Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA053963

Local Sample ID:	NASH071
Subject ID:	SU000954
Subject Type:	Human clinical study
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Age Or Age Range:	23-83
Gender:	Male and Female
Human Ethnicity:	Mixed

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Sample Preparation:

Sampleprep ID:	SP000961
Sampleprep Summary:	Plasma sample extraction. One hundred microliters of thawed plasma containing 4 µl of internal standard mix were extracted with 500 µl cold (-20°C) 70% CH3OH by incubation in an ice bath for 30 min. After mixing by vortex at 4°C for 1 min and centrifugation (4°C, 18,000 g, 10 min), the solvent was evaporated from the supernatant. The residue was dissolved in 200 µl of resuspension solvent, vortexed to mix (1 min at 4°C), and centrifuged (4°C, 18,000 g, 10 min) to remove any insoluble material. GPLs: GPLs from liver samples were extracted and analyzed by MS essentially as described in (20, 21). Extraction and analysis of plasma samples was according to previously published procedures (22). Cardiolipin, coenzyme Q, and dolichol: Lipid extractions were performed based on the Bligh and Dyer method with minor modifications (23-25). FAs and eicosanoids: FFAs were extracted essentially as previously described after supplementation with deuterated internal standards (Cayman Chemicals) (26, 27). Eicosanoids were isolated via solid phase extraction, utilizing 25 deuterated internal standards (28, 29). Sterols and oxysterols: Sterols and oxysterols were extracted using previously described methods (30). Neutral lipids: Cholesteryl esters (CEs), TAGs, and DAGs were extracted from weighed liver tissue (0.5-1 mg) suspended in 0.5 ml PBS that had been homogenized by sonication. Extractions of plasma (0.05 ml diluted to 0.1 ml with PBS), urine (1 ml), and tissue sonicates were carried out using 1 ml hexane:methyl t-butyl ether (1:1, v/v), essentially as previously described (31). Sphingolipids: Sphingolipids from liver, plasma, and urine were extracted following previously published procedures (32, 33), with the exception that methylene chloride was substituted for chloroform for the single-phase extraction of sphingoid bases. 20. Ivanova P. T., Milne S. B., Byrne M. O., Xiang Y., Brown H. A. 2007. Glycerophospholipid identification and quantitation by electrospray ionization mass spectrometry. Methods Enzymol. 432: 21-57. 21. Myers D. S., Ivanova P. T., Milne S. B., Brown H. A. 2011. Quantitative analysis of glycerophospholipids by LC-MS: acquisition, data handling, and interpretation. Biochim. Biophys. Acta. 1811: 748-757. 22. Quehenberger O., Armando A. M., Brown H. A., Milne S. B., Myers D. S., Merrill A. H., Jr, Bandyopadhyay S., Jones K. N., Kelly S., Shaner R. L., et al. 2010. Lipidomics reveals a remarkable diversity of lipids in human plasma. J. Lipid Res. 51: 3299-3305. 23. Guan Z., Li S., Smith D., Shaw W., Raetz C. 2007. Identification of N-acylphosphatidylserine molecules in eukaryotic cells. Biochemistry. 46: 14500-14513. 24. Tan B. K., Bogdanov M., Zhao J., Dowhan W., Raetz C. R., Guan Z. 2012. Discovery of cardiolipin synthase utilizing phosphatidylethanolamine and phosphatidylglycerol as substrates. Proc. Natl. Acad. Sci. USA. 109: 16504-16509. 25. Wen R., Lam B., Guan Z. 2013. Aberrant dolichol chain lengths as biomarkers for retinitis pigmentosa caused by impaired dolichol biosynthesis. J. Lipid Res. 54: 3516-3522. 26. Quehenberger O., Armando A., Dumlao D., Stephens D. L., Dennis E. A. 2008. Lipidomics analysis of essential fatty acids in macrophages. Prostaglandins Leukot. Essent. Fatty Acids. 79: 123-129. 27. Quehenberger O., Armando A. M., Dennis E. A. 2011. High sensitivity quantitative lipidomics analysis of fatty acids in biological samples by gas chromatography-mass spectrometry. Biochim. Biophys. Acta. 1811: 648-656. 28. Deems R., Buczynski M. W., Bowers-Gentry R., Harkewicz R., Dennis E. A. 2007. Detection and quantitation of eicosanoids via high performance liquid chromatography-electrospray ionization-mass spectrometry. Methods Enzymol. 432: 59-82. 29. Dumlao D. S., Buczynski M. W., Norris P. C., Harkewicz R., Dennis E. A. 2011. High-throughput lipidomic analysis of fatty acid derived eicosanoids and N-acylethanolamines. Biochim. Biophys. Acta. 1811: 724-736. 30. McDonald J. G., Smith D. D., Stiles A. R., Russell D. W. 2012. A comprehensive method for extraction and quantitative analysis of sterols and secosteroids from human plasma. J. Lipid Res. 53: 1399-1409. 31. Hutchins P. M., Barkley R. M., Murphy R. C. 2008. Separation of cellular nonpolar neutral lipids by normal-phase chromatography and analysis by electrospray ionization mass spectrometry. J. Lipid Res. 49: 804-813. 32. Shaner R. L., Allegood J. C., Park H., Wang E., Kelly S., Haynes C. A., Sullards M. C., Merrill A. H., Jr 2009. Quantitative analysis of sphingolipids for lipidomics using triple quadrupole and quadrupole linear ion trap mass spectrometers. J. Lipid Res. 50: 1692-1707. 33. Sullards M. C., Liu Y., Chen Y., Merrill A. H., Jr 2011. Analysis of mammalian sphingolipids by liquid chromatography tandem mass spectrometry (LC-MS/MS) and tissue imaging mass spectrometry (TIMS). Biochim. Biophys. Acta. 1811: 838-853.

Sampleprep ID:

SP000961

Sampleprep Summary:

Plasma sample extraction. One hundred microliters of thawed plasma containing 4 µl of internal standard mix were extracted with 500 µl cold (-20°C) 70% CH3OH by incubation in an ice bath for 30 min. After mixing by vortex at 4°C for 1 min and centrifugation (4°C, 18,000 g, 10 min), the solvent was evaporated from the supernatant. The residue was dissolved in 200 µl of resuspension solvent, vortexed to mix (1 min at 4°C), and centrifuged (4°C, 18,000 g, 10 min) to remove any insoluble material. GPLs: GPLs from liver samples were extracted and analyzed by MS essentially as described in (20, 21). Extraction and analysis of plasma samples was according to previously published procedures (22). Cardiolipin, coenzyme Q, and dolichol: Lipid extractions were performed based on the Bligh and Dyer method with minor modifications (23-25). FAs and eicosanoids: FFAs were extracted essentially as previously described after supplementation with deuterated internal standards (Cayman Chemicals) (26, 27). Eicosanoids were isolated via solid phase extraction, utilizing 25 deuterated internal standards (28, 29). Sterols and oxysterols: Sterols and oxysterols were extracted using previously described methods (30). Neutral lipids: Cholesteryl esters (CEs), TAGs, and DAGs were extracted from weighed liver tissue (0.5-1 mg) suspended in 0.5 ml PBS that had been homogenized by sonication. Extractions of plasma (0.05 ml diluted to 0.1 ml with PBS), urine (1 ml), and tissue sonicates were carried out using 1 ml hexane:methyl t-butyl ether (1:1, v/v), essentially as previously described (31). Sphingolipids: Sphingolipids from liver, plasma, and urine were extracted following previously published procedures (32, 33), with the exception that methylene chloride was substituted for chloroform for the single-phase extraction of sphingoid bases.
20. Ivanova P. T., Milne S. B., Byrne M. O., Xiang Y., Brown H. A. 2007. Glycerophospholipid identification and quantitation by electrospray ionization mass spectrometry. Methods Enzymol. 432: 21-57.
21. Myers D. S., Ivanova P. T., Milne S. B., Brown H. A. 2011. Quantitative analysis of glycerophospholipids by LC-MS: acquisition, data handling, and interpretation. Biochim. Biophys. Acta. 1811: 748-757.
22. Quehenberger O., Armando A. M., Brown H. A., Milne S. B., Myers D. S., Merrill A. H., Jr, Bandyopadhyay S., Jones K. N., Kelly S., Shaner R. L., et al. 2010. Lipidomics reveals a remarkable diversity of lipids in human plasma. J. Lipid Res. 51: 3299-3305.
23. Guan Z., Li S., Smith D., Shaw W., Raetz C. 2007. Identification of N-acylphosphatidylserine molecules in eukaryotic cells. Biochemistry. 46: 14500-14513.
24. Tan B. K., Bogdanov M., Zhao J., Dowhan W., Raetz C. R., Guan Z. 2012. Discovery of cardiolipin synthase utilizing phosphatidylethanolamine and phosphatidylglycerol as substrates. Proc. Natl. Acad. Sci. USA. 109: 16504-16509.
25. Wen R., Lam B., Guan Z. 2013. Aberrant dolichol chain lengths as biomarkers for retinitis pigmentosa caused by impaired dolichol biosynthesis. J. Lipid Res. 54: 3516-3522.
26. Quehenberger O., Armando A., Dumlao D., Stephens D. L., Dennis E. A. 2008. Lipidomics analysis of essential fatty acids in macrophages. Prostaglandins Leukot. Essent. Fatty Acids. 79: 123-129.
27. Quehenberger O., Armando A. M., Dennis E. A. 2011. High sensitivity quantitative lipidomics analysis of fatty acids in biological samples by gas chromatography-mass spectrometry. Biochim. Biophys. Acta. 1811: 648-656.
28. Deems R., Buczynski M. W., Bowers-Gentry R., Harkewicz R., Dennis E. A. 2007. Detection and quantitation of eicosanoids via high performance liquid chromatography-electrospray ionization-mass spectrometry. Methods Enzymol. 432: 59-82.
29. Dumlao D. S., Buczynski M. W., Norris P. C., Harkewicz R., Dennis E. A. 2011. High-throughput lipidomic analysis of fatty acid derived eicosanoids and N-acylethanolamines. Biochim. Biophys. Acta. 1811: 724-736.
30. McDonald J. G., Smith D. D., Stiles A. R., Russell D. W. 2012. A comprehensive method for extraction and quantitative analysis of sterols and secosteroids from human plasma. J. Lipid Res. 53: 1399-1409.
31. Hutchins P. M., Barkley R. M., Murphy R. C. 2008. Separation of cellular nonpolar neutral lipids by normal-phase chromatography and analysis by electrospray ionization mass spectrometry. J. Lipid Res. 49: 804-813.
32. Shaner R. L., Allegood J. C., Park H., Wang E., Kelly S., Haynes C. A., Sullards M. C., Merrill A. H., Jr 2009. Quantitative analysis of sphingolipids for lipidomics using triple quadrupole and quadrupole linear ion trap mass spectrometers. J. Lipid Res. 50: 1692-1707.
33. Sullards M. C., Liu Y., Chen Y., Merrill A. H., Jr 2011. Analysis of mammalian sphingolipids by liquid chromatography tandem mass spectrometry (LC-MS/MS) and tissue imaging mass spectrometry (TIMS). Biochim. Biophys. Acta. 1811: 838-853.