Summary of study ST000915

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000633. The data can be accessed directly via it's Project DOI: 10.21228/M8V961 This work is supported by NIH grant, U2C- DK119886.

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Study IDST000915
Study TitleBiomarkers of NAFLD progression: a lipidomics approach to an epidemic. Part 1:Liver
Study TypeLipidomics Study
Study SummaryThe spectrum of nonalcoholic fatty liver disease (NAFLD) includes steatosis, nonalcoholic steatohepatitis (NASH), and cirrhosis. Recognition and timely diagnosis of these different stages, particularly NASH, is important for both potential reversibility and limitation of complications. Liver biopsy remains the clinical standard for definitive diagnosis. Diagnostic tools minimizing the need for invasive procedures or that add information to histologic data are important in novel management strategies for the growing epidemic of NAFLD. We describe an 'omics' approach to detecting a reproducible signature of lipid metabolites, aqueous intracellular metabolites, SNPs, and mRNA transcripts in a double-blinded study of patients with different stages of NAFLD that involves profiling liver biopsies, plasma, and urine samples. Using linear discriminant analysis, a panel of 20 plasma metabolites that includes glycerophospholipids, sphingolipids, sterols, and various aqueous small molecular weight components involved in cellular metabolic pathways, can be used to differentiate between NASH and steatosis. This identification of differential biomolecular signatures has the potential to improve clinical diagnosis and facilitate therapeutic intervention of NAFLD.
Institute
LIPID MAPS
DepartmentBioengineering
Last NameFahy
First NameEoin
Address9500 Gilman, La Jolla, CA, 92093, USA
Emailefahy@ucsd.edu
Phone858-534-4076
Submit Date2018-01-14
Publicationshttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4340319/
Analysis Type DetailMS
Release Date2018-04-05
Release Version1
Eoin Fahy Eoin Fahy
https://dx.doi.org/10.21228/M8V961
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR000633
Project DOI:doi: 10.21228/M8V961
Project Title:Biomarkers of NAFLD progression: a lipidomics approach to an epidemic
Project Type:Lipidomics Study
Project Summary:The spectrum of nonalcoholic fatty liver disease (NAFLD) includes steatosis, nonalcoholic steatohepatitis (NASH), and cirrhosis. Recognition and timely diagnosis of these different stages, particularly NASH, is important for both potential reversibility and limitation of complications. Liver biopsy remains the clinical standard for definitive diagnosis. Diagnostic tools minimizing the need for invasive procedures or that add information to histologic data are important in novel management strategies for the growing epidemic of NAFLD. We describe an 'omics' approach to detecting a reproducible signature of lipid metabolites, aqueous intracellular metabolites, SNPs, and mRNA transcripts in a double-blinded study of patients with different stages of NAFLD that involves profiling liver biopsies, plasma, and urine samples. Using linear discriminant analysis, a panel of 20 plasma metabolites that includes glycerophospholipids, sphingolipids, sterols, and various aqueous small molecular weight components involved in cellular metabolic pathways, can be used to differentiate between NASH and steatosis. This identification of differential biomolecular signatures has the potential to improve clinical diagnosis and facilitate therapeutic intervention of NAFLD.
Institute:University of California, San Diego
Department:Bioengineering
Last Name:Fahy
First Name:Eoin
Address:9500 Gilman, La Jolla, CA, 92093, USA
Email:efahy@ucsd.edu
Phone:858-534-4076
Funding Source:NIGMS Grant GM U54069338
Publications:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4340319/
Contributors:LIPID MAPS Consortium

Subject:

Subject ID:SU000953
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

Factors:

Subject type: Human clinical study; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Diagnosis
SA053802NASH055Cirrhosis
SA053803NASH049Cirrhosis
SA053804NASH048Cirrhosis
SA053805NASH064Cirrhosis
SA053806NASH068Cirrhosis
SA053807NASH005Cirrhosis
SA053808NASH069Cirrhosis
SA053809NASH047Cirrhosis
SA053810NASH065Cirrhosis
SA053811NASH052Cirrhosis
SA053812NASH016Cirrhosis
SA053813NASH013Cirrhosis
SA053814NASH040Cirrhosis
SA053815NASH007Cirrhosis
SA053816NASH022Cirrhosis
SA053817NASH009Cirrhosis
SA053818NASH026Cirrhosis
SA053819NASH029Cirrhosis
SA053820NASH028Cirrhosis
SA053821NASH027Cirrhosis
SA053822NASH072NASH
SA053823NASH057NASH
SA053824NASH044NASH
SA053825NASH074NASH
SA053826NASH088NASH
SA053827NASH039NASH
SA053828NASH090NASH
SA053829NASH087NASH
SA053830NASH084NASH
SA053831NASH031NASH
SA053832NASH018NASH
SA053833NASH012NASH
SA053834NASH038NASH
SA053835NASH010NASH
SA053836NASH019NASH
SA053837NASH015NASH
SA053838NASH037NASH
SA053839NASH035NASH
SA053840NASH030NASH
SA053841NASH021NASH
SA053842NASH067Normal
SA053843NASH070Normal
SA053844NASH066Normal
SA053845NASH060Normal
SA053846NASH077Normal
SA053847NASH053Normal
SA053848NASH054Normal
SA053849NASH086Normal
SA053850NASH091Normal
SA053851NASH051Normal
SA053852NASH089Normal
SA053853NASH085Normal
SA053854NASH080Normal
SA053855NASH082Normal
SA053856NASH078Normal
SA053857NASH014Normal
SA053858NASH017Normal
SA053859NASH020Normal
SA053860NASH006Normal
SA053861NASH004Normal
SA053862NASH050Normal
SA053863NASH003Normal
SA053864NASH024Normal
SA053865NASH011Normal
SA053866NASH043Normal
SA053867NASH045Normal
SA053868NASH034Normal
SA053869NASH042Normal
SA053870NASH046Normal
SA053871NASH041Normal
SA053872NASH036Normal
SA053873NASH075Steatosis
SA053874NASH073Steatosis
SA053875NASH071Steatosis
SA053876NASH076Steatosis
SA053877NASH081Steatosis
SA053878NASH083Steatosis
SA053879NASH062Steatosis
SA053880NASH079Steatosis
SA053881NASH002Steatosis
SA053882NASH032Steatosis
SA053883NASH023Steatosis
SA053884NASH001Steatosis
SA053885NASH033Steatosis
SA053886NASH056Steatosis
SA053887NASH059Steatosis
SA053888NASH058Steatosis
SA053889NASH061Steatosis
Showing results 1 to 88 of 88

Collection:

Collection ID:CO000947
Collection Summary:Human samples were collected according to a protocol approved by Vanderbilt University Medical Center's Internal Review Board (#120829) and under informed written patients' consent prior to inclusion in this study. Sample sizes were selected to minimize the invasive procedures. Plasma samples were obtained from patients' blood collected during standard of care surgical procedures. Urine samples were collected from patients' Foley catheters placed for standard of care procedure. Liver samples were obtained from the excess tissue collected as part of the standard of care liver biopsies performed at the time of surgery that would otherwise be discarded. Subsequently, studies at University of California, San Diego were conducted under further auspices of University of California, San Diego Internal Review Board #121220.
Sample Type:Liver

Treatment:

Treatment ID:TR000967
Treatment Summary:-

Sample Preparation:

Sampleprep ID:SP000960
Sampleprep Summary:Liver sample extraction. Approximately 10 mg of frozen liver was homogenized in 500 µl of cold (-20°C) 70% CH3OH using a tight-fit glass homogenizer (Kimble/Kontes Glass Co., Vineland, NJ) for about 1 min on ice in the presence of 4 µl of internal standard mix. The suspension was vortexed for 10 s and left 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 supernatant was collected and the solvent was evaporated. 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.

Combined analysis:

Analysis ID AN001485 AN001486 AN001487 AN001488 AN001489 AN001490
Analysis type MS MS MS MS MS MS
Chromatography type Core G Core J Core K Core E Core I Core H
Chromatography system Multiple Multiple Multiple Multiple Multiple Multiple
Column Multiple Multiple Multiple Multiple Multiple Multiple
MS Type Other Other Other Other Other Other
MS instrument type - - - - - -
MS instrument name
Ion Mode UNSPECIFIED UNSPECIFIED UNSPECIFIED UNSPECIFIED UNSPECIFIED UNSPECIFIED
Units pmol/mg nmol/mg pmol/mg pmol/mg pmol/mg pmol/mg

Chromatography:

Chromatography ID:CH001042
Chromatography Summary:FFAs were analyzed by stable isotope dilution GC-MS after derivatization, essentially as described previously (26, 27). This method quantifies 33 FAs including all major and minor saturated FAs, monounsaturated FAs, and PUFAs containing 12 to 26 carbons. Eicosanoids were analyzed by a stable isotope dilution LC/MS method utilizing 26 deuterated internal standards (28, 29). The metabolites were quantified after separation by reverse phase chromatography on a 2.1 × 100 mm BEH Shield column, 1.7 µM (Waters, Milford, MA) employing an Acquity UPLC system (Waters). Detection and quantification were performed on an AB SCIEX 6500 QTrap mass spectrometer equipped with an IonDrive Turbo V source (AB SCIEX, Framingham, MA), operated in negative ionization mode via MRM, using standard curves generated from 145 authentic quantification standards (34). The method analyzes an additional 13 metabolites based on authentic primary standards, but which cannot be quantified due to the lack of appropriate internal standards. Data analysis was performed using MultiQuant 2.1 software (AB SCIEX). 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.
Instrument Name:Multiple
Column Name:Multiple
Chromatography Type:Core G
  
Chromatography ID:CH001043
Chromatography Summary:Sterols and oxysterols were measured using methods previously described (30). Plasma total cholesterol was measured using a Vitros 250 chemistry system (Ortho-Clinical Diagnostics, Rochester, NY). Plasma free cholesterol and liver free and total cholesterol were measured using methods adapted from (30). 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.
Instrument Name:Multiple
Column Name:Multiple
Chromatography Type:Core J
  
Chromatography ID:CH001044
Chromatography Summary:Cardiolipin analysis was achieved with normal phase LC coupled with high-resolution MS performed on a TripleTOF 5600 system (AB SCIEX, Foster City, CA) (24). Dolichol and coenzyme Q were analyzed by reverse phase LC coupled with multiple reaction monitoring (MRM) MS utilizing a 4000 Q-Trap hybrid triple quadrupole linear ion-trap mass spectrometer (AB SCIEX) (22, 23). For dolichol analysis, MRM was performed in negative ion mode, with the precursor ions being the (M+acetate)- adduct ions and the product ions being the acetate ions (m/z 59). For coenzyme Q analysis, MRM was carried out in positive ion mode, with ammonium adducts (M+NH4)+ as precursor ions and the proton adducts of the quinone ring of coenzyme Q (m/z 197) as product ions. For quantitation, an internal standard mixture composed of a cardiolipin mix (Avanti Polar Lipids, Inc.), nor-dolichol (13-22) (Avanti Polar Lipids, Inc.), and yeast coenzyme Q6 (Sigma) was added during the first step of lipid extraction (22). 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.
Instrument Name:Multiple
Column Name:Multiple
Chromatography Type:Core K
  
Chromatography ID:CH001045
Chromatography Summary:The organic solvent extraction layer containing CEs, TAGs, and DAGs was taken to dryness, then derivatized with 2,5-difluorophenylisocyanate to convert DAGs to urethane derivatives (35). The derivatized extract was separated by normal phase LC as previously described (35). The CEs eluting first from the LC column were detected by 20 specific MRM transitions corresponding to each [M+NH4]+ ion being collisionally activated to m/z 369.3. During elution of TAGs, the mass spectrometer was set to carry out full mass scanning from m/z 400-1,000. The DAGs were detected by neutral loss scanning of 190 Da. 35. Leiker T. J., Barkley R. M., Murphy R. C. 2011. Analysis of diacylglycerol molecular species in cellular lipid extracts by normal-phase LC-electrospray mass spectrometry. Int. J. Mass Spectrom. 305: 103-109
Instrument Name:Multiple
Column Name:Multiple
Chromatography Type:Core E
  
Chromatography ID:CH001046
Chromatography Summary:Sphingolipids were analyzed by LC-MS/MS essentially as described in (32, 33) with minor modifications to include the 1-deoxy-sphingolipids as generally described in (36). 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. 36. Zitomer N. C., Mitchell T., Voss K. A., Bondy G. S., Pruett S. T., Garnier-Amblard E. C., Liebeskind L. S., Park H., Wang E., Sullards M. C., et al. 2009. Ceramide synthase inhibition by fumonisin B1 causes accumulation of 1-deoxysphinganine: a novel category of bioactive 1-deoxysphingoid bases and 1-deoxydihydroceramides biosynthesized by mammalian cell lines and animals. J. Biol. Chem. 284: 4786-4795.
Instrument Name:Multiple
Column Name:Multiple
Chromatography Type:Core I
  
Chromatography ID:CH001047
Chromatography Summary:Extracted GPLs from liver and plasma were analyzed by MS, as described elsewhere (20, 21). 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.
Instrument Name:Multiple
Column Name:Multiple
Chromatography Type:Core H

MS:

MS ID:MS001368
Analysis ID:AN001485
Instrument Type:-
MS Type:Other
Ion Mode:UNSPECIFIED
  
MS ID:MS001369
Analysis ID:AN001486
Instrument Type:-
MS Type:Other
Ion Mode:UNSPECIFIED
  
MS ID:MS001370
Analysis ID:AN001487
Instrument Type:-
MS Type:Other
Ion Mode:UNSPECIFIED
  
MS ID:MS001371
Analysis ID:AN001488
Instrument Type:-
MS Type:Other
Ion Mode:UNSPECIFIED
  
MS ID:MS001372
Analysis ID:AN001489
Instrument Type:-
MS Type:Other
Ion Mode:UNSPECIFIED
  
MS ID:MS001373
Analysis ID:AN001490
Instrument Type:-
MS Type:Other
Ion Mode:UNSPECIFIED
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