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 plateletassociated 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.

Low sphingosine-1-phosphate plasma levels are predictive for increased mortality in patients with liver cirrhosis

“…In contrast to our data, one study detected increased serum S1P levels in patients with liver fibrosis [13]. However, due to the activation of platelets during the clotting process, serum is supposed to be an inadequate sample matrix to reflect S1P levels at the time of blood sampling [14]. Another study, including 15 patients with chronic hepatitis C infection, revealed a decrease of S1P levels with increasing progression of liver fibrosis [15], which supports our findings.…”

Sphingosine‐1‐Phosphate Modulates the Effect of Estrogen in Human Osteoblasts

“…Determining tissue expression of S1P under varying conditions will be a challenging target of further work and will be important to evaluate the role of this new pathway in the regulation of bone differentiation. Measurements of S1P in serum indicate that it circulates at concentrations of 500 to 1000 nM, albeit significantly protein bound and in some circumstances released by platelet activation. Carriers of S1P include albumin and apolipoprotein M; in serum, S1P is important in vascular homeostasis and immune cell response .…”

Accurate quantification of sphingosine-1-phosphate in normal and Fabry disease plasma, cells and tissues by LC-MS/MS with 13 C-encoded natural S1P as internal standard

“…Interpretation of literature reports is complicated by the fact that quantification of plasma S1P is notoriously cumbersome and over time a vast number of different procedures have been employed for its measurement. The use of different analytical techniques by researchers might have contributes to the variations in reported ranges of S1P in plasma of normal individuals (200 to 840 nM) [4,5,8,21,22]. The quantification of S1P is challenging due to its zwitterion character, even more so the determination of relatively low levels in a complex matrix of other lipids.…”