Rapid Evaluation of 25 key sphingolipids and phosphosphingolipids in human plasma by LC-MSMS (original) (raw)
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Chemistry and Physics of Lipids, 2008
BACKGROUND: Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are ubiquitous lipid messengers found in the blood and most cell types. Both lysophospholipids are ligands of G protein-coupled receptors and mediate important physiological processes. Moreover, lysophospholipids are potential biomarkers for various diseases, including atherosclerosis and cancer. Because existing methodologies are of limited value for systematic evaluations of S1P and LPA in clinical studies, we developed a fast and simple quantification method that uses liquid chromatography-tandem mass spectrometry (LC-MS/MS).
Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2011
Sphingolipids comprise bioactive molecules which are known to play important roles both as intracellular and extracellular signalling molecules. Here we used a previously developed hydrophilic interaction chromatography tandem mass spectrometry (HILIC-MS/MS) method to profile plasma sphingolipids. Method validation showed sufficient precision and sensitivity for application in large clinical studies. Sample stability testing demonstrated that immediate plasma separation is important to achieve reliable results. Analysis of plasma from 25 healthy blood donors revealed a comprehensive overview of free sphingoid base, sphingosylphosphorylcholine (SPC), hexosylceramide (HexCer), lactosylceramide (LacCer), and ceramide-1-phosphate (Cer1P) species level. Besides the major sphingoid base sphingosine (d18:1), we found d16:1 and d18:2 species in most of these lipid classes. Interestingly, pronounced differences were detected in the species profiles of HexCer and LacCer. Additionally, sphingolipids were quantified in lipoprotein fractions prepared by fast performance liquid chromatography (FPLC). HexCer and LacCer showed similar distributions with about 50% in LDL, 40% in HDL and less than 10% in the VLDL fraction. More than 90% of sphingoid base phosphates were found in HDL and albumin containing fractions. In summary, HILIC-MS/MS provides a valuable tool to profile minor sphingolipid species in plasma and in lipoprotein fractions. Comparing profiles from tissues or blood cells, these species profiles may help to address the origin of plasma sphingolipids.
Journal of Chromatography B, 2012
Bioactive glycerophospho-and sphingolipids species are involved in the regulation of numerous biological processes and implicated in the pathophysiology of various diseases. Here we review electrospray ionization tandem mass spectrometric (ESI-MS/MS) methods for the analysis of these bioactive lipid species in blood including lysophosphatidic acid (LPA), lysophosphatidylcholine (LPC), bis(monoacylglycero)phosphate (BMP), ceramide (Cer), sphingosine-1-phosphate (S1P) and sphingosylphosphorylcholine (SPC). Beside direct tandem mass spectrometric and liquid chromatography coupled approaches, we present an overview of concentrations of these bioactive lipids in plasma. The analytical strategies are discussed together with aspects of sample preparation, quantification and sample stability.
A rapid and quantitative LC-MS/MS method to profile sphingolipids
The Journal of Lipid Research, 2010
including ceramide, sphingosine (SPH), and SPH-1phosphate (S1P), have been shown to be involved in different cell functions such as proliferation, differentiation, growth arrest, and apoptosis (2). Especially the counterregulatory functions of ceramide and S1P, resembling the sphingolipid rheostat, indicate that not only a single metabolite concentration but rather the relative levels of these lipids are important to determine the cell fate (2-5). Sphingolipids are associated with several diseases such as cancer, obesity, and atherosclerosis (1, 2, 6-9). Structural diversity and inter-conversion of these sphingolipid metabolites represent technical challenges. Nevertheless, to understand the differential role of sphingolipids in a regulatory network, it is imperative to use specifi c and quantitative methods. During the last decade, LC-MS/MS has become a powerful tool for sphingolipid analysis (10-21). However, either these methods do not cover a broad spectrum of sphingolipid metabolites or they show disadvantages like laborious sample preparation, time-consuming LC-separation, or separation of analytes and internal standards (ISs). Therefore, we applied, as previously described for lysophosphatidic acid and sphingoid base phosphates, hydrophilic interaction chromatography (HILIC) coupled to MS (18) to achieve coelution of sphingolipid species and their ISs. We present a fast and simple LC-MS/MS method for the quantifi cation of hexosylceramide (HexCer), lactosylceramide (LacCer), sphingosine (SPH), sphinganine (SPA), phyto-SPH (PhytoSPH), di-and trimethyl-SPH (Di-; TrimetSPH), sphingosylphosphorylcholine (SPC), ceramide-1-phosphate (Cer1P), and dihydroceramide-1phosphate (dhCer1P). This method was validated and ap-Abstract Sphingolipids comprise a highly diverse and complex class of molecules that serve not only as structural components of membranes but also as signaling molecules. To understand the differential role of sphingolipids in a regulatory network, it is important to use specifi c and quantitative methods. We developed a novel LC-MS/MS method for the rapid, simultaneous quantifi cation of sphingolipid metabolites, including sphingosine, sphinganine, phyto-sphingosine, di-and trimethyl-sphingosine, sphingosylphosphorylcholine, hexosylceramide, lactosylceramide, ceramide-1-phosphate, and dihydroceramide-1-phosphate. Appropriate internal standards (ISs) were added prior to lipid extraction. In contrast to most published methods based on reversed phase chromatography, we used hydrophilic interaction liquid chromatography and achieved good peak shapes, a short analysis time of 4.5 min, and, most importantly , coelution of analytes and their respective ISs. To avoid an overestimation of species concentrations, peak areas were corrected regarding isotopic overlap where necessary. Quantifi cation was achieved by standard addition of naturally occurring sphingolipid species to the sample matrix. The method showed excellent precision, accuracy, detection limits, and robustness. As an example, sphingolipid species were quantifi ed in fi broblasts treated with myriocin or sphingosine-kinase inhibitor. In summary, this method represents a valuable tool to evaluate the role of sphingolipids in the regulation of cell functions.
Journal of Lipid Research, 2003
Sphingosine (SPH) comprises the backbone of sphingolipids and is known as a second messenger involved in the modulation of cell growth, differentiation, and apoptosis. The currently available methods for the quantification of SPH are, in part, complicated, time-consuming, insensitive, or unselective. Therefore, a fast and convenient methodology for the quantification of SPH and the biosynthetic intermediate sphinganine (SPA) was developed. The method is based on an HPLC separation coupled to electrospray ionization tandem mass spectrometry (MS/MS). Quantitation is achieved by the use of a constant concentration of a non-naturally occurring internal standard, 17-carbon chain SPH (C17-SPH), together with a calibration curve established by spiking different concentrations of naturally occurring sphingoid bases. SPH and SPA coeluted with C17-SPH, which allows an accurate correction of the analyte response. Interference of the SPH ؉ 2 isotope with SPA quantification was corrected by an experimentally determined factor. The limits of detection were 9 fmol for SPH and 21 fmol for SPA. The overall coefficients of variation were 8% and 13% for SPH and SPA, respectively. The developed HPLC-tandem mass spectrometry methodology, with an analysis time of 3.5 min, simple sample preparation, and automated data analysis, allows high-throughput quantification of sphingoid bases from crude lipid extracts and is a valuable tool for studies of cellular sphingolipid metabolism and signaling. -Lieser, B., G. Liebisch, W. Drobnik, and G. Schmitz. Quantification of sphingosine and sphinganine from crude lipid extracts by HPLC electrospray ionization tandem mass spectrometry. J. Lipid Res. 2003. 44:
The Dark Side of Sphingolipids: Searching for Potential Cardiovascular Biomarkers
Biomolecules
Cardiovascular diseases (CVDs) are the leading cause of death and illness in Europe and worldwide, responsible for a staggering 47% of deaths in Europe. Over the past few years, there has been increasing evidence pointing to bioactive sphingolipids as drivers of CVDs. Among them, most studies place emphasis on the cardiovascular effect of ceramides and sphingosine-1-phosphate (S1P), reporting correlation between their aberrant expression and CVD risk factors. In experimental in vivo models, pharmacological inhibition of de novo ceramide synthesis averts the development of diabetes, atherosclerosis, hypertension and heart failure. In humans, levels of circulating sphingolipids have been suggested as prognostic indicators for a broad spectrum of diseases. This article provides a comprehensive review of sphingolipids’ contribution to cardiovascular, cerebrovascular and metabolic diseases, focusing on the latest experimental and clinical findings. Cumulatively, these studies indicate th...
Diagnosis of sphingolipidoses: a new simultaneous measurement of lysosphingolipids by LC-MS/MS
Clinical chemistry and laboratory medicine, 2016
Lysosphingolipids (LysoSLs) are derivatives of sphingolipids which have lost the amide-linked acyl chain. More recently, LysoSLs have been identified as storage compounds in several sphingolipidoses, including Gaucher, Fabry and Niemann-Pick diseases. To date, different methods have been developed to measure each individual lysosphingolipid in plasma. This report describes a rapid liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) assay for simultaneous quantification of several LysoSLs in plasma. We analyzed the following compounds: hexosylsphingosine (HexSph), globotriaosylsphingosine (LysoGb3), lysosphingomyelin (LysoSM) and lysosphingomyelin-509 (LysoSM-509). The sample preparation requires only 100 μL of plasma and consists of an extraction with a mixture of MeOH/acetone/H2O (45:45:10, v/v). The method validation showed high sensitivity, an excellent accuracy and precision. Reference ranges were determined in healthy adult and pediatric population. The resul...
Lipids in Health and Disease
Background: Lipoproteins are major players in the development and progression of atherosclerotic plaques leading to coronary stenosis and myocardial infarction. Epidemiological, genetic and experimental observations have implicated the association of sphingolipids and intermediates of sphingolipid synthesis in atherosclerosis. We aimed to investigate relationships between quantitative changes in serum sphingolipids, the regulation of the metabolism of lipoproteins (LDL, HDL), and endophenotypes of coronary artery disease (CAD). Methods: We carried out untargeted liquid chromatographymass spectrometry (UPLC-MS) lipidomics of serum samples of subjects belonging to a cross-sectional study and recruited on the basis of absence or presence of angiographically-defined CAD, and extensively characterized for clinical and biochemical phenotypes. Results: Among the 2998 spectral features detected in the serum samples, 1328 metabolic features were significantly correlated with at least one of the clinical or biochemical phenotypes measured in the cohort. We found evidence of significant associations between 34 metabolite signals, corresponding to a set of sphingomyelins, and serum HDL cholesterol. Many of these metabolite associations were also observed with serum LDL and total cholesterol levels but not as much with serum triglycerides. Conclusion: Among patients with CAD, sphingolipids in the form of sphingomyelins are directly correlated with serum levels of lipoproteins and total cholesterol. Results from this study support the fundamental role of sphingolipids in modulating lipid serum levels, highlighting the importance to identify novel targets in the sphingolipid metabolic pathway for anti-atherogenic therapies.
Hyphenated HPTLC has been used to analyze several neutral sphingolipids acting as lysosomal storage disease (LSD) biomarkers. Automated multiple development (AMD) provides separation of lipid peaks, which are detected and quantified using fluorescence detection by intensity changes (FDIC) after primuline post-impregnation. A final online transfer to a mass spectrometer by means of an elution-based interface allows their identification using electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI).Given that the increases in fluorescent emission detected by FDIC are produced by non-specific, electrostatic interactions between the primuline and hydrocarbon chains in the ceramide backbones of sphingolipids, it is a non-destructive detection technique,
Mass spectrometric analysis of neutral sphingolipids: Methods, applications, and limitations
Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2011
Sphingolipids represent an important class among lipids, especially when considering their vital roles in lipid metabolism. Thus, a variety of methods have been created to accomplish their analysis and the term "sphingolipidomics" has recently been coined to underline the motivation to enable a comprehensive analysis of all sphingolipid species including the acidic and the neutral ones. In this review, we summarize selected mainly biomedical based mass spectrometric approaches for the analysis of neutral sphingolipids regarding their advantages, applications and limitations. To underline some practical aspects of method development, we focus on a new method recently developed in our laboratory, which enables separation, detection, and mass spectrometric profiling of ceramide, hexosylceramide, lactosylceramide, globotriaosylceramide, globotetraosylceramide, sphingomyelin species, and cholesterol in one run. This method can be applied to investigate impairments of neutral sphingolipid metabolism in a variety of disorders such as sphingolipidoses and be employed to screen for sphingolipid profile changes as induced by knockout experiments or related studies. This article is part of a Special Issue entitled: Lipodomics and Imaging Mass Spectrometry.