KARAKTERISERING AV EN PLASMABUREN SIGNALMOLEKYL SOM STIMULERAR FRISÄTTNING AV SFINGOSIN-1-FOSFAT FRÅN RÖDA BLODKROPPAR

Abstract

Bioactive sphingolipid Sphingosine-1-phosphate (S1P) is found in blood, where it plays an important part in, for example, angiogenesis. S1P is synthesized in hepatocytes, vascular endothelium and blood cells, then transported with high-density lipoprotein (HDL) or albumin in plasma. S1P can accumulate in, and be released from red blood cells. In experiments, red blood cells (human) were removed from the plasma to examine under which conditions they released S1P. S1P only released from the blood cells when incubated in plasma, not other types of media. This poses the question, what kind of molecule in plasma controls the release of S1P from red blood cells? Previous experiments shows that the molecule might be a protein. The purpose of this study is to characterize and, if possible, identify the signalling molecule. The method consisted of several parts, initially with protein precipitation of the plasma. Precipitated and whole plasma was then fractionated with spin columns with different filter sizes (50kD, 100kD, 300kD) to determine if the size of the protein affects the release of S1P. Blood samples were taken from volunteers and the red blood cells were separated out before being added to untreated or treated plasma. After incubation, lipids were extracted from the samples and then analyzed by LC-MS. Albumin depleted plasma was also incubated with red blood cells to investigate the effect it might have on the release of S1P. Results shows S1P release in the samples with plasma fractionated with all three filter sizes but not from the samples where the plasma proteins are filtered out. It also shows that S1P was not released when incubated with albumin depleted plasma. The conclusion from this study is that the molecule that signals the release of S1P is likely a protein bound to albumin, and this complex is larger than 300kD.