Quantitative measurement of different ceramide species from crude cellular extracts by electrospray ionization tandem mass spectrometry (ESI-MS/MS)

Liebisch, Gerhard; Drobnik, Wolfgang; Reil, Markus; Trümbach, Barbara; Arnecke, Ralf; Olgemöller, Bernhard; Roscher, Adelbert A.; Schmitz, Gerd

doi:10.1016/s0022-2275(20)33398-8

Cited by 263 publications

(46 citation statements)

References 23 publications

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“…Each precursor ion was separated in the first quadrupole (Q1), fragmented in the second quadrupole (Q2), and the product ions were scanned in the third quadrupole (Q3). We found that, as reported before [20,22,25,32], one typical fragment for ceramides is the m/z 264. This is generated with the loss of the acyl group and two molecules of water.…”

Section: Optimization Of Tandem Mass Spectrometrysupporting

confidence: 86%

“…Tandem mass spectrometry can be a useful method to quantify different forms of ceramide simultaneously. In case of multiple reaction monitoring mode, a prior separation step is not essential as the different transitions are specific to the molecules [20][21][22][23]. However, if tandem mass spectrometry is coupled to high-performance liquid chromatography, the selectivity improves and the method is more robust since the components are separated from the impurities.…”

Section: Introductionmentioning

confidence: 99%

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Simultaneous Quantitative Determination of Different Ceramide and Diacylglycerol Species in Cultured Cells by Using Liquid Chromatography–Electrospray Tandem Mass Spectrometry

Somogyi

Donkó

Sarnyai

et al. 2020

Period. Polytech. Chem. Eng.

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A sensitive, reproducible reverse-phased high performance liquid chromatography electrospray tandem mass spectrometry (HPLC-ESI-MS/MS) method with simple sample preparation was developed for the simultaneous determination of a wide range of ceramides, diacylglycerols (DAGs) in cultured cells. Chromatographic separation of the compounds was achieved in a 14-minute run using a C8 column with a gradient elution by methanol and 10 mM ammonium acetate buffer as mobile phase at a flow rate of 0.5 ml/min. Various ceramides, DAGs were detected with a triple quadrupol system in multiple reaction monitoring mode, which is based on a soft positive electrospray ionization. The usual sample preparation process was shortened by the application of pure methanol for the extraction instead of the widely used methanol/chloroform mixture. C17:0 ceramide which does not occur in the cell samples, was used as an internal standard. The sample preparation process was optimized and the methodology was tested on a human hepatocarcinoma cell culture. Our results clearly showed accumulation of some ceramides and DAGs in the cells treated with BSA-conjugated palmitate for 8 hours. Since both ceramides and DAGs are important lipid intermediates and signal messengers, alteration in their cellular levels have major impact on cell functions, and thus our novel analytic method can be widely used in lipotoxicity research. The presented technique can be further developed to measure other intermediates of ceramide synthesis and other derivatives of DAGs as well.

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Section: Optimization Of Tandem Mass Spectrometrysupporting

confidence: 86%

Section: Introductionmentioning

confidence: 99%

Simultaneous Quantitative Determination of Different Ceramide and Diacylglycerol Species in Cultured Cells by Using Liquid Chromatography–Electrospray Tandem Mass Spectrometry

Somogyi

Donkó

Sarnyai

et al. 2020

Period. Polytech. Chem. Eng.

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“…The following neutral losses were applied: Phosphatidylethanolamine (PE) 141, phosphatidylserine (PS) 185, and phosphatidylinositol (PI) 277 [68]. Sphingosine based ceramides were analyzed using a fragment ion of m/z 264 [69].…”

Section: Lipid Analysismentioning

confidence: 99%

Liver Lipids of Patients with Hepatitis B and C and Associated Hepatocellular Carcinoma

Haberl

Weiss

Peschel

et al. 2021

IJMS

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Hepatocellular carcinoma (HCC) still remains a difficult to cure malignancy. In recent years, the focus has shifted to lipid metabolism for the treatment of HCC. Very little is known about hepatitis B virus (HBV) and C virus (HCV)-related hepatic lipid disturbances in non-malignant and cancer tissues. The present study showed that triacylglycerol and cholesterol concentrations were similar in tumor adjacent HBV and HCV liver, and were not induced in the HCC tissues. Higher levels of free cholesterol, polyunsaturated phospholipids and diacylglycerol species were noted in non-tumorous HBV compared to HCV liver. Moreover, polyunsaturated phospholipids and diacylglycerols, and ceramides declined in tumors of HBV infected patients. All of these lipids remained unchanged in HCV-related HCC. In HCV tumors, polyunsaturated phosphatidylinositol levels were even induced. There were no associations of these lipid classes in non-tumor tissues with hepatic inflammation and fibrosis scores. Moreover, these lipids did not correlate with tumor grade or T-stage in HCC tissues. Lipid reprogramming of the three analysed HBV/HCV related tumors mostly resembled HBV-HCC. Indeed, lipid composition of non-tumorous HCV tissue, HCV tumors, HBV tumors and HBV/HCV tumors was highly similar. The tumor suppressor protein p53 regulates lipid metabolism. The p53 and p53S392 protein levels were induced in the tumors of HBV, HCV and double infected patients, and this was significant in HBV infection. Negative correlation of tumor p53 protein with free cholesterol indicates a role of p53 in cholesterol metabolism. In summary, the current study suggests that therapeutic strategies to target lipid metabolism in chronic viral hepatitis and associated cancers have to consider disease etiology.

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“…A variety of techniques have been previously used for the separation and analysis of ceramides, including thin layer chromatography, gas liquid chromatography (GLC), or HPLC (12)(13)(14)(15)(16) and, more recently, mass spectrometry (MS) (10,17,18). Molecular species of free ceramides, as regards the fatty acid composition, have been identified and quantitated in different cell types such as human platelets (18), erythrocytes (19), leukemia U937 cells (20), or skin fibroblasts (17). However, all the reported methods required either a multistep preparation of the lipid sample (e.g., pre-purification of the ceramide before anthroylation and HPLC) (20), or expensive mass spectrometry equipment (17).…”

mentioning

confidence: 99%

“…Molecular species of free ceramides, as regards the fatty acid composition, have been identified and quantitated in different cell types such as human platelets (18), erythrocytes (19), leukemia U937 cells (20), or skin fibroblasts (17). However, all the reported methods required either a multistep preparation of the lipid sample (e.g., pre-purification of the ceramide before anthroylation and HPLC) (20), or expensive mass spectrometry equipment (17). A comparative and recent review of the current methods emphasized the lack of a simple technique allowing the simultaneous measurement of sphingomyelin and ceramides in biological samples (21).…”

mentioning

confidence: 99%

Coupled assay of sphingomyelin and ceramide molecular species by gas liquid chromatography

Vieu

Tercé

Chevy

et al. 2002

Journal of Lipid Research

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This study reports a single-step analysis of the molecular species of endogenous ceramides and of the ceramide moiety of sphingomyelins in biological samples, using gas liquid chromatography (GLC). Silylated sphingomyelins were quantitatively converted to monosilylated ceramide upon injection into GLC, whereas the free ceramides were di-silylated on the primary and secondary alcohol function, as confirmed by mass spectrometry. The reproducible shift of the retention times between the mono-and disilylated derivatives enables simultaneous quantification of the variety of sphingomyelin and ceramide molecular species. Overlapping diacylglycerols were first removed by a mild alkaline treatment of the lipid extract. The lowest detection limit (5 pmol) did not allow for identification of free ceramides in human plasma, but 17 molecular species of ceramides derived from sphingomyelins were quantified, from NC16:0 up to NC24:1. By contrast, three major free ceramides (NC16:0, NC24:0, and NC24:1) were quantified in HEPG2 and Chinese hamster ovary (CHO) cells. Upon induction of apoptosis in CHO cells by C6-ceramide, we could follow the disappearance of the C6-ceramide, its partial conversion to C6-sphingomyelin, and the prominent increase of NC16:0 ceramide. Thus, our method represents a unique procedure of simultaneous analysis of sphingomyelin and ceramide molecular species able to monitor the variation of the different pools in biological samples.

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Quantitative measurement of different ceramide species from crude cellular extracts by electrospray ionization tandem mass spectrometry (ESI-MS/MS)

Abstract: Quantitative measurement of different ceramide species from crude cellular extracts by electrospray ionization tandem mass spectrometry (ESI-MS/MS).

Cited by 263 publications

References 23 publications

Simultaneous Quantitative Determination of Different Ceramide and Diacylglycerol Species in Cultured Cells by Using Liquid Chromatography–Electrospray Tandem Mass Spectrometry

Simultaneous Quantitative Determination of Different Ceramide and Diacylglycerol Species in Cultured Cells by Using Liquid Chromatography–Electrospray Tandem Mass Spectrometry

Liver Lipids of Patients with Hepatitis B and C and Associated Hepatocellular Carcinoma

Coupled assay of sphingomyelin and ceramide molecular species by gas liquid chromatography

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