Novel methods for the quantification of (2E)-hexadecenal by liquid chromatography with detection by either ESI QTOF tandem mass spectrometry or fluorescence measurement

Abstract

Sphingosine-1-phosphate lyase (SPL) is the only known enzyme that irreversibly cleaves sphingosine-1-phosphate (S1P) into phosphoethanolamine and (2E)-hexadecenal during the final step of sphingolipid catabolism. Because S1P is involved in a wide range of physiological and diseased processes, determining the activity of the degrading enzyme is of great interest. Therefore, we developed two procedures based on liquid chromatography (LC) for analysing (2E)-hexadecenal, which is one of the two S1P degradation products. After separation, two different quantification methods were performed, tandem mass spectrometry (MS) and fluorescence detection. However, (2E)-hexadecenal as a long-chain aldehyde is not ionisable by electrospray ionisation (ESI) for MS quantification and has an insufficient number of corresponding double bonds for fluorescence detection. Therefore, we investigated 2-diphenylacetyl-1,3-indandione-1-hydrazone (DAIH) as a derivatisation reagent. DAIH transforms the aldehyde into an ionisable and fluorescent analogue for quantitative analysis. Our conditions were optimised to obtain the outstanding limit of detection (LOD) of 1 fmol per sample (30 μL) for LC–MS/MS and 0.75 pmol per sample (200 μL) for LC determination with fluorescence detection. We developed an extraction procedure to separate and concentrate (2E)-hexadecenal from biological samples for these measurements. To confirm our new methods, we analysed the (2E)-hexadecenal level of different cell lines and human plasma for the first time ever. Furthermore, we treated HT-29 cells with different concentrations of 4-deoxypyridoxine (DOP), which competitively inhibits pyridoxal-5-phosphate (P5P), an essential cofactor for SPL activity, and observed a significant decrease in (2E)-hexadecenal relative to the untreated cells.

Introduction

The name “sphingolipid” denotes a ubiquitous, heterogeneous class of compounds with various structural and biochemical properties. Once it became known that sphingolipids are involved in many signalling pathways, their importance escalated. It was initially recognised that sphingolipid signalling is important for tumour development and other human ailments, such as diabetes, heart disease, neurological disorders and immune dysfunctions [1], [2], [3], [4], [5], [6], [7], [8], [9]. A comprehensive understanding of the key role sphingolipids play in essential cellular events, such as differentiation, migration, apoptosis and inflammation, necessitates elucidation of the generation and action of these lipids. Although most of the degradation pathways have been mapped, knowledge on the metabolic organisation and inter-relations of the enzymes involved in sphingolipid metabolism is still required [10]. The transformation of bioactive sphingolipids has a unique metabolic entry point, serine palmitoyl transferase (SPT), that forms the first sphingolipid via the de novo pathway and a unique exit enzyme, S1P lyase that breaks down S1P into non-sphingolipid molecules. The various steps in between these two constitute a highly complex network that connects the metabolism of many sphingolipids with S1P as one of the most important substances [11]. S1P is a major regulator of many biological events, e.g., it enhances cell proliferation, activates migration and facilitates angiogenesis [12]. It can be generated from sphingosine through the activity of two sphingosine kinases. There are also two possible degradation pathways for the lipid. On the one hand, S1P can be dephosphorylated to regenerate sphingosine through a reversible process induced by specific intracellular S1P phosphatases. On the other hand, the membrane-bound enzyme S1P lyase can irreversibly cleave S1P to generate phosphoethanolamine and (2E)-hexadecenal, which can in turn be reduced to palmitate and reincorporated into the lipid metabolic pathway [13], [14]. Because of the diverse roles S1P plays in physiology and pathophysiology, it is important to determine the activity of its degrading enzyme [15], [16]. For this purpose, we developed a modern tandem mass spectrometry (MS) procedure for the sensitive quantification of the SPL-product (2E)-hexadecenal, using DAIH derivatisation. In this manner, it is possible to identify the degradation pathway of S1P and make conclusions about the degree of it. Additionally, an easily practicable quantification procedure for (2E)-hexacecenal was established using instruments (LC/fluorescence detector) that are typically available in most laboratories. The developed methods provide means to investigate S1P degradation and its control in biological systems.