A high-performance liquid chromatographic method to measure sphingosine 1-phosphate and related compounds from sphingosine kinase assays and other biological samples.
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.
Caligan
T B
TB
Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Peters
K
K
Ou
J
J
Wang
E
E
Saba
J
J
Merrill
A H
AH
D001792:Blood Platelets / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D008246:Lysophospholipids; D010710:Phosphates / Q000032:analysis / Q000737:chemistry; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000032:analysis* / Q000097:blood; D009764:o-Phthalaldehyde / Q000032:analysis / Q000737:chemistry; D000099157:Sphingosine Kinase
D008246:Lysophospholipids; D010710:Phosphates; D017853:Phosphotransferases (Alcohol Group Acceptor); D013110:Sphingosine; D009764:o-Phthalaldehyde; D000099157:Sphingosine Kinase; C060506:sphingosine 1-phosphate