title: 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.
authors.affiliation: Department of Translational Medicine, Lund University, Skåne University Hospital, Inga Marie Nilssons gata 53, 205 02, Malmö, Sweden.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Frej
authors.affiliation: Department of Clinical Chemistry and Pharmacology, Lund University, Skåne University Hospital, Klinikgatan 19, 221 85, Lund, Sweden.
authors.forename: Anders
authors.initials: A
authors.lastname: Andersson
authors.affiliation: Department of Clinical Chemistry, Skåne University Hospital, Inga Marie Nilssons gata 53, 205 02, Malmö, Sweden.
authors.forename: Benny
authors.initials: B
authors.lastname: Larsson
authors.affiliation: Department of Translational Medicine, Lund University, Skåne University Hospital, Inga Marie Nilssons gata 53, 205 02, Malmö, Sweden.
authors.forename: Li Jun
authors.initials: LJ
authors.lastname: Guo
authors.affiliation: Department of Translational Medicine, Lund University, Skåne University Hospital, Inga Marie Nilssons gata 53, 205 02, Malmö, Sweden.
authors.forename: Eva
authors.initials: E
authors.lastname: Norström
authors.affiliation: Department of Translational Medicine, Lund University, Skåne University Hospital, Inga Marie Nilssons gata 53, 205 02, Malmö, Sweden.
authors.forename: Kaisa E
authors.initials: KE
authors.lastname: Happonen
authors.affiliation: Department of Translational Medicine, Lund University, Skåne University Hospital, Inga Marie Nilssons gata 53, 205 02, Malmö, Sweden. bjorn.dahlback@med.lu.se.
authors.forename: Björn
authors.initials: B
authors.lastname: Dahlbäck
mesh_terms: D001053:Apolipoproteins / Q000737:chemistry; D000075243:Apolipoproteins M; D001777:Blood Coagulation / Q000502:physiology; D001792:Blood Platelets / Q000737:chemistry / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods / Q000592:standards*; D006801:Humans; D054834:Lipocalins / Q000737:chemistry; D008075:Lipoproteins, HDL / Q000737:chemistry; D059625:Liquid-Liquid Extraction / Q000379:methods; D008246:Lysophospholipids / Q000096:biosynthesis / Q000097:blood*; D010949:Plasma / Q000737:chemistry*; D015539:Platelet Activation / Q000502:physiology; D011485:Protein Binding; D044967:Serum / Q000737:chemistry*; D012709:Serum Albumin / Q000737:chemistry; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000097:blood; D053719:Tandem Mass Spectrometry / Q000379:methods / Q000592:standards*
chemical_list: C400523:APOM protein, human; D001053:Apolipoproteins; D000075243:Apolipoproteins M; D054834:Lipocalins; D008075:Lipoproteins, HDL; D008246:Lysophospholipids; D012709:Serum Albumin; C060506:sphingosine 1-phosphate; D013110:Sphingosine
keywords: Apolipoprotein; Liquid chromatography; Mass spectrometry; Sphingolipid