Summary of Study ST001842

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 PR000667. The data can be accessed directly via it's Project DOI: 10.21228/M8FX07 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.

Study ID	ST001842
Study Title	Identification of unique metabolite networks between Latino and Caucasian patients with nonalcoholic fatty liver disease (NAFLD) (part I)
Study Summary	Nonalcoholic fatty liver disease (NAFLD) is a spectrum of liver pathology ranging from simple steatosis to nonalcoholic steatohepatitis (NASH); the latter is characterized by inflammation and fibrosis. Risk factors for NALFD include obesity, diabetes, hyperlipidemia, and hypertension—all of which are features of metabolic syndrome. NAFLD is a very heterogeneous disease, as it presents in different patterns in males and females and in patients from different ethnicities, with unclear predictors for development and severity of disease. Previous studies have shown that NAFLD is 1.4 times more frequent in Hispanics than in Caucasians. One of the major challenges in NAFLD is the lack of accurate, noninvasive biomarkers for the detection of the most aggressive presentation, NASH. The gold standard for the diagnosis is liver biopsy, which is an invasive procedure associated with possible complications. Noninvasive diagnosis of NASH is a major unmet medical need and there are no ethnicity-specific biomarkers that can diagnose this condition and predict its progression. Therefore, the main gap in knowledge that this proposal and line of research will address is the characterizing the different plasma and liver metabolomics profile of patients with fatty liver from two ethnicities (Latinos vs. Caucasians) and of both sexes. The overall hypothesis of the present study is that the higher incidence of nonalcoholic fatty liver (NAFL) in Latino patients is reflected in a different plasma and liver metabolomics profile compared to Caucasian patients with further sex-related differences. Characterization of metabolite networks can aid in identifying the mechanistic underpinnings of sex and ethnic driven differences in NAFL which could help diagnose and establish a prognosis of this condition, especially in the critical transition from NAFL to the more aggressive nonalcoholic steatohepatitis (NASH).To address this hypothesis, plasma metabolomics profile of samples from male and female Latino and Caucasian bariatric surgery patients with NAFL and from healthy subjects will be compared. Metabolomics findings will be related with liver pathology and liver transcriptome profiles from intraoperatively obtained liver biopsies using correlation, network, and pathway analysis.
Institute	University of California, Davis
Department	Department of Internal Medicine, Division of Gastroenterology and Hepatology
Laboratory	Medici Lab
Last Name	Medici
First Name	Valentina
Address	4150 V Street - PSSB Suite 3500 - 95817 Sacramento CA
Email	vmedici@ucdavis.edu
Phone	(916) 734 3751
Submit Date	2021-06-10
Raw Data Available	Yes
Raw Data File Type(s)	cdf
Analysis Type Detail	GC-MS
Release Date	2021-07-05
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR000667
Project DOI:	doi: 10.21228/M8FX07
Project Title:	Identification of unique metabolite networks between Latino and Caucasian patients with nonalcoholic fatty liver disease (NAFLD)
Project Summary:	Nonalcoholic fatty liver disease (NAFLD) is a spectrum of liver pathology ranging from simple steatosis to nonalcoholic steatohepatitis (NASH); the latter is characterized by inflammation and fibrosis. Risk factors for NALFD include obesity, diabetes, hyperlipidemia, and hypertension—all of which are features of metabolic syndrome. NAFLD is a very heterogeneous disease, as it presents in different patterns in males and females and in patients from different ethnicities, with unclear predictors for development and severity of disease. Previous studies have shown that NAFLD is 1.4 times more frequent in Hispanics than in Caucasians. One of the major challenges in NAFLD is the lack of accurate, noninvasive biomarkers for the detection of the most aggressive presentation, NASH. The gold standard for the diagnosis is liver biopsy, which is an invasive procedure associated with possible complications. Noninvasive diagnosis of NASH is a major unmet medical need and there are no ethnicity-specific biomarkers that can diagnose this condition and predict its progression. Therefore, the main gap in knowledge that this proposal and line of research will address is the characterizing the different plasma and liver metabolomics profile of patients with fatty liver from two ethnicities (Latinos vs. Caucasians) and of both sexes. The overall hypothesis of the present study is that the higher incidence of nonalcoholic fatty liver (NAFL) in Latino patients is reflected in a different plasma and liver metabolomics profile compared to Caucasian patients with further sex-related differences. Characterization of metabolite networks can aid in identifying the mechanistic underpinnings of sex and ethnic driven differences in NAFL which could help diagnose and establish a prognosis of this condition, especially in the critical transition from NAFL to the more aggressive nonalcoholic steatohepatitis (NASH).To address this hypothesis, plasma metabolomics profile of samples from male and female Latino and Caucasian bariatric surgery patients with NAFL and from healthy subjects will be compared. Metabolomics findings will be related with liver pathology and liver transcriptome profiles from intraoperatively obtained liver biopsies using correlation, network, and pathway analysis.
Institute:	University of California, Davis
Department:	Department of Internal Medicine, Division of Gastroenterology and Hepatology
Laboratory:	Medici Lab
Last Name:	Medici
First Name:	Valentina
Address:	GI and Hepatology Division Academic Office - 4150 V Street - PSSB Suite 3500 - 95817 Sacramento CA
Email:	vmedici@ucdavis.edu
Phone:	(916) 734 3751

Subject:

Subject ID:	SU001919
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Age Or Age Range:	23-73
Gender:	Male and female
Human Race:	Hispanic and Caucasian

Factors:

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

mb_sample_id	local_sample_id	Diagnosis	Ethnicity	Organ
SA171464	HC126_033	Healthy Control	Caucasian	plasma
SA171465	M2_035	Healthy Control	Caucasian	plasma
SA171466	K9_038	Healthy Control	Caucasian	plasma
SA171467	HC105_037	Healthy Control	Caucasian	plasma
SA171468	HC117_032	Healthy Control	Caucasian	plasma
SA171469	HC121_036	Healthy Control	Caucasian	plasma
SA171470	HC122_034	Healthy Control	Caucasian	plasma
SA171471	K10_039	Healthy Control	Caucasian	plasma
SA171472	2_019	Healthy Control	Hispanic	plasma
SA171473	236_028	Healthy Control	Hispanic	plasma
SA171474	151_024	Healthy Control	Hispanic	plasma
SA171475	HC128_040	Healthy Control	Hispanic	plasma
SA171476	10_020	Healthy Control	Hispanic	plasma
SA171477	316_029	Healthy Control	Hispanic	plasma
SA171478	80_022	Healthy Control	Hispanic	plasma
SA171479	175_025	Healthy Control	Hispanic	plasma
SA171480	110_023	Healthy Control	Hispanic	plasma
SA171481	204_027	Healthy Control	Hispanic	plasma
SA171482	343_030	Healthy Control	Hispanic	plasma
SA171483	197_026	Healthy Control	Hispanic	plasma
SA171484	HC127_031	Healthy Control	Hispanic	plasma
SA171485	26_021	Healthy Control	Hispanic	plasma
SA171486	B-1342_050	NAFLD	Caucasian	liver
SA171487	BSU-0197_043	NAFLD	Caucasian	liver
SA171488	BSU-0187_046	NAFLD	Caucasian	liver
SA171489	B-1297_041	NAFLD	Caucasian	liver
SA171490	B-0286_044	NAFLD	Caucasian	liver
SA171491	B-0254_048	NAFLD	Caucasian	liver
SA171492	BSU-0146_042	NAFLD	Caucasian	liver
SA171493	B-1513_047	NAFLD	Caucasian	liver
SA171494	B-1441_049	NAFLD	Caucasian	liver
SA171495	B-1501_045	NAFLD	Caucasian	liver
SA171496	P-0432_003	NAFLD	Caucasian	plasma
SA171497	P-3194_001	NAFLD	Caucasian	plasma
SA171498	P-3266_007	NAFLD	Caucasian	plasma
SA171499	P-0185_002	NAFLD	Caucasian	plasma
SA171500	P-1006_004	NAFLD	Caucasian	plasma
SA171501	P-3182_008	NAFLD	Caucasian	plasma
SA171502	P-0788_006	NAFLD	Caucasian	plasma
SA171503	P-0361_005	NAFLD	Caucasian	plasma
SA171504	B-1432_060	NAFLD	Hispanic	liver
SA171505	B-0269_057	NAFLD	Hispanic	liver
SA171506	B-1156_059	NAFLD	Hispanic	liver
SA171507	B-0945_053	NAFLD	Hispanic	liver
SA171508	B-0193_051	NAFLD	Hispanic	liver
SA171509	B-0262_052	NAFLD	Hispanic	liver
SA171510	B-1148_055	NAFLD	Hispanic	liver
SA171511	B-1184_054	NAFLD	Hispanic	liver
SA171512	B-1405_058	NAFLD	Hispanic	liver
SA171513	B-1371_056	NAFLD	Hispanic	liver
SA171514	P-3242_018	NAFLD	Hispanic	plasma
SA171515	P-3227_016	NAFLD	Hispanic	plasma
SA171516	P-2546_011	NAFLD	Hispanic	plasma
SA171517	P-3008_013	NAFLD	Hispanic	plasma
SA171518	P-0320_009	NAFLD	Hispanic	plasma
SA171519	P-3200_014	NAFLD	Hispanic	plasma
SA171520	P-3043_012	NAFLD	Hispanic	plasma
SA171521	P-1010_010	NAFLD	Hispanic	plasma
SA171522	P-3011_017	NAFLD	Hispanic	plasma
SA171523	P-0796_015	NAFLD	Hispanic	plasma

Showing results 1 to 60 of 60

Showing results 1 to 60 of 60

Collection:

Collection ID:	CO001912
Collection Summary:	Blood was collected as a part of a routine/pre-operation check up, not more than 2 weeks prior to operation day (bariatric surgery) for the NAFLD group and among normal BMI healthy volunteers for the control group. All volunteers were fasted 10-12 hours before collection. Liver was collected and frozen.
Sample Type:	Blood, Liver

Treatment:

Treatment ID:	TR001931
Treatment Summary:	Subjects were divided into two groups either: Healthy control or Nonalcoholic fatty liver disease (NAFLD)

Sample Preparation:

Sampleprep ID:	SP001925
Sampleprep Summary:	Sample preparation of blood plasma or serum samples for CSH, HILIC and GC analysis Purpose: This SOP describes sample extraction and preparation of blood plasma or serum for lipid profiling on the CSH, and HILIC platform by liquid chromatography/ mass spectrometry (LC-MS) as well as primary metabolomics platform on GC/MS. This method is to be used when there is low sample volume for separate extractions, and when more than one platform is to be used in a project. References: Matyash V, Liebisch G, Kurzchalia TV, Shevchenko A and Schwudke D (2008) Lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics. J Lip Res 2008, 49: 1137-1146 Starting material: Plasma/serum: 20 µL sample volume or aliquot Equipment: Centrifuge Eppendorf 5415 D Calibrated pipettes 20-200µL and 100-1000µL Multi-Tube Vortexer (VWR VX-2500) Orbital Mixing Chilling/Heating Plate (Torrey Pines Scientific Instruments) Speed vacuum concentration system (Labconco Centrivap cold trap) Chemicals: Product Manufacturer & Part Number Eppendorf tubes 1.5 mL, uncolored Eppendorf 022363204 Eppendorf tubes 2 mL, uncolored Eppendorf 022363352 Crushed ice UC Davis Water, LC/MS Grade Fisher Optima W6-4 MTBE, HPLC Grade Acros Organics 389050010 Methanol, LC/MS Grade Fisher A456-4 Bioreclamation human plasma (disodium EDTA) Bioreclamation HMPLEDTA Acetonitrile, HPLC Grade Fisher Optima A955-4 Iso-Propanol, HPLC Grade Fisher A461-4 Sample Preparation: Preparation of extraction solvent Combine 120 mL of chilled MeOH/QC mix with 400 mL of chilled MTBE/Cholesterol Ester 22:1 in a clean 500 mL stock bottle. Mix thoroughly by swirling or stir plate and store at -20°C until use. *See SOP “QC mix for LC-MS lipid analysis” for preparation of MeOH/QC mix and MTBE/Cholesterol Ester 22:1. Preparation of Clean Up solvent For 1 L of extraction solvent, combine 375 mL of acetonitrile, 375 mL of isopropanol, and 250 mL water in a 1 L bottle conditioned with the aforementioned chemicals. If a different total volume of extraction solvent is needed, simply mix acetonitrile, isopropanol, and water in volumes in proportion 3:3:2. Purge the extraction solution mix for 5 min with nitrogen with small bubbles. Make sure that the nitrogen line is flushed out of air before using it for degassing the extraction solvent solution. Store at -20°C until use. Note: if solvent freezes, sonicate until thawed and mix before use. Extraction Thaw raw samples/controls at room temperature (or in the refrigerator at 4˚C) and either invert the tube or vortex 10 sec at low speed to homogenize. Aliquot 20 μL of plasma sample into a 1.5 mL Eppendorf tube. Keep all samples on ice. Add 975 µL ice-cold 3:10 (v/v) MeOH/MTBE + QC mix/CE 22:1 extraction solvent mixture to each aliquot, keeping the extraction solvent on ice during the procedure. Vortex samples for 10 seconds, then shake for 5 minutes at 4°C on the orbital mixer. Add 188 µL room temperature LC/MS grade water to each tube. Vortex tubes for 20 seconds and then centrifuge for 2 min at 14,000 rcf. Transfer the upper organic phase to two separate tubes (350 µL/each tube) for lipidomics analysis. Transfer 75 µL of the remaining organic phase to a 2, 15, or 50 mL tube for pools, depending on number of samples in the study. Transfer the bottom aqueous phase to two separate tubes (110 µL/each tube) for HILIC/GC-TOF analysis. Dry down one tube from each phase by centrivap, keeping the undried tubes as backups. Store all tubes at -20˚C until ready for analysis. Clean up step for GC only (and pooling) Resuspend the dried aliquot with 500 μL 3:3:2 (v/v/v) ACN:IPA:H2O (degassed as given above) and vortex for about 10 sec. Centrifuge for 2 min at 14000 rcf. Remove 450 uL supernatant to a clean 1.5 mL eppendorf tube. Tranfering remainder to a 2, 15, 50 mL Tube, dependent on number of samples. Aliquot out 1.9 mL uL of supernatant to new 2ml eppendorf tubes. Centrifuge for 2 min at 14000 rcf Aliquot out 4x450 uL of supernatant into clean 1.5 mL Eppendorf tubes. Evaporate to comeplete dryness in the Labconco Centruvap cold trap concentrator. Submit to derivatization . Pooling (CSH platform only) Transfer multiple 350 µL aliquots of pooled samples to 1.5 mL Eppendorf tubes, one aliquot for every 10 samples in the study. If there is still pool remaining, prepare additional aliquots for backup. Evaporate to complete dryness in the Labconco Centrivap cold trap concentrator. Store all tubes at -20°C until ready for analysis. Quality assurance For every 10 samples, extract a method blank (20 µL of H2O) and a sample control (20 µL human Bioreclamation or analogous species plasma) in addition to samples. For large studies (>100 samples), for every 100 samples a NIST plasma extract should be prepared in the same manner as positive controls. Disposal of waste Collect all chemicals in appropriate bottles and follow the disposal rules. Collect residual plasma/serum samples in specifically designed red ‘biohazard’ waste bags. Extraction of Mammalian Tissue Samples: Liver 1. References: Fiehn O, Kind T (2006) Metabolite profiling in blood plasma. In: Metabolomics: Methods and Protocols. Weckwerth W (ed.), Humana Press, Totowa NJ (in press) 2.Starting material: Liver sample: weigh 4mg per sample into 2mL Eppendorf tubes. 3. Equipment: Centrifuge (Eppendorf 5415 D) Calibrated pipettes 1-200μl and 100-1000μl Eppendorf tubes 2mL, clear (Cat. No. 022363204) Centrifuge tubes 50mL, polypropylene Eppendorff Tabletop Centrifuge (Proteomics core Lab.) ThermoElectron Neslab RTE 740 cooling bath at –20°C MiniVortexer (VWR) Orbital Mixing Chilling/Heating Plate (Torrey Pines Scientific Instruments) Speed vacuum concentration system (Labconco Centrivap cold trap) Turex mini homogenizer 4. Chemicals Acetonitrile, LCMS grade (JT Baker; Cat. No.9829-02) Isopropanol, HPLC grade (JT Baker; Cat. No. 9095-02) Methanol Acetone Crushed ice 18 MΩ pure water (Millipore) Nitrogen line with pipette tip pH paper 5-10 (EMD Chem. Inc.) 5. Procedure Preparation of extraction mix and material before experiment: Switch on bath to pre-cool at –20°C (±2°C validity temperature range) Check pH of acetonitrile and isopropanol (pH7) using wetted pH paper Make the extraction solution by mixing acetonitrile, isopropanol and water in proportions 3 : 3 : 2 De-gas the extraction solution for 5 min with nitrogen. Make sure that the nitrogen line was flushed out of air before using it for degassing the extraction solvent solution Sample Preparation Weigh 4mg tissue sample in to a 2mL Eppendorf tube. Add 1mL extraction solvent to the tissue sample and homogenize for 45 seconds ensuring that sample resembles a powder. In between samples, clean the homogenizer in solutions of methanol, acetone, water, and the extraction solvent in the order listed. Vortex samples for 10 seconds, then 5 minutes on 4°C shaker. Centrifuge the samples for 2 minutes at 14,000 rcf. Aliquot 500µL supernatant for analysis, and 500µL for a backup. Store backup aliquots in the -20°C freezer. Evaporate one 500µl analysis aliquot in the Labconco Centrivap cold trap concentrator to complete dryness (typically overnight). The dried aliquot is then re-suspended with 500μl 50% acetonitrile (degassed as given) Centrifuge for 2 minutes at 14,000 rcf using the centrifuge Eppendorf 5415. Remove supernatant to a new Eppendorf tube. Evaporate the supernatant to dryness in the the Labconco Centrivap cold trap concentrator. Submit to derivatization. The residue should contain membrane lipids because these are supposedly not soluble enough to be found in the 50% acetonitrile solution. Therefore, this ‘membrane residue’ is now taken for membrane lipidomic fingerprinting using the nanomate LTQ ion trap mass spectrometer. Likely, a good solvent to redissolve the membrane lipids is e.g. 75% isopropanol (degassed as given above). If the ‘analysis’ aliquot is to be used for semi lipophilic compounds such as tyrosine pathway intermediates (incl. dopamine, serotonine etc, i.e. polar aromatic compounds), then these are supposedly to be found together with the ‘GCTOF’ aliquot. We can assume that this mixture is still too complex for Agilent chipLCMS. Therefore, in order to develop and validate target analysis for such aromatic compounds, we should use some sort of Solid Phase purification. We re-suspend the dried ‘GCTOF’ aliquot in 300 l water (degassed as before) to take out sugars, aliphatic amino acids, hydroxyl acids and similar logP compounds. The residue should contain our target aromatics .We could also try to adjust pH by using low concentration acetate or phosphate buffer. The residue could then be taken up in 50% acetonitrile and used for GCTOF and Agilent chipMS experiments. The other aliquot should be checked how much of our target compounds would actually be found in the ‘sugar’ fraction. 6. Problems To prevent contamination disposable material is used. Control pH from extraction mix. 7. Quality assurance For each sequence of sample extractions, perform one blank negative control extraction by applying the total procedure (i.e. all materials and plastic ware) without biological sample. 8. Disposal of waste Collect all chemicals in appropriate bottles and follow the disposal rules.

Sampleprep ID:

SP001925

Sampleprep Summary:

Sample preparation of blood plasma or serum samples for CSH, HILIC and GC analysis Purpose: This SOP describes sample extraction and preparation of blood plasma or serum for lipid profiling on the CSH, and HILIC platform by liquid chromatography/ mass spectrometry (LC-MS) as well as primary metabolomics platform on GC/MS. This method is to be used when there is low sample volume for separate extractions, and when more than one platform is to be used in a project. References: Matyash V, Liebisch G, Kurzchalia TV, Shevchenko A and Schwudke D (2008) Lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics. J Lip Res 2008, 49: 1137-1146 Starting material: Plasma/serum: 20 µL sample volume or aliquot Equipment: Centrifuge Eppendorf 5415 D Calibrated pipettes 20-200µL and 100-1000µL Multi-Tube Vortexer (VWR VX-2500) Orbital Mixing Chilling/Heating Plate (Torrey Pines Scientific Instruments) Speed vacuum concentration system (Labconco Centrivap cold trap) Chemicals: Product Manufacturer & Part Number Eppendorf tubes 1.5 mL, uncolored Eppendorf 022363204 Eppendorf tubes 2 mL, uncolored Eppendorf 022363352 Crushed ice UC Davis Water, LC/MS Grade Fisher Optima W6-4 MTBE, HPLC Grade Acros Organics 389050010 Methanol, LC/MS Grade Fisher A456-4 Bioreclamation human plasma (disodium EDTA) Bioreclamation HMPLEDTA Acetonitrile, HPLC Grade Fisher Optima A955-4 Iso-Propanol, HPLC Grade Fisher A461-4 Sample Preparation: Preparation of extraction solvent Combine 120 mL of chilled MeOH/QC mix with 400 mL of chilled MTBE/Cholesterol Ester 22:1 in a clean 500 mL stock bottle. Mix thoroughly by swirling or stir plate and store at -20°C until use. *See SOP “QC mix for LC-MS lipid analysis” for preparation of MeOH/QC mix and MTBE/Cholesterol Ester 22:1. Preparation of Clean Up solvent For 1 L of extraction solvent, combine 375 mL of acetonitrile, 375 mL of isopropanol, and 250 mL water in a 1 L bottle conditioned with the aforementioned chemicals. If a different total volume of extraction solvent is needed, simply mix acetonitrile, isopropanol, and water in volumes in proportion 3:3:2. Purge the extraction solution mix for 5 min with nitrogen with small bubbles. Make sure that the nitrogen line is flushed out of air before using it for degassing the extraction solvent solution. Store at -20°C until use. Note: if solvent freezes, sonicate until thawed and mix before use. Extraction Thaw raw samples/controls at room temperature (or in the refrigerator at 4˚C) and either invert the tube or vortex 10 sec at low speed to homogenize. Aliquot 20 μL of plasma sample into a 1.5 mL Eppendorf tube. Keep all samples on ice. Add 975 µL ice-cold 3:10 (v/v) MeOH/MTBE + QC mix/CE 22:1 extraction solvent mixture to each aliquot, keeping the extraction solvent on ice during the procedure. Vortex samples for 10 seconds, then shake for 5 minutes at 4°C on the orbital mixer. Add 188 µL room temperature LC/MS grade water to each tube. Vortex tubes for 20 seconds and then centrifuge for 2 min at 14,000 rcf. Transfer the upper organic phase to two separate tubes (350 µL/each tube) for lipidomics analysis. Transfer 75 µL of the remaining organic phase to a 2, 15, or 50 mL tube for pools, depending on number of samples in the study. Transfer the bottom aqueous phase to two separate tubes (110 µL/each tube) for HILIC/GC-TOF analysis. Dry down one tube from each phase by centrivap, keeping the undried tubes as backups. Store all tubes at -20˚C until ready for analysis. Clean up step for GC only (and pooling) Resuspend the dried aliquot with 500 μL 3:3:2 (v/v/v) ACN:IPA:H2O (degassed as given above) and vortex for about 10 sec. Centrifuge for 2 min at 14000 rcf. Remove 450 uL supernatant to a clean 1.5 mL eppendorf tube. Tranfering remainder to a 2, 15, 50 mL Tube, dependent on number of samples. Aliquot out 1.9 mL uL of supernatant to new 2ml eppendorf tubes. Centrifuge for 2 min at 14000 rcf Aliquot out 4x450 uL of supernatant into clean 1.5 mL Eppendorf tubes. Evaporate to comeplete dryness in the Labconco Centruvap cold trap concentrator. Submit to derivatization . Pooling (CSH platform only) Transfer multiple 350 µL aliquots of pooled samples to 1.5 mL Eppendorf tubes, one aliquot for every 10 samples in the study. If there is still pool remaining, prepare additional aliquots for backup. Evaporate to complete dryness in the Labconco Centrivap cold trap concentrator. Store all tubes at -20°C until ready for analysis. Quality assurance For every 10 samples, extract a method blank (20 µL of H2O) and a sample control (20 µL human Bioreclamation or analogous species plasma) in addition to samples. For large studies (>100 samples), for every 100 samples a NIST plasma extract should be prepared in the same manner as positive controls. Disposal of waste Collect all chemicals in appropriate bottles and follow the disposal rules. Collect residual plasma/serum samples in specifically designed red ‘biohazard’ waste bags. Extraction of Mammalian Tissue Samples: Liver 1. References: Fiehn O, Kind T (2006) Metabolite profiling in blood plasma. In: Metabolomics: Methods and Protocols. Weckwerth W (ed.), Humana Press, Totowa NJ (in press) 2.Starting material: Liver sample: weigh 4mg per sample into 2mL Eppendorf tubes. 3. Equipment: Centrifuge (Eppendorf 5415 D) Calibrated pipettes 1-200μl and 100-1000μl Eppendorf tubes 2mL, clear (Cat. No. 022363204) Centrifuge tubes 50mL, polypropylene Eppendorff Tabletop Centrifuge (Proteomics core Lab.) ThermoElectron Neslab RTE 740 cooling bath at –20°C MiniVortexer (VWR) Orbital Mixing Chilling/Heating Plate (Torrey Pines Scientific Instruments) Speed vacuum concentration system (Labconco Centrivap cold trap) Turex mini homogenizer 4. Chemicals Acetonitrile, LCMS grade (JT Baker; Cat. No.9829-02) Isopropanol, HPLC grade (JT Baker; Cat. No. 9095-02) Methanol Acetone Crushed ice 18 MΩ pure water (Millipore) Nitrogen line with pipette tip pH paper 5-10 (EMD Chem. Inc.) 5. Procedure Preparation of extraction mix and material before experiment: Switch on bath to pre-cool at –20°C (±2°C validity temperature range) Check pH of acetonitrile and isopropanol (pH7) using wetted pH paper Make the extraction solution by mixing acetonitrile, isopropanol and water in proportions 3 : 3 : 2 De-gas the extraction solution for 5 min with nitrogen. Make sure that the nitrogen line was flushed out of air before using it for degassing the extraction solvent solution Sample Preparation Weigh 4mg tissue sample in to a 2mL Eppendorf tube. Add 1mL extraction solvent to the tissue sample and homogenize for 45 seconds ensuring that sample resembles a powder. In between samples, clean the homogenizer in solutions of methanol, acetone, water, and the extraction solvent in the order listed. Vortex samples for 10 seconds, then 5 minutes on 4°C shaker. Centrifuge the samples for 2 minutes at 14,000 rcf. Aliquot 500µL supernatant for analysis, and 500µL for a backup. Store backup aliquots in the -20°C freezer. Evaporate one 500µl analysis aliquot in the Labconco Centrivap cold trap concentrator to complete dryness (typically overnight). The dried aliquot is then re-suspended with 500μl 50% acetonitrile (degassed as given) Centrifuge for 2 minutes at 14,000 rcf using the centrifuge Eppendorf 5415. Remove supernatant to a new Eppendorf tube. Evaporate the supernatant to dryness in the the Labconco Centrivap cold trap concentrator. Submit to derivatization. The residue should contain membrane lipids because these are supposedly not soluble enough to be found in the 50% acetonitrile solution. Therefore, this ‘membrane residue’ is now taken for membrane lipidomic fingerprinting using the nanomate LTQ ion trap mass spectrometer. Likely, a good solvent to redissolve the membrane lipids is e.g. 75% isopropanol (degassed as given above). If the ‘analysis’ aliquot is to be used for semi lipophilic compounds such as tyrosine pathway intermediates (incl. dopamine, serotonine etc, i.e. polar aromatic compounds), then these are supposedly to be found together with the ‘GCTOF’ aliquot. We can assume that this mixture is still too complex for Agilent chipLCMS. Therefore, in order to develop and validate target analysis for such aromatic compounds, we should use some sort of Solid Phase purification. We re-suspend the dried ‘GCTOF’ aliquot in 300 l water (degassed as before) to take out sugars, aliphatic amino acids, hydroxyl acids and similar logP compounds. The residue should contain our target aromatics .We could also try to adjust pH by using low concentration acetate or phosphate buffer. The residue could then be taken up in 50% acetonitrile and used for GCTOF and Agilent chipMS experiments. The other aliquot should be checked how much of our target compounds would actually be found in the ‘sugar’ fraction. 6. Problems To prevent contamination disposable material is used. Control pH from extraction mix. 7. Quality assurance For each sequence of sample extractions, perform one blank negative control extraction by applying the total procedure (i.e. all materials and plastic ware) without biological sample. 8. Disposal of waste Collect all chemicals in appropriate bottles and follow the disposal rules.

Combined analysis:

Analysis ID	AN002985
Analysis type	MS
Chromatography type	GC
Chromatography system	LECO Pegasus IV GC-time of flight mass spectrometers
Column	Rtx-5Sil MS
MS Type	EI
MS instrument type	GC-TOF
MS instrument name	Leco Pegasus IV TOF
Ion Mode	UNSPECIFIED
Units	normalized peak height

Chromatography:

Chromatography ID:	CH002214
Chromatography Summary:	Primary metabolism by GCTOF
Instrument Name:	LECO Pegasus IV GC-time of flight mass spectrometers
Column Name:	Rtx-5Sil MS
Chromatography Type:	GC

MS:

MS ID:	MS002775
Analysis ID:	AN002985
Instrument Name:	Leco Pegasus IV TOF
Instrument Type:	GC-TOF
MS Type:	EI
MS Comments:	GC-TOF Method: Instruments: Gerstel CIS4 –with dual MPS Injector/ Agilent 6890 GC- Pegasus III TOF MS Injector conditions: Agilent 6890 GC is equipped with a Gerstel automatic liner exchange system (ALEX) that includes a multipurpose sample (MPS2) dual rail, and a Gerstel CIS cold injection system (Gerstel, Muehlheim, Germany) with temperature program as follows: 50°C to 275°C final temperature at a rate of 12 °C/s and hold for 3 minutes. Injection volume is 0.5 μl with 10 μl/s injection speed on a splitless injector with purge time of 25 seconds. Liner (Gerstel #011711-010-00) is changed after every 10 samples, (using the Maestro1 Gerstel software vs. 1.1.4.18). Before and after each injection, the 10 μl injection syringe is washed three times with 10 μl ethyl acetate. Gas Chromatography conditions: A 30 m long, 0.25 mm i.d. Rtx-5Sil MS column (0.25 μm 95% dimethyl 5% diphenyl polysiloxane film) with additional 10 m integrated guard column is used (Restek, Bellefonte PA). 99.9999% pure Helium with built-in purifier (Airgas, Radnor PA) is set at constant flow of 1 ml/min. The oven temperature is held constant at 50°C for 1 min and then ramped at 20°C/min to 330°C at which it is held constant for 5 min. Mass spectrometer settings: A Leco Pegasus IV time of flight mass spectrometer is controlled by the Leco ChromaTOF software vs. 2.32 (St. Joseph, MI). The transfer line temperature between gas chromatograph and mass spectrometer is set to 280°C. Electron impact ionization at 70V is employed with an ion source temperature of 250°C. Acquisition rate is 17 spectra/second, with a scan mass range of 85-500 Da.
Ion Mode:	UNSPECIFIED