Blood sphingolipidomics in healthy humans: impact of sample collection methodology

We used a HPLC-MS/MS methodology for determination of a basic metabolomic profile (18:1,18:0 sphingoid backbone, C14-C26 N-acyl part) of “normal” sphingolipid levels in human serum and plasma. Blood was collected from healthy males and nonpregnant females under fasting and nonfasting conditions with and without anticoagulants. Sphingolipids analyzed included sphingoid bases, sphingosine and dihydrosphingosine, their 1-phosphates (S1P and dhS1P), molecular species (Cn-) of ceramide (Cer), sphingomyelin (SM), hexosylceramide (HexCer), lactosylceramide (LacCer), and Cer 1-phosphate (Cer1P). SM, LacCer, HexCer, Cer, and Cer1P constituted 87.7, 5.8, 3.4, 2.8, and 0.15% of total sphingolipids, respectively. The abundant circulating SM was C16-SM (64.0 µM), and it increased with fasting (100 µM). The abundant LacCer was C16-LacCer (10.0 µM) and the abundant HexCer was C24-HexCer (2.5 µM). The abundant Cer, C24-Cer (4.0 µM), was not influenced by fasting; however, levels of C16-C20 Cers were decreased in response to fasting. S1P levels were higher in serum than plasma (0.68 µM vs. 0.32 µM). We also determined levels of sphingoid bases and SM species in isolated lipoprotein classes. HDL3 was the major carrier of S1P, dhS1P, and Sph, and LDL was the major carrier of Cer and dhSph. Per particle, VLDL contained the highest levels of SM, Cer, and S1P. HPLC-MS/MS should provide a tool for clinical testing of circulating bioactive sphingolipids in human blood. We used a HPLC-MS/MS methodology for determination of a basic metabolomic profile (18:1,18:0 sphingoid backbone, C14-C26 N-acyl part) of “normal” sphingolipid levels in human serum and plasma. Blood was collected from healthy males and nonpregnant females under fasting and nonfasting conditions with and without anticoagulants. Sphingolipids analyzed included sphingoid bases, sphingosine and dihydrosphingosine, their 1-phosphates (S1P and dhS1P), molecular species (Cn-) of ceramide (Cer), sphingomyelin (SM), hexosylceramide (HexCer), lactosylceramide (LacCer), and Cer 1-phosphate (Cer1P). SM, LacCer, HexCer, Cer, and Cer1P constituted 87.7, 5.8, 3.4, 2.8, and 0.15% of total sphingolipids, respectively. The abundant circulating SM was C16-SM (64.0 µM), and it increased with fasting (100 µM). The abundant LacCer was C16-LacCer (10.0 µM) and the abundant HexCer was C24-HexCer (2.5 µM). The abundant Cer, C24-Cer (4.0 µM), was not influenced by fasting; however, levels of C16-C20 Cers were decreased in response to fasting. S1P levels were higher in serum than plasma (0.68 µM vs. 0.32 µM). We also determined levels of sphingoid bases and SM species in isolated lipoprotein classes. HDL3 was the major carrier of S1P, dhS1P, and Sph, and LDL was the major carrier of Cer and dhSph. Per particle, VLDL contained the highest levels of SM, Cer, and S1P. HPLC-MS/MS should provide a tool for clinical testing of circulating bioactive sphingolipids in human blood. Sphingolipids, once considered mainly structural components of cell membranes, have emerged as key signaling molecules involved in the regulation of a variety of cellular functions, including cell growth and differentiation, proliferation, and apoptotic cell death (1Cuvillier O. Pirianov G. Kleuser B. Vanek P.G. Coso O.A. Gutkind S. Spiegel S. Suppression of ceramide-mediated programmed cell death by sphingosine-1-phosphate.Nature. 1996; 381: 800-803Crossref PubMed Scopus (1352) Google Scholar, 2Hannun Y.A. Functions of ceramide in coordinating cellular responses to stress.Science. 1996; 274: 1855-1859Crossref PubMed Scopus (1497) Google Scholar, 3Mathias S. Pena L.A. Kolesnick R.N. Signal transduction of stress via ceramide.Biochem. J. 1998; 335: 465-480Crossref PubMed Scopus (620) Google Scholar, 4Spiegel S. Merrill Jr, A.H. Sphingolipid metabolism and cell growth regulation.FASEB J. 1996; 10: 1388-1397Crossref PubMed Scopus (650) Google Scholar). Sphingolipids are distributed in the cytoplasmic and plasma membranes and interact with cholesterol to form membrane lipid microdomains that allow signal transduction. Complex membrane sphingolipids such as sphingomyelin (SM) and glycosphingolipids modulate the behavior of growth factor receptors and extracellular matrix proteins and also serve as binding sites for some microorganisms, toxins, and viruses (5Vesper H. Schmelz E.M. Nikolova-Karakashian M.N. Dillehay D.L. Lynch D.V. Merrill Jr, A.H. Sphingolipids in food and the emerging importance of sphingolipids to nutrition.J. Nutr. 1999; 129: 1239-1250Crossref PubMed Scopus (367) Google Scholar). Maintenance of the membrane structure is crucial for mechanical stabilization, and any local or global changes in lipid asymmetry can induce a variety of cellular responses. Sphingolipids are the most structurally diverse as well as complex category of lipids due to their numerous variations in the sphingoid bases, N-acyl linked fatty acids, and head groups (5Vesper H. Schmelz E.M. Nikolova-Karakashian M.N. Dillehay D.L. Lynch D.V. Merrill Jr, A.H. Sphingolipids in food and the emerging importance of sphingolipids to nutrition.J. Nutr. 1999; 129: 1239-1250Crossref PubMed Scopus (367) Google Scholar). Several sphingolipid metabolites, especially ceramide (Cer), sphingosine (Sph), sphingosine 1-phosphate (S1P), and Cer 1-phosphate (Cer1P), have been identified as bioactive signaling molecules that control cell growth and death (1Cuvillier O. Pirianov G. Kleuser B. Vanek P.G. Coso O.A. Gutkind S. Spiegel S. Suppression of ceramide-mediated programmed cell death by sphingosine-1-phosphate.Nature. 1996; 381: 800-803Crossref PubMed Scopus (1352) Google Scholar, 2Hannun Y.A. 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B. of bioactive sphingolipids by 2009; PubMed Scopus Google Scholar). the a for of the sphingolipids: sphingoid bases Sph, sphingoid S1P, and molecular species (Cn-) of Cer Cer1P SM hexosylceramide and lactosylceramide a basic metabolomic of human serum and plasma The was to establish a profile of sphingolipids in “normal” human serum and plasma with and to a for circulating sphingolipid species to used for and clinical in and The was by the the of and was from were in the and with or were and or were also the were for healthy levels of lipid and and including and were also for and and were the and and the testing was the clinical of are in were to for the fasting of the fasting were with to of The was of and and contained from fat as determined by a The nonfasting was collected the of the profile of LDL VLDL Blood total total concentration are in a are serum was collected in serum with plasma was collected in plasma with or as Blood was also collected in with as plasma the to the was the were to provide the the of collected in and of the the were S. for serum and plasma Res. 2009; 8: PubMed Scopus Google Scholar). serum were for the to and for to the plasma were on for were for serum and plasma were were and lipid and were from and Sphingolipid were from or from a and with of The were from and to for serum and plasma (100 including from and were for was to in with The were also with of in for The were with of and and for on a The was to The plasma or serum was with and of was to of The were and for The was and to the the and by was for SM and the was to with The were in of The were to with in were for on the The 1 for SM determination was to under The were in 1 and of in and The were to for the were to the and were the of and for The was including any the was to under The were in and to in were for on the of sphingolipids were by HPLC-MS/MS The of a and to a with in were under a of in in and of 1 in in on a J. J. B. of bioactive sphingolipids by 2009; PubMed Scopus Google Scholar). Sphingolipids analyzed included sphingoid bases and their 1-phosphates (S1P and dhS1P), and of Cer, SM, HexCer, LacCer, and Quantitative were on for from to with a of were to serum and plasma The were also analyzed by the HPLC-MS/MS in a The was of to analyzed The concentration was determined by the on the Sphingolipids with were the of of the and sphingolipids with are in The was to the of serum and plasma were to the of and Lipid of serum and plasma to and were The for the and and were by the of from and for to The was by the from the Blood was collected a from healthy were not for any or and without of or of the were and the was by the The was from or in the of a lipoprotein of and to the of a major on and LDL that cholesterol Blood was and plasma was VLDL were isolated from plasma with with by for on a a LDL was isolated with by for and HDL3 were isolated by of plasma with with in a for The were and isolated was and by in a were µM by a and levels in lipoprotein were by the (100 of and and were and analyzed HPLC-MS/MS as The of the Sph, S1P, dhS1P, and of lipoprotein was the sphingolipid concentration lipoprotein used the molecular of and for and and the in the lipoprotein as and for and Quantitative of Blood and and Scholar, of the of the molecular of PubMed Scopus Google Scholar, J. in and Scholar). The of lipoprotein concentration in plasma was as by by Full Text Full Text PDF PubMed Scopus Google Scholar). of the and the for the were the and HDL3 and respectively. two groups were by and than two groups by or by for or are as for sphingolipids in lipoprotein from plasma are as of total sphingolipid that SM was the sphingolipid in serum and in males and and the of sphingolipids sphingolipid in serum and plasma was as SM by LacCer HexCer Cer S1P and Cer1P and and The most SM species was was higher in the serum of fasting with nonfasting µM vs. µM) The most abundant SM was however, not the response to fasting conditions as with in also to the of SM species in plasma with the for the are not of sphingolipid to total sphingolipid are from serum fasting or nonfasting from nonfasting or from fasting or nonfasting or in a are from serum fasting or nonfasting from nonfasting or from fasting or nonfasting or The highest to SM was LacCer, it was abundant than SM in serum and plasma The most LacCer species in and was C16-LacCer µM) nonfasting levels of and with fasting males the in LacCer levels as females levels of and than fasting the was higher in nonfasting than fasting with the in females in are for plasma with and are for serum and plasma with anticoagulants. We analyzed the molecular species of HexCer, and the HexCer species profile in serum and plasma of fasting and nonfasting The highest HexCer species in serum and plasma was C24-HexCer (2.5 µM), with fasting and nonfasting was higher in nonfasting than fasting in serum and plasma was used as was in the plasma of nonfasting than fasting in also to the of HexCer species in serum and plasma from not We analyzed the molecular species of Cer, and and S1P and under fasting in the Cer species was C24-Cer (4.0 µM), with in levels of Cer species serum and plasma or in serum S1P levels were higher than plasma S1P levels (0.68 µM vs. The higher levels for S1P in serum were mainly a of of S1P the K. S. a bioactive sphingolipid in is a of human plasma and PubMed Scopus Google Scholar). levels were also higher than plasma levels µM vs. were in Cer species or sphingoid bases and their 1-phosphates and We the of fasting on the levels of Cer species and sphingoid bases and their was of fasting on the levels of the C24-Cer in males and females levels of and Cers and were under fasting than nonfasting conditions in males and were in levels of sphingoid bases and S1P fasting and nonfasting the Cer1P and were the most abundant in males and females fasting serum levels were than nonfasting levels of not in response to fasting in are for however, are also for plasma The in levels of serum and plasma in higher levels of sphingolipids were in females than sphingolipids included and total that levels of and were higher in females than with species a of higher in females than levels of in females with males by Full Text Full Text PDF PubMed Scopus Google in for the higher sphingolipid levels in in levels of serum are from under fasting in a are from under fasting We analyzed the to the of and with the of on sphingolipid levels in collected human blood. in the from serum in sphingolipid levels with and We also the of and with is the most used in clinical in the and a to levels of sphingolipids with was higher in levels of molecular species of HexCer than the of on levels of LacCer, S1P, and was from that of in and also in levels of S1P and were higher in serum than plasma to of of on sphingolipid are males and females under fasting from from from in a are males and females under fasting from from from including human serum and plasma are and to of the of and on the of sphingolipids in serum and plasma. and plasma from fasting were used to the of and were in sphingolipid levels the not in however, and of a of and of also the in the in was a of levels for of sphingolipid levels in plasma the and of and on of sphingolipid of from in sphingolipid levels in a of from in sphingolipid levels was also used to sphingolipid of isolated and of and The of SM, Cer, and S1P, and the of and in lipoprotein are in the lipoprotein the VLDL contained the highest levels of SM, Cer, and S1P. The lipoprotein particle, contained the levels of SM and Cer, S1P levels were higher than and LDL also in and HDL3 and the in their and The in the LDL was higher than the VLDL to mainly due to the of Cer in the LDL of SM, Cer, and S1P in human lipoprotein lipoprotein particle, molecular for and HDL3 are and of in the lipoprotein are and for and are plasma for Cer and S1P in and plasma Blood was from or in the of and lipoprotein particle, molecular for and HDL3 are and of in the lipoprotein are and for and Quantitative of Blood and and Scholar, of the of the molecular of PubMed Scopus Google Scholar, J. in and Scholar). in a are plasma for Cer and S1P in and plasma Blood was from or in the of and of sphingolipids on lipoprotein in human plasma are in of S1P was on HDL3 HDL3 were the major of and Sph, with and of total and dhS1P, respectively. The major carrier of Cer and was with and of total Cer and The lipoprotein the to plasma sphingolipids was mainly concentration in plasma is the of SM, Cer, Sph, S1P, and in human and sphingolipid concentration in lipoprotein from was by the lipoprotein concentration are plasma for Cer, Sph, S1P, and in and plasma Blood was from or in the of and sphingolipid concentration in lipoprotein from was by the lipoprotein concentration by Full Text Full Text PDF PubMed Scopus Google Scholar). in a are plasma for Cer, Sph, S1P, and in and plasma Blood was from or in the of and The of the of the molecular species of SM, Cer, and and and their in lipoprotein are in The levels of SM species in VLDL were higher than LDL and levels of SM species in were higher than HDL3 levels of Cer species in VLDL were higher than LDL and levels of Cer species in were higher than HDL3 the most abundant SM species in lipoprotein was C16-SM C24-Cer was the Cer with the highest levels Cer species in by and in VLDL contained higher levels of Sph, S1P, and than LDL levels of S1P, not Sph, or dhS1P, were higher in the HDL3 than in the The major of was to establish a profile of sphingolipids in “normal” human serum and plasma with and a for circulating sphingolipid species to used for and clinical in and The structural of sphingolipids, and their in and in that for lipids Sphingolipids groups in their are in and in are in and have and most sphingolipids necessary for their The of with a profile of the of lipid molecules the a for of major sphingolipids on and sphingoid bases and sphingoid (S1P and dhS1P), and molecular species of Cer, SM, HexCer, and LacCer a basic metabolomic of human serum and plasma The of that C16-SM and and levels of C16-C20 and decreased in response to fasting. Thus, in were to in levels of sphingolipids in response to fasting. should changes in sphingolipid levels and also of fasting and The also in levels of serum and plasma levels of and total and were in females than in levels of in females with males in for the higher sphingolipid levels in and to in sphingolipid levels males and are for in clinical the and a to levels of sphingolipids with was higher in levels of molecular species of HexCer than anticoagulants. the from serum in sphingolipid levels with and Thus, the for sphingolipids was not that serum S1P levels were higher than plasma S1P is for of however, a to S1P K. S. a bioactive sphingolipid in is a of human plasma and PubMed Scopus Google Scholar). a and S1P K. and of sphingolipids in J. 1999; PubMed Scopus Google Scholar, K. S. a bioactive sphingolipid in is a of human plasma and PubMed Scopus Google Scholar). 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