/2) > or = 18 h) when EDTA was present and could be detected in picomole amounts. The HPLC retention time of the sphingoid base 1-phosphates could be shifted by adjusting the mobile phase to pH 5.5, which is useful in separating overlapping compounds (such as sphingosine 1-phosphate and 4-D-hydroxysphinganine) and in confirming the identity of sphingoid base 1-phosphates in biological samples. The extraction procedure and HPLC method facilitated assays of sphingosine kinase with different sphingoid bases as substrates and/or inhibitors and enabled the quantitation of sphingoid base 1-phosphates in human plasma, serum, and platelets as well as in strains of Saccharomyces cerevisae with mutations in sphingolipid metabolism.

Full Text

Duke Authors

Cited Authors

  • Caligan, TB; Peters, K; Ou, J; Wang, E; Saba, J; Merrill, AH

Published Date

  • May 15, 2000

Published In

Volume / Issue

  • 281 / 1

Start / End Page

  • 36 - 44

PubMed ID

  • 10847608

Pubmed Central ID

  • 10847608

International Standard Serial Number (ISSN)

  • 0003-2697

Digital Object Identifier (DOI)

  • 10.1006/abio.2000.4555

Language

  • eng

Conference Location

  • United States