Quantification of sphingosine 1-phosphate by validated LC-MS/MS method revealing strong correlation with apolipoprotein M in plasma but not in serum due to platelet activation during blood coagulation

Abstract

Sphingosine 1-phosphate (S1P) is a signalling sphingolipid affecting multiple cellular functions of vascular and immune systems. It circulates at submicromolar levels bound to HDL-associated apolipoprotein M (apoM) or to albumin. S1P in blood is mainly produced by platelets and erythrocytes, making blood sampling for S1P quantification delicate. Standardisation of sampling is thereby of great importance to obtain robust data. By optimising and characterising the extraction procedure and the LC-MS/MS analysis, we have developed and validated a highly specific and sensitive method for S1P quantification. Blood was collected from healthy individuals (n = 15) to evaluate the effects of differential blood sampling on S1P levels. To evaluate correlation between S1P and apoM in different types of plasma and serum, apoM was measured by ELISA. The method showed good accuracy and precision in the range of 0.011 to 0.9 μM with less than 0.07 % carryover. We found that the methanol precipitation used to extract S1P co-extracted apoM and several other HDL-proteins from plasma. The platelet-associated S1P was released during coagulation, thus increasing the S1P concentration to double in serum as compared to that in plasma. Gel filtration chromatography revealed that the platelet-released S1P was mainly bound to albumin. This explains why the strong correlation between S1P and apoM levels in plasma is lost upon the clotting process and hence not observed in serum. We have developed, characterised and validated an efficient, highly sensitive and specific method for the quantification of S1P in biological material.

Keywords: Apolipoprotein; Liquid chromatography; Mass spectrometry; Sphingolipid.

Fig. 1

Evaluation of ion suppression and elution profile for C17S1P, S1P and deuterium-marked S1P. A Elution profile of S1P extracted from human plasma with C17S1P as IS. A, B The dark blue and red traces represent the quantification (m/z 380/264) and qualification ions for S1P (eluted after 3.8 min), respectively. The green and grey traces represent the quantification (m/z 366/250) and the qualification ions (m/z 366/82) for C17S1P (eluted after 3.4 min), respectively. The light blue line represents carbon C¹³ isotopes of palmitoyl-oleoyl-phosphatidylcholine (m/z 763/185) that elute after 6–7 min. B–D Ion suppression analysis in human plasma. B Elution timepoints for C17S1P, S1P and palmitoyl-oleoyl-phosphatidylcholine, C quantification ion of S1P (m/z 380/264) and D quantification ion for C17S1P (m/z 366/250). E Elution profile of S1P extracted from human plasma with d7S1P as IS. The chromatogram shows ions for S1P and d7S1P both eluting after 3.7 min and carbon C¹³ isotopes of palmitoyl-oleoyl-phosphatidylcholine, eluting after 6–7 min. F Plotted standard curve of CS. G S1P signal in 0.011 μM CS (LLOQ); H, I carryover evaluation. Representative chromatograms of 0.9 μM CS (H) and blank (I)

Fig. 2

HDL-proteins in methanol extract. A, B Citrate-plasma was extracted by methanol precipitation and compared to unextracted citrate-plasma. A Total protein analysis by silver staining. B Apolipoprotein content analysed by western blotting

Fig. 3

Platelet contamination affecting S1P analysis and the S1P correlation to apoM. Citrate-plasma was collected from 15 healthy individuals and centrifuged at different protocols. A Platelet concentration was measured by flow cytometry, B S1P by LC-MS/MS and C apoM by ELISA. D Correlation analysis between S1P and apoM. Results are plotted as individual values. Statistical analysis between groups was made by Friedman’s test, and correlation was calculated by Spearman’s rank correlation coefficient (r _S). *p < 0.05; **p < 0.01, ****p < 0.0001

Fig. 4

S1P and apoM concentrations in different plasma types and in serum. Citrate-, Li-hep- and EDTA-plasma and serum were collected from 15 healthy individuals and centrifuged for 2000g in 20 min. A S1P was analysed by LC-MS/MS. B apoM was analysed by ELISA; C correlation analysis between S1P and apoM. Results are plotted as individual values. Statistical analysis between groups was made by Friedman’s test, and correlation was calculated by Spearman’s rank correlation coefficient (r _S). *p < 0.05; ***p < 0.001, ****p < 0.0001

Fig. 5

S1P released during blood coagulation binds to albumin. Citrate-plasma or serum pooled from 10 individuals (total volume 500 μL) was subjected to gel filtration chromatography. S1P was extracted from the collected fractions and analysed in LC-MS/MS. ApoM was measured by ELISA and albumin was measured by a commercial kit. Area under the curve was calculated to evaluate the S1P content eluted with either apoM or albumin