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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-015-9008-4) contains supplementary material, which is available to authorized users.
Highlights
Funding by the Swedish Research Council, the Swedish Heart-Lung Foundation and Söderberg’s Foundation.Electronic supplementary material The online version of this article contains supplementary material, which is available to authorized users.Sphingosine 1-phosphate (S1P) is a sphingolipid with pleiotropic functions [1, 2]
S1P is carried by apolipoprotein M in the lipoproteins and by albumin; approximately 60 % is normally present in high-density lipoproteins (HDL), 10 % in low-density lipoproteins (LDL) and 30 % is bound to albumin [4,5,6]
We demonstrate that apolipoprotein M (apoM) correlates with S1P only in platelet-poor plasma (PPP) but not in platelet-rich plasma (PRP) or serum and that S1P released from platelets during blood coagulation mainly binds to albumin and not to apoM
Summary
Funding by the Swedish Research Council, the Swedish Heart-Lung Foundation and Söderberg’s Foundation.Electronic supplementary material The online version of this article (doi:10.1007/s00216-015-9008-4) contains supplementary material, which is available to authorized users.Sphingosine 1-phosphate (S1P) is a sphingolipid with pleiotropic functions [1, 2]. S1P is present in plasma in the submicromolar range and can be produced by platelets, erythrocytes and endothelial cells. Platelets are not the main source of S1P in plasma and thrombocytopenic mice have normal S1P levels [8]. Erythrocytes, which are the most important source of plasma S1P, can phosphorylate sphingosine to generate as well as store S1P but cannot produce S1P de novo [9, 10]. These features make the handling of blood samples taken for S1P analysis important and delicate; guidelines for and knowledge on pre-analytical standardisation are limited [11, 12]
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