Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
2019938 | Prostaglandins & Other Lipid Mediators | 2007 | 9 Pages |
Abstract
Sphingosine 1-phosphate (S1P), a lysophospholipid mediator that signals through G protein-coupled receptors, regulates a wide plethora of biological responses such as angiogenesis and immune cell trafficking. Detection and quantification of S1P in biological samples is challenging due to its unique physicochemical nature and occurrence in trace quantities. In this report, we describe a new method to selectively enrich S1P and dihydro-S1P from biological samples by the Fe3+ gel immobilized metal affinity chromatography (IMAC). The eluted S1P from IMAC was dephosphorylated, derivatized with o-phthalaldehyde (OPA), and detected by high-performance liquid chromatography (HPLC) coupled to a fluorescence detector. IMAC purification of S1P was linear for a wide range of S1P concentration. Using this assay, secretion of endogenous S1P from endothelial cells, fibroblasts and colon cancer cells was demonstrated. We also show that dihydro-S1P was the major sphingoid base phosphate secreted from HUVEC over expressed with Sphk1 cDNA. Pharmcological antagonists of ABC transporters, glyburide and MK-571 attenuated endogenous S1P release. This assay was also used to demonstrate that plasma S1P levels were not altered in mice deficient for ABC transporters, Abca1, Abca7 and Abcc1/Mrp1. IMAC-based affinity-enrichment coupled with a HPLC-based separation and detection system is a rapid and sensitive method to accurately quantify S1P.
Keywords
MEFIMACHUVECS1PSphingosine 1-phosphate (S1P)sphingosine 1-phosphateABC transportersHuman umbilical vein endothelial cellsEndothelial cellsmouse embryonic fibroblastHigh-performance liquid chromatography (HPLC)HPLChigh-performance liquid chromatographyImmobilized metal affinity chromatography (IMAC)immobilized metal affinity chromatography
Related Topics
Life Sciences
Biochemistry, Genetics and Molecular Biology
Biochemistry
Authors
Yong-Moon Lee, Krishnan Venkataraman, Sun-Il Hwang, David K. Han, Timothy Hla,