An improved high-performance liquid chromatographic method for the determination of sphingosine-1-phosphate in complex biological materials

Sphingosine-1-phosphate (SPP) has been proposed to act both as an intracell ular second messenger and as an extracellular mediator via specific cell su rface receptors. Based on the increasing diverse cellular roles methods to quantify endogenous and exogenous SPP are highly required. Here, we report a rapid HPLC method that allows quantification of SPP in th e picomolar range even in complex biological systems. A two-step lipid extr action serves to separate SPP from most interfering phospholipids and sphin golipids. Importantly, dihydrosphingosine-1-phosphate (dihydro-SPP), not de tectable in all cultured cells and biological samples in considerable amoun ts, possesses equal extraction properties and therefore is an ideal interna l standard. Following extraction SPP and dihydro-SPP are converted to fluor escent isoindol derivatives by ortho-phthaldialdehyde (OPA) and separated b y HPLC using a gradient program with methanol and 0.07 M K2HPO4 as eluents. With this procedure we were able to obtain reproducible measurements of SP P over a broad range from 0.5 pM to 0.2 nM. The identity of SPP and dihydro -SPP was confirmed by the use of the ion pair reagent tetraammoniumsulfate, which induced a shift of both peaks but did not alter peak areas. Moreover , enzymatic conversions to sphingosine and sphinganine by bovine intestinal mucose alkaline phosphatase (AP) excluded the existence of overlapping com pounds. Levels of SPP were determined in a variety of biological samples like serum , thrombocytes, primary keratinocytes and several cell lines. Furthermore, we were able to detect increases of intracellular SPP levels in human kerat inocytes after exposure to 1 alpha ,25-dihydroxyvitamin D-3 (1,25-(OH)(2)D- 3) for which a stimulation of sphingosine kinase activity has been recogniz ed.