High-Throughput Analysis of Sphingosine 1-Phosphate, Sphinganine 1-Phosphate, and Lysophosphatidic Acid in Plasma Samples by Liquid Chromatography–Tandem Mass Spectrometry

Scherer, Max; Schmitz, Gerd; Liebisch, Gerhard

doi:10.1373/clinchem.2008.113779

Cited by 139 publications

(153 citation statements)

References 18 publications

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“…To analyze polar sphingolipids from one lipid extract, we tested a butanolic extraction previously described for S1P analysis ( 18 ). The extraction effi ciency was determined in fi broblast homogenate by adding a sphingolipid standard mixture before and after extraction ( Table 2 ).…”

Section: Extraction Effi Ciency and Matrix Effectsmentioning

confidence: 99%

“…Analysis of S1P, ceramide, and sphingomyelin S1P was analyzed by LC-MS/MS as described previously ( 18 ). Ceramide and sphingomyelin species were analyzed by fl ow injection analysis ESI-MS/MS ( 25,26 ).…”

Section: Quantifi Cation Of Sphingolipid Speciesmentioning

confidence: 99%

“…To obtain the full range of sphingolipid concentrations, we additionally analyzed S1P, sphingomyelin, and ceramide species using previously described methods ( 18,25,26,28 ).…”

Section: Analysis Of Fi Broblasts Treated With Myriocin/skimentioning

confidence: 99%

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A rapid and quantitative LC-MS/MS method to profile sphingolipids

Scherer

Leuthäuser-Jaschinski

Ecker

et al. 2010

Journal of Lipid Research

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128

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including ceramide, sphingosine (SPH), and SPH-1-phosphate (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-1-phosphate (dhCer1P). This method was validated and apAbstract 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, sphingol...

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Section: Extraction Effi Ciency and Matrix Effectsmentioning

confidence: 99%

Section: Quantifi Cation Of Sphingolipid Speciesmentioning

confidence: 99%

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A rapid and quantitative LC-MS/MS method to profile sphingolipids

Scherer

Leuthäuser-Jaschinski

Ecker

et al. 2010

Journal of Lipid Research

Self Cite

128

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“…The correction of isotopic overlap of lipid species as well as data analysis by self programmed Excel Macros, was performed for all lipid classes. Sphingoid bases, hexosyl-and lactosylceramides (LacCER) were quantified by hydrophilic interaction chromatography (HILIC) coupled with ESI-MS/MS in a similar way as described previously (66).…”

Section: Lipid Species Analysis By Electrospray Ionization Tandem Masmentioning

confidence: 99%

Protein kinase C-Î´ isoform mediates lysosome labilization in DNA damage-induced apoptosis

Parent

Scherer²,

Liebisch³

et al. 2011

Int J Oncol

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Abstract.A lysosomal pathway, characterized by the partial rupture or labilization of lysosomal membranes (LLM) and cathepsin release into the cytosol, is evoked during the early events of 20-S-camptothecin lactone (CPT)-induced apoptosis in human cancer cells, including human histiocytic lymphoma U-937 cells. These lysosomal events begin rapidly and simultaneously with mitochondrial permeabilization and caspase activation within 3 h after drug treatment. Recently, in a comparative proteomics analysis performed on highlyenriched lysosomal extracts, we identified proteins whose translocation to lysosomes correlated with LLM induction after CPT treatment, including protein kinase C-‰ (PKC-‰).In this study, we show that the PKC-‰ translocation to lysosomes is required for LLM, as silencing its expression with RNA interference or suppressing its activity with the inhibitor, rottlerin, prevents CPT-induced LLM. PKC-‰ translocation to lysosomes is associated with lysosomal acidic sphingomyelinase (ASM) phosphorylation and activation, which in turn leads to an increase in ceramide (CER) content in lysosomes. The accumulation of endogenous CER in lysosomes is a critical event for CPT-induced LLM as suppressing PKC-‰ or ASM activity reduces both the CPTmediated CER generation in lysosomes and CPT-induced LLM. These findings reveal a novel mechanism by which PKC-‰ mediates ASM phosphorylation/activation and CER accumulation in lysosomes in CPT-induced LLM, rapidly activating the lysosomal pathway of apoptosis after CPT treatment.

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“…However, recently high-throughput methods have also been developed in the fields of lipidomics 2 and glycomics 3 which now enable exciting new insights into these biological traits.…”

Section: Introductionmentioning

confidence: 99%

Glycomics meets lipidomics—associations of N-glycans with classical lipids, glycerophospholipids, and sphingolipids in three European populations

et al. 2011

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Recently, high-throughput technologies have been made available which allow the measurement of a broad spectrum of glycomics and lipidomics parameters in many samples. The aim of this study was to apply these methods and investigate associations between 46 glycan and 183 lipid traits measured in blood of 2041 Europeans from three different local populations (Croatia -VIS cohort; Sweden -NSPHS cohort; Great Britain -ORCADES cohort). N-glycans have been analyzed with High Performance Liquid Chromatography (HPLC) and lipids with Electrospray Ionization Tandem Mass Spectrometry (ESI-MS/MS) covering sterol lipids, glycerolipids, glycerophospholipids and sphingolipids in eight subclasses. Overall, 8418 associations were calculated using linear mixed effect models adjusted for pedigree, sex, age and multiple testing. We found 330 significant correlations in VIS. Pearson's correlation coefficient r ranged from À0.27 to 0.34 with corresponding p-values between 1.45 Â 10 À19 and 4.83 Â 10 À6 , indicating statistical significance. A total of 71 correlations in VIS could be replicated in NSPHS (r = [À0.19; 0.35], p = [4.16 Â 10 À18 ; 9.38 Â 10 À5 ]) and 31 correlations in VIS were also found in ORCADES (r = [À0.20; 0.24], p = [2.69 Â 10 À10 ; 7.55 Â 10 À5 ]). However, in total only 10 correlations between a subset of triantennary glycans and unsaturated phosphatidylcholine, saturated ceramide, and sphingomyelin lipids in VIS (r = [0.18; 0.34], p = [2.98 Â 10 À21 ; 1.69 Â 10 À06 ]) could be replicated in both NSPHS and ORCADES. In summary, the results show strong and consistent associations between certain glycans and lipids in all populations, but also population-specific correlations which may be caused by environmental and genetic differences. These associations point towards potential interactive metabolic pathways.

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High-Throughput Analysis of Sphingosine 1-Phosphate, Sphinganine 1-Phosphate, and Lysophosphatidic Acid in Plasma Samples by Liquid Chromatography–Tandem Mass Spectrometry

Cited by 139 publications

References 18 publications

A rapid and quantitative LC-MS/MS method to profile sphingolipids

A rapid and quantitative LC-MS/MS method to profile sphingolipids

Protein kinase C-Î´ isoform mediates lysosome labilization in DNA damage-induced apoptosis

Glycomics meets lipidomics—associations of N-glycans with classical lipids, glycerophospholipids, and sphingolipids in three European populations

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