Summary of study ST000916

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 IDST000916
Study TitleBiomarkers of NAFLD progression: a lipidomics approach to an epidemic. Part 2:Plasma
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/
Raw Data AvailableYes
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

Select appropriate tab below to view additional metadata details:


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: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

Factors:

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

mb_sample_id local_sample_id Diagnosis
SA053890NASH055Cirrhosis
SA053891NASH049Cirrhosis
SA053892NASH048Cirrhosis
SA053893NASH064Cirrhosis
SA053894NASH068Cirrhosis
SA053895NASH005Cirrhosis
SA053896NASH069Cirrhosis
SA053897NASH047Cirrhosis
SA053898NASH065Cirrhosis
SA053899NASH052Cirrhosis
SA053900NASH016Cirrhosis
SA053901NASH013Cirrhosis
SA053902NASH040Cirrhosis
SA053903NASH007Cirrhosis
SA053904NASH022Cirrhosis
SA053905NASH009Cirrhosis
SA053906NASH026Cirrhosis
SA053907NASH029Cirrhosis
SA053908NASH028Cirrhosis
SA053909NASH027Cirrhosis
SA053910NASH072NASH
SA053911NASH057NASH
SA053912NASH044NASH
SA053913NASH074NASH
SA053914NASH088NASH
SA053915NASH039NASH
SA053916NASH090NASH
SA053917NASH087NASH
SA053918NASH084NASH
SA053919NASH031NASH
SA053920NASH018NASH
SA053921NASH012NASH
SA053922NASH038NASH
SA053923NASH010NASH
SA053924NASH019NASH
SA053925NASH015NASH
SA053926NASH037NASH
SA053927NASH035NASH
SA053928NASH030NASH
SA053929NASH021NASH
SA053930NASH067Normal
SA053931NASH070Normal
SA053932NASH066Normal
SA053933NASH060Normal
SA053934NASH077Normal
SA053935NASH053Normal
SA053936NASH054Normal
SA053937NASH086Normal
SA053938NASH091Normal
SA053939NASH051Normal
SA053940NASH089Normal
SA053941NASH085Normal
SA053942NASH080Normal
SA053943NASH082Normal
SA053944NASH078Normal
SA053945NASH014Normal
SA053946NASH017Normal
SA053947NASH020Normal
SA053948NASH006Normal
SA053949NASH004Normal
SA053950NASH050Normal
SA053951NASH003Normal
SA053952NASH024Normal
SA053953NASH011Normal
SA053954NASH043Normal
SA053955NASH045Normal
SA053956NASH034Normal
SA053957NASH042Normal
SA053958NASH046Normal
SA053959NASH041Normal
SA053960NASH036Normal
SA053961NASH075Steatosis
SA053962NASH073Steatosis
SA053963NASH071Steatosis
SA053964NASH076Steatosis
SA053965NASH081Steatosis
SA053966NASH083Steatosis
SA053967NASH062Steatosis
SA053968NASH079Steatosis
SA053969NASH002Steatosis
SA053970NASH032Steatosis
SA053971NASH023Steatosis
SA053972NASH001Steatosis
SA053973NASH033Steatosis
SA053974NASH056Steatosis
SA053975NASH059Steatosis
SA053976NASH058Steatosis
SA053977NASH061Steatosis
Showing results 1 to 88 of 88

Collection:

Collection ID:CO000948
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:Plasma

Treatment:

Treatment ID:TR000968
Treatment Summary:-

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.

Combined analysis:

Analysis ID AN001491 AN001492 AN001493 AN001494 AN001495 AN001496
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/ml nmol/ml pmol/ml nmol/ml pmol/ml pmol/ml

Chromatography:

Chromatography ID:CH001048
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:CH001049
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:CH001050
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:CH001051
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:CH001052
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:CH001053
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:MS001374
Analysis ID:AN001491
Instrument Type:-
MS Type:Other
Ion Mode:UNSPECIFIED
  
MS ID:MS001375
Analysis ID:AN001492
Instrument Type:-
MS Type:Other
Ion Mode:UNSPECIFIED
  
MS ID:MS001376
Analysis ID:AN001493
Instrument Type:-
MS Type:Other
Ion Mode:UNSPECIFIED
  
MS ID:MS001377
Analysis ID:AN001494
Instrument Type:-
MS Type:Other
Ion Mode:UNSPECIFIED
  
MS ID:MS001378
Analysis ID:AN001495
Instrument Type:-
MS Type:Other
Ion Mode:UNSPECIFIED
  
MS ID:MS001379
Analysis ID:AN001496
Instrument Type:-
MS Type:Other
Ion Mode:UNSPECIFIED
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