Summary of Study ST001844

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	ST001844
Study Title	Identification of unique metabolite networks between Latino and Caucasian patients with nonalcoholic fatty liver disease (NAFLD) (part III)
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)	wiff
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:	SU001921
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	organ
SA171584	Medici_B-1513_047_posHILIC__022	Liver
SA171585	Medici_B-1441_049_posHILIC__015	Liver
SA171586	Medici_B-1342_050_posHILIC__008	Liver
SA171587	Medici_B-1297_041_posHILIC__023	Liver
SA171588	Medici_BSU-0146_042_posHILIC__009	Liver
SA171589	Medici_BSU-0187_046_posHILIC__002	Liver
SA171590	Medici_B-0193_051_posHILIC__004	Liver
SA171591	Medici_B-0262_052_posHILIC__020	Liver
SA171592	Medici_BSU-0197_043_posHILIC__005	Liver
SA171593	Medici_B-0286_044_posHILIC__017	Liver
SA171594	Medici_B-1501_045_posHILIC__018	Liver
SA171595	Medici_B-1148_055_posHILIC__006	Liver
SA171596	Medici_B-0945_053_posHILIC__019	Liver
SA171597	Medici_B-0254_048_posHILIC__021	Liver
SA171598	Medici_B-1184_054_posHILIC__003	Liver
SA171599	Medici_B-1156_059_posHILIC__010	Liver
SA171600	Medici_B-1432_060_posHILIC__011	Liver
SA171601	Medici_B-1371_056_posHILIC__014	Liver
SA171602	Medici_B-1405_058_posHILIC__016	Liver
SA171603	Medici_B-0269_057_posHILIC__007	Liver
SA171604	Medici_plasma_316_029_posHILIC__059	Plasma
SA171605	Medici_plasma_343_030_posHILIC__037	Plasma
SA171606	Medici_plasma_80_022_posHILIC__045	Plasma
SA171607	Medici_plasma_26_021_posHILIC__058	Plasma
SA171608	Medici_plasma_175_025_posHILIC__069	Plasma
SA171609	Medici_plasma_HC127_031_posHILIC__067	Plasma
SA171610	Medici_plasma_197_026_posHILIC__041	Plasma
SA171611	Medici_plasma_2_019_posHILIC__066	Plasma
SA171612	Medici_plasma_204_027_posHILIC__070	Plasma
SA171613	Medici_plasma_236_028_posHILIC__029	Plasma
SA171614	Medici_plasma_151_024_posHILIC__034	Plasma
SA171615	Medici_plasma_K10_039_posHILIC__032	Plasma
SA171616	Medici_plasma_K9_038_posHILIC__060	Plasma
SA171617	Medici_plasma_M2_035_posHILIC__044	Plasma
SA171618	Medici_plasma_HC126_033_posHILIC__033	Plasma
SA171619	Medici_plasma_HC122_034_posHILIC__053	Plasma
SA171620	Medici_plasma_HC105_037_posHILIC__061	Plasma
SA171621	Medici_plasma_HC117_032_posHILIC__048	Plasma
SA171622	Medici_plasma_HC121_036_posHILIC__055	Plasma
SA171623	Medici_plasma_HC128_040_posHILIC__035	Plasma
SA171624	Medici_plasma_P-3242_018_posHILIC__047	Plasma
SA171625	Medici_plasma_P-3008_013_posHILIC__042	Plasma
SA171626	Medici_plasma_P-3011_017_posHILIC__031	Plasma
SA171627	Medici_plasma_P-3043_012_posHILIC__054	Plasma
SA171628	Medici_plasma_P-3200_014_posHILIC__068	Plasma
SA171629	Medici_plasma_P-2546_011_posHILIC__049_reinject	Plasma
SA171630	Medici_plasma_P-2546_011_posHILIC__049	Plasma
SA171631	Medici_plasma_P-0320_009_posHILIC__065	Plasma
SA171632	Medici_plasma_P-0796_015_posHILIC__046	Plasma
SA171633	Medici_plasma_P-1010_010_posHILIC__043	Plasma
SA171634	Medici_plasma_P-3227_016_posHILIC__030	Plasma
SA171635	Medici_plasma_P-0185_002_posHILIC__056	Plasma
SA171636	Medici_plasma_P-3194_001_posHILIC__036	Plasma
SA171637	Medici_plasma_P-3266_007_posHILIC__073	Plasma
SA171638	Medici_plasma_10_020_posHILIC__072	Plasma
SA171639	Medici_plasma_P-3182_008_posHILIC__071	Plasma
SA171640	Medici_plasma_P-1006_004_posHILIC__057	Plasma
SA171641	Medici_plasma_P-0361_005_posHILIC__038	Plasma
SA171642	Medici_plasma_P-0432_003_posHILIC__062	Plasma
SA171643	Medici_plasma_P-0788_006_posHILIC__074	Plasma
SA171644	Medici_plasma_110_023_posHILIC__050	Plasma

Showing results 1 to 61 of 61

Showing results 1 to 61 of 61

Collection:

Collection ID:	CO001914
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 obtained during surgery (no preservatives, flash-frozen)
Sample Type:	Blood, Liver

Treatment:

Treatment ID:	TR001933
Treatment Summary:	Subjects were divided into two groups either: Healthy control or Nonalcoholic fatty liver disease (NAFLD)

Sample Preparation:

Sampleprep ID:	SP001927
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:

SP001927

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	AN002987
Analysis type	MS
Chromatography type	HILIC
Chromatography system	Agilent 1290 Infinity
Column	Waters Acquity BEH Amide (150 x 2.1mm,1.7um)
MS Type	EI
MS instrument type	TTOF
MS instrument name	ABI Sciex 6600 TripleTOF
Ion Mode	UNSPECIFIED
Units	normalized peak height

Chromatography:

Chromatography ID:	CH002216
Chromatography Summary:	Biogenic amines by LC TTOF HILIC
Instrument Name:	Agilent 1290 Infinity
Column Name:	Waters Acquity BEH Amide (150 x 2.1mm,1.7um)
Column Temperature:	45
Flow Gradient:	0 min 100% (A); 0-2 min 100% (A); 2-7.7 min 30% (A); 7.7-9.5 min 60% (A); 9.5-10.3 min 70% (A); 10.3-12.8 min 0% (A); 12.8-16.8 min 0% (A)
Flow Rate:	0.4 mL/min
Solvent A:	100% water; 0.125% formic acid; 10 mM ammonium formate
Solvent B:	95% acetonitrile/5% water; 0.125% formic acid; 10 mM ammonium formate
Chromatography Type:	HILIC

MS:

MS ID:	MS002777
Analysis ID:	AN002987
Instrument Name:	ABI Sciex 6600 TripleTOF
Instrument Type:	TTOF
MS Type:	EI
MS Comments:	The LC/QTOFMS analyses are performed using an Agilent 1290 Infinity LC system (G4220A binary pump, G4226A autosampler, and G1316C Column Thermostat) coupled to a SCIEX Triple TOF mass spectrometer. Polar compounds are separated on an Acquity UPLC BEH Amide Column, 130Å, 1.7 µm, 2.1 mm X 150 mm maintained at 45°C at a flow-rate of 0.4 mL/min. Solvent pre-heating (Agilent G1316) was used. The mobile phases consist of: Water, 10 mM Ammonium Formate, 0.125% Formic Acid (A) and Acetonitrile: Water (95/5, v/v), 10 mM Ammonium Formate, 0.125% Formic Acid (B) The gradient is as follows: 0 min 100% (A); 0–2 min 100% (A); 2–7.7 min 30% (A); 7.7–9.5 min 60% (A); 9.5–10.3 min 70% (A); 10.3–12.8 min 0% (A); 12.8–16.8 min 0% (A. A sample volume of 1 µL for positive mode and 3 µL for negative mode is used for the injection. Sample temperature is maintained at 4°C in the autosampler. SCIEX Triple TOF 6600mass spectrometers are operated with electrospray ionization (ESI) performing full scan in the mass range m/z 50–1200. Number of cycles in MS1 is 1667 with cycle time of 500ms and accumulation time 475ms. Mass spectrometer parameters are as follows (positive mode) Gas Temp 300°C, gas pressures in psi units with : GS1 and GS2 50 psi, CUR: 35. ISVF is 4500V and DP and CE are 10V and 100 V.
Ion Mode:	UNSPECIFIED