Summary of Study ST001152

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 PR000771. The data can be accessed directly via it's Project DOI: 10.21228/M8197Z This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST001152
Study TitleMetabolomic Analysis of Liver Tissues for Characterization of Hepatocellular Carcinoma
Study SummaryHepatocellular carcinoma (HCC) is the most common type of primary liver cancer causing more than half a million annual deaths world-wide. Understanding the molecular mechanisms contributing to HCC development and progression is highly desirable for improved surveillance, diagnosis and treatment. Liver tissue metabolomics has the potential to reflect the physiological changes behind HCC development. Also, it allows researchers to investigate racial disparities in HCC. The use of both gas chromatography – mass spectrometry (GC-MS) and liquid chromatography – mass spectrometry (LC-MS) platforms helps increase the metabolome coverage, allowing researchers to better unravel the relationships of metabolites and HCC. The objective of this study is to identify HCC-associated metabolites by analysis of liver tissues from HCC patients using both GC-MS and LC-MS platforms. Paired tumor and non-tumor tissues from 40 patients were analyzed by GC-MS and LC-MS. The patients consist of 14 African-Americans (AA), 10 Asian-Americans (AS), and 16 European-Americans (EA). The levels of the metabolites extracted from the solid liver tissue of the HCC area and adjacent non-HCC area were compared. Among the analytes detected by GC-MS and LC-MS with significant alterations, 17 were selected based on availability of putative metabolite identifications. These metabolites belong to TCA cycle, glycolysis, purines, and lipid metabolism, and have been previously reported in liver metabolomics studies where high correlation with HCC progression was implied. We demonstrated that metabolites that are related to HCC pathogenesis can be identified through metabolomics analysis of liver tissues by both GC-MS and LC-MS. In addition, this analysis has led to the identification of metabolites associated with HCC in a race-specific manner.
Institute
Georgetown University
DepartmentOncology
LaboratoryRessom Lab
Last NameDi Poto
First NameCristina
Address3970 Reservoir Rd. NW, Research Bldg., Room W325
Emailcd329@georgetown.edu
Phone2026872926
Submit Date2019-03-07
Num Groups4
Total Subjects40
Raw Data AvailableYes
Raw Data File Type(s)cdf, raw(Waters)
Analysis Type DetailGC-MS/LC-MS
Release Date2020-03-03
Release Version1
Cristina Di Poto Cristina Di Poto
https://dx.doi.org/10.21228/M8197Z
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR000771
Project DOI:doi: 10.21228/M8197Z
Project Title:Metabolomic Analysis of Liver Tissues for Characterization of Hepatocellular Carcinoma
Project Summary:Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer causing more than half a million annual deaths world-wide. Understanding the molecular mechanisms contributing to HCC development and progression is highly desirable for improved surveillance, diagnosis and treatment. Liver tissue metabolomics has the potential to reflect the physiological changes behind HCC development. Also, it allows researchers to investigate racial disparities in HCC. The use of both gas chromatography – mass spectrometry (GC-MS) and liquid chromatography – mass spectrometry (LC-MS) platforms helps increase the metabolome coverage, allowing researchers to better unravel the relationships of metabolites and HCC. The objective of this study is to identify HCC-associated metabolites by analysis of liver tissues from HCC patients using both GC-MS and LC-MS platforms. Paired tumor and non-tumor tissues from 40 patients were analyzed by GC-MS and LC-MS. The patients consist of 14 African-Americans (AA), 10 Asian-Americans (AS), and 16 European-Americans (EA). The levels of the metabolites extracted from the solid liver tissue of the HCC area and adjacent non-HCC area were compared. Among the analytes detected by GC-MS and LC-MS with significant alterations, 17 were selected based on availability of putative metabolite identifications. These metabolites belong to TCA cycle, glycolysis, purines, and lipid metabolism, and have been previously reported in liver metabolomics studies where high correlation with HCC progression was implied. We demonstrated that metabolites that are related to HCC pathogenesis can be identified through metabolomics analysis of liver tissues by both GC-MS and LC-MS. In addition, this analysis has led to the identification of metabolites associated with HCC in a race-specific manner.
Institute:Georgetown University
Department:Oncology
Laboratory:Ressom Lab
Last Name:Ressom
First Name:Habtom
Address:3970 Reservoir Rd., NW, Research Bldg, Room W325, Washington, DC, 20007, USA
Email:hwr@georgetown.edu
Phone:2026872283

Subject:

Subject ID:SU001217
Subject Type:Human
Subject Species:Homo sapiens
Taxonomy ID:9606

Factors:

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

mb_sample_id local_sample_id Group RACE
SA079912HCC/CIRR 07_ADJ/CADJ/C Black
SA079913HCC/CIRR 08_ADJ/CADJ/C Black
SA079914HCC/CIRR 02_ADJ/CADJ/C Black
SA079915HCC/CIRR 01_ADJ/CADJ/C Black
SA079916HCC/CIRR 06_ADJ/CADJ/C Black
SA079917HCC/CIRR 09_ADJ/CADJ/C White
SA079918HCC/CIRR 10_ADJ/CADJ/C White
SA079919HCC/CIRR 05_ADJ/CADJ/C White
SA079920HCC/CIRR 03_ADJ/CADJ/C White
SA079921HCC/CIRR 04_ADJ/CADJ/C White
SA079922HCC/NOR 24_ADJ/NADJ/N Asian
SA079923HCC/NOR 23_ADJ/NADJ/N Asian
SA079924HCC/NOR 30_ADJ/NADJ/N Asian
SA079925HCC/NOR 29_ADJ/NADJ/N Asian
SA079926HCC/NOR 25_ADJ/NADJ/N Asian
SA079927HCC/NOR 27_ADJ/NADJ/N Asian
SA079928HCC/NOR 28_ADJ/NADJ/N Asian
SA079929HCC/NOR 26_ADJ/NADJ/N Asian
SA079930HCC/NOR 17_ADJ/NADJ/N Asian
SA079931HCC/NOR 22_ADJ/NADJ/N Asian
SA079932HCC/NOR 07_ADJ/NADJ/N Black
SA079933HCC/NOR 12_ADJ/NADJ/N Black
SA079934HCC/NOR 08_ADJ/NADJ/N Black
SA079935HCC/NOR 10_ADJ/NADJ/N Black
SA079936HCC/NOR 04_ADJ/NADJ/N Black
SA079937HCC/NOR 16_ADJ/NADJ/N Black
SA079938HCC/NOR 03_ADJ/NADJ/N Black
SA079939HCC/NOR 21_ADJ/NADJ/N Black
SA079940HCC/NOR 19_ADJ/NADJ/N Black
SA079941HCC/NOR 20_ADJ/NADJ/N White
SA079942HCC/NOR 01_ADJ/NADJ/N White
SA079943HCC/NOR 13_ADJ/NADJ/N White
SA079944HCC/NOR 09_ADJ/NADJ/N White
SA079945HCC/NOR 15_ADJ/NADJ/N White
SA079946HCC/NOR 14_ADJ/NADJ/N White
SA079947HCC/NOR 18_ADJ/NADJ/N White
SA079948HCC/NOR 11_ADJ/NADJ/N White
SA079949HCC/NOR 06_ADJ/NADJ/N White
SA079950HCC/NOR 05_ADJ/NADJ/N White
SA079951HCC/NOR 02_ADJ/NADJ/N White
SA079952Blank_BBlank -
SA079953Blank_ABlank -
SA079954Blank_CBlank -
SA079955Blank_DBlank -
SA079956Blank_EBlank -
SA079957HCC/CIRR 02_HCC/CHCC/C Black
SA079958HCC/CIRR 08_HCC/CHCC/C Black
SA079959HCC/CIRR 07_HCC/CHCC/C Black
SA079960HCC/CIRR 01_HCC/CHCC/C Black
SA079961HCC/CIRR 06_HCC/CHCC/C Black
SA079962HCC/CIRR 10_HCC/CHCC/C White
SA079963HCC/CIRR 09_HCC/CHCC/C White
SA079964HCC/CIRR 05_HCC/CHCC/C White
SA079965HCC/CIRR 04_HCC/CHCC/C White
SA079966HCC/CIRR 03_HCC/CHCC/C White
SA079967HCC/NOR 23_HCC/NHCC/N Asian
SA079968HCC/NOR 30_HCC/NHCC/N Asian
SA079969HCC/NOR 29_HCC/NHCC/N Asian
SA079970HCC/NOR 24_HCC/NHCC/N Asian
SA079971HCC/NOR 27_HCC/NHCC/N Asian
SA079972HCC/NOR 26_HCC/NHCC/N Asian
SA079973HCC/NOR 25_HCC/NHCC/N Asian
SA079974HCC/NOR 28_HCC/NHCC/N Asian
SA079975HCC/NOR 22_HCC/NHCC/N Asian
SA079976HCC/NOR 17_HCC/NHCC/N Asian
SA079977HCC/NOR 07_HCC/NHCC/N Black
SA079978HCC/NOR 12_HCC/NHCC/N Black
SA079979HCC/NOR 08_HCC/NHCC/N Black
SA079980HCC/NOR 10_HCC/NHCC/N Black
SA079981HCC/NOR 04_HCC/NHCC/N Black
SA079982HCC/NOR 16_HCC/NHCC/N Black
SA079983HCC/NOR 03_HCC/NHCC/N Black
SA079984HCC/NOR 21_HCC/NHCC/N Black
SA079985HCC/NOR 19_HCC/NHCC/N Black
SA079986HCC/NOR 02_HCC/NHCC/N White
SA079987HCC/NOR 09_HCC/NHCC/N White
SA079988HCC/NOR 05_HCC/NHCC/N White
SA079989HCC/NOR 06_HCC/NHCC/N White
SA079990HCC/NOR 01_HCC/NHCC/N White
SA079991HCC/NOR 18_HCC/NHCC/N White
SA079992HCC/NOR 11_HCC/NHCC/N White
SA079993HCC/NOR 15_HCC/NHCC/N White
SA079994HCC/NOR 14_HCC/NHCC/N White
SA079995HCC/NOR 13_HCC/NHCC/N White
SA079996HCC/NOR 20_HCC/NHCC/N White
SA079997QC_ADJ/C_4QC_ADJ/C -
SA079998QC_ADJ/C_3QC_ADJ/C -
SA079999QC_ADJ/C_1QC_ADJ/C -
SA080000QC_ADJ/C_5QC_ADJ/C -
SA080001QC_ADJ/C_2QC_ADJ/C -
SA080002QC_ADJ/C_6QC_ADJ/C -
SA080003QC_ADJ/N_7QC_ADJ/N -
SA080004QC_ADJ/N_8QC_ADJ/N -
SA080005QC_ADJ/N_6QC_ADJ/N -
SA080006QC_ADJ/N_4QC_ADJ/N -
SA080007QC_ADJ/N_3QC_ADJ/N -
SA080008QC_ADJ/N_5QC_ADJ/N -
SA080009QC_ADJ/N_9QC_ADJ/N -
SA080010QC_ADJ/N_1QC_ADJ/N -
SA080011QC_ADJ/N_15QC_ADJ/N -
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Collection:

Collection ID:CO001211
Collection Summary:Adult patients were recruited at MedStar Georgetown University Hospital (MGUH). All participants provided informed consent to a protocol approved by the Institutional Review Board (IRB) at Georgetown University. Following the participant’s informed consent signature and enrollment, tissue samples were collected at the time of the surgical procedure and stored under liquid nitrogen until the day of metabolite extraction. HCC cases were diagnosed based on well-established diagnostic imaging criteria and/or histology. Clinical stages for HCC cases were determined based on the TNM staging system.
Sample Type:Liver

Treatment:

Treatment ID:TR001232
Treatment Summary:Tissue samples were collected at the time of the surgical procedure and stored under liquid nitrogen until the day of metabolite extraction.

Sample Preparation:

Sampleprep ID:SP001225
Sampleprep Summary:10 mg of liver tissue was homogenized on ice with 1 mL of pre-chilled methanol:water (1:1) containing five internal standards: 0.001 ppm debrisoquine, 0.004 ppm 4-nitro benzoic acid, 0.0004 ppb stearoyl (d35)-2-hydroxy-glycerophosphocholine, 0.2 ppb D-erythro-sphingosine (d7)-1-phosphate, and 2 ppm Myristic-d27 acid. Homogenized samples were centrifuged at 14.500 g, at 4°C for 15 minutes. Supernatant (S1) was collected and divided in two aliquots (one for GC-MS and one for LC-MS analysis), while the remaining pellet (P1) was kept at -80°C until further extraction. For the extraction of medium to polar compounds, 1:1 volume of pre-chilled acetonitrile was added to the two aliquots (GC&LC), vortex-mixed and kept on ice for 20 minutes. Samples were centrifuged again at 14.500 g, at 4°C for 15 minutes, and supernatant (S2) was collected and concentrated to dryness in a speedvac system operated at room temperature. Pellets (P2 and P3) and supernatants were stored at -80°C. For the extraction of low-polar compounds, P1 was resuspended with 500μl of pre-chilled dichloromethane:methanol (3:1) while P2, and P3 with 125 μL of the same mix. Pellets were sonicated on ice for 90sec, combined and centrifuged at 14.500 g, at 4°C for 20 minutes. Supernatant (S3) was collected, split in two aliquots (one for GC-MS and one for LC-MS analysis) and a 1:1 volume of pre-chilled acetonitrile was added to both aliquots (GC&LC), vortexed and kept on ice for 20 minutes. Samples were then centrifuged at 14.500 g for 15 min at 4°C while pellets were kept at -80 °C for protein quantitation. Finally, supernatants (S4) were concentrated to dryness by speedvac and kept at -80°C until the day of analysis. Blank samples were prepared together with the human samples by adding all the reagents to an empty tube and following the same sample preparation steps. Dried supernatant (S2) collected for GC-MS analysis underwent derivatization step. Samples were derivatized in each batch prior to injection following a two-stage process of oximation and trimethylsilylation (-Si(CH3)3). Briefly, 20µL of a 20mg/mL methoxyamine hydrochloride in pyridine were added to the dried extracts, vortexed and incubated at 30°C for 90 minutes. After returning the samples to room temperature, 80µL of MSTFA were added, vortex-mixed and incubated at 30°C for 30 minutes. Samples were then centrifuged at 14,500 g for 15 minutes, and 60µL of the supernatant were transferred into 250µL clear glass autosampler vials. Finally, 20µL of 0.006 µg/µL C18 Methyl Stearate in hexane were added to the vial prior to injection. For quality assessment, Myristic-d27 acid was spiked into the working solution to verify tissue metabolites extraction and derivatization steps. C18 Methyl Stearate was added just before GC-MS analysis to monitor each sample injection. QC samples (QCs) were generated by pooling together the supernatant obtained after derivatization of samples of each biological group in each batch separately. A retention index (RI) standard sample was prepared by mixing fatty acid methyl esters (FAMEs) with alkanes. Specifically, FAMEs C8, C9, C10, C12, C14, C16, C18, C20, C22, C24, C26, C28 and C30 linear chain length were individually dissolved in chloroform at concentrations of 0.8 mg/mL (C8 – C16) and 0.4 mg/ml (C18 – C30) to generate FAME-1 stock solutions. 100 µL of each FAME-1 were mixed together and 1.2 mL of chloroform were added for a final volume of 2.5mL generating FAME-2 solution. Alkanes, containing all even CnH2n+2 from C10 to C40, were purchased as a mixture at a concentration of 50mg/L in n-heptane. Alkane mixture were mixed with FAME-2 markers and hexane at a ratio of 1:2:17 and vortex-mixed prior to injection into the GC-MS. Dried supernatants (S2 & S4) collected from the first and second extraction for LC-MS analysis were reconstituted with 125 μL of methanol:acetonitrile:water (50:25:25) each, and combined for a total volume of 250 μL. d35-lysophosphocholine and d7-sphingosine-1-phosphate were included to determine the quality of the metabolite extraction. Debrisoquine and 4-nitrobenzoic acid were used to assess equipment performance. QCs were generated by pooling together the supernatant obtained after resuspension in appropriate solvent of each biological group.

Combined analysis:

Analysis ID AN001900 AN001901 AN001902
Analysis type MS MS MS
Chromatography type GC Reversed phase Reversed phase
Chromatography system Agilent 7890A Waters Acquity Waters Acquity
Column Agilent DB5-MS (30m x 0.25mm, 0.25um) Waters Acquity CSH C18 (100 x 2.1mm,1.7um) Waters Acquity CSH C18 (100 x 2.1mm,1.7um)
MS Type EI ESI ESI
MS instrument type GC-TOF QTOF QTOF
MS instrument name Leco Pegasus HT TOF Waters Synapt-G2-Si Waters Synapt-G2-Si
Ion Mode POSITIVE POSITIVE NEGATIVE
Units Da Da Da

Chromatography:

Chromatography ID:CH001376
Instrument Name:Agilent 7890A
Column Name:Agilent DB5-MS (30m x 0.25mm, 0.25um)
Chromatography Type:GC
  
Chromatography ID:CH001377
Instrument Name:Waters Acquity
Column Name:Waters Acquity CSH C18 (100 x 2.1mm,1.7um)
Chromatography Type:Reversed phase

MS:

MS ID:MS001756
Analysis ID:AN001900
Instrument Name:Leco Pegasus HT TOF
Instrument Type:GC-TOF
MS Type:EI
MS Comments:Software Chromatof
Ion Mode:POSITIVE
  
MS ID:MS001757
Analysis ID:AN001901
Instrument Name:Waters Synapt-G2-Si
Instrument Type:QTOF
MS Type:ESI
MS Comments:Software XCMS
Ion Mode:POSITIVE
  
MS ID:MS001758
Analysis ID:AN001902
Instrument Name:Waters Synapt-G2-Si
Instrument Type:QTOF
MS Type:ESI
MS Comments:Software XCMS
Ion Mode:NEGATIVE
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