A novel method to quantify sphingosine 1-phosphate by immobilized metal affinity chromatography (IMAC)

Sphingosine I-phosphate (S I P), 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 S I P 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 S I P and dihydro-S I P from biological samples by the Fe3+ gel immobilized metal affinity chromatography (IMAC). The eluted S I P 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 S I P was linear for a wide range of S I P concentration. Using this assay, secretion of endogenous S I P from endothelial cells, fibroblasts and colon cancer cells was demonstrated. We also show that dihydro-S I P 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 S I P release. This assay was also used to demonstrate that plasma S I P 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 S I P. (c) 2007 Elsevier Inc. All rights reserved.