Summary of Study ST001809

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 PR001143. The data can be accessed directly via it's Project DOI: 10.21228/M8ZH7D 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	ST001809
Study Title	The Metabolic Benefits of Short Cycles of Very Low Caloric Intake are Dependent on Diet Composition in Middle-Aged Mice
Study Summary	Diet composition, calories, and fasting times contribute to maintenance of health. Here, middle-aged mice were maintained for 5 months on 4:10 feeding cycles, consisting of 4 days of very low-calorie intake (VLCI) achieved with either standard laboratory chow (SD) or a fasting mimicking diet (FMD), followed by 10 days of ad libitum access to SD. Fat and lean mass loss was accompanied with improved performance, glucoregulation, and metabolic flexibility independent of diet composition. However, only the 4:10/SD cycles elicited a long-lasting metabolomic reprograming in serum and liver that was preserved six days after refeeding. Challenged with an obesogenic diet, cycles of VLCI achieved with either high-fat diet (HFD) or FMD during the low-calorie period did not prevent diet-induced obesity nor did they elicited a long-lasting metabolic memory, despite achieving modest metabolic flexibility. Our results highlight the importance of diet composition in mediating the metabolic benefits of short cycles of VLCI.
Institute	National Institutes of Health
Department	Experimental Gerontology Section and Translational Gerontology Branch, NIA
Last Name	de Cabo
First Name	Rafael
Address	251 Bayview Blvd. Suite 100/Room 5C214. Baltimore, MD 21224
Email	deCaboRa@grc.nia.nih.gov
Phone	+1-410-558-8510
Submit Date	2021-05-27
Raw Data Available	Yes
Raw Data File Type(s)	cdf
Analysis Type Detail	GC-MS
Release Date	2021-09-15
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR001143
Project DOI:	doi: 10.21228/M8ZH7D
Project Title:	The Metabolic Benefits of Short Cycles of Very Low Caloric Intake are Dependent on Diet Composition in Middle-Aged Mice
Project Summary:	Diet composition, calories, and fasting times contribute to maintenance of health. Here, middle-aged mice were maintained for 5 months on 4:10 feeding cycles, consisting of 4 days of very low-calorie intake (VLCI) achieved with either standard laboratory chow (SD) or a fasting mimicking diet (FMD), followed by 10 days of ad libitum access to SD. Fat and lean mass loss was accompanied with improved performance, glucoregulation, and metabolic flexibility independent of diet composition. However, only the 4:10/SD cycles elicited a long-lasting metabolomic reprograming in serum and liver that was preserved six days after refeeding. Challenged with an obesogenic diet, cycles of VLCI achieved with either high-fat diet (HFD) or FMD during the low-calorie period did not prevent diet-induced obesity nor did they elicited a long-lasting metabolic memory, despite achieving modest metabolic flexibility. Our results highlight the importance of diet composition in mediating the metabolic benefits of short cycles of VLCI.
Institute:	National Institutes of Health
Department:	Experimental Gerontology Section and Translational Gerontology Branch, NIA
Last Name:	de Cabo
First Name:	Rafael
Address:	251 Bayview Blvd. Suite 100/Room 5C214. Baltimore, MD 21224
Email:	deCaboRa@grc.nia.nih.gov
Phone:	+1-410-558-8510

Subject:

Subject ID:	SU001886
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

Factors:

Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id	local_sample_id	treatment
SA167967	L-FMD075_164	ALHF-Liver
SA167968	L-FMD063_155	ALHF-Liver
SA167969	L-FMD064_156	ALHF-Liver
SA167970	L-FMD061_153	ALHF-Liver
SA167971	L-FMD060_152	ALHF-Liver
SA167972	L-FMD058_150	ALHF-Liver
SA167973	L-FMD059_151	ALHF-Liver
SA167974	L-FMD065_157	ALHF-Liver
SA167975	L-FMD062_154	ALHF-Liver
SA167976	L-FMD067_158	ALHF-Liver
SA167977	L-FMD073_163	ALHF-Liver
SA167978	L-FMD072_162	ALHF-Liver
SA167979	L-FMD070_161	ALHF-Liver
SA167980	L-FMD068_159	ALHF-Liver
SA167981	L-FMD069_160	ALHF-Liver
SA167982	S-FMD064_111	ALHF-Serum
SA167983	S-FMD065_112	ALHF-Serum
SA167984	S-FMD063_110	ALHF-Serum
SA167985	S-FMD060_107	ALHF-Serum
SA167986	S-FMD067_113	ALHF-Serum
SA167987	S-FMD061_108	ALHF-Serum
SA167988	S-FMD062_109	ALHF-Serum
SA167989	S-FMD058_105	ALHF-Serum
SA167990	S-FMD075_119	ALHF-Serum
SA167991	S-FMD059_106	ALHF-Serum
SA167992	S-FMD073_118	ALHF-Serum
SA167993	S-FMD072_117	ALHF-Serum
SA167994	S-FMD069_115	ALHF-Serum
SA167995	S-FMD070_116	ALHF-Serum
SA167996	S-FMD068_114	ALHF-Serum
SA167936	L-FMD013_063	AL-Liver
SA167937	L-FMD012_062	AL-Liver
SA167938	L-FMD014_064	AL-Liver
SA167939	L-FMD016_066	AL-Liver
SA167940	L-FMD001_052	AL-Liver
SA167941	L-FMD011_061	AL-Liver
SA167942	L-FMD015_065	AL-Liver
SA167943	L-FMD018_067	AL-Liver
SA167944	L-FMD005_055	AL-Liver
SA167945	L-FMD002_053	AL-Liver
SA167946	L-FMD010_060	AL-Liver
SA167947	L-FMD006_056	AL-Liver
SA167948	L-FMD004_054	AL-Liver
SA167949	L-FMD009_059	AL-Liver
SA167950	L-FMD007_057	AL-Liver
SA167951	L-FMD008_058	AL-Liver
SA167952	S-FMD007_005	AL-Serum
SA167953	S-FMD008_006	AL-Serum
SA167954	S-FMD009_007	AL-Serum
SA167955	S-FMD005_003	AL-Serum
SA167956	S-FMD002_001	AL-Serum
SA167957	S-FMD010_008	AL-Serum
SA167958	S-FMD006_004	AL-Serum
SA167959	S-FMD015_013	AL-Serum
SA167960	S-FMD018_015	AL-Serum
SA167961	S-FMD004_002	AL-Serum
SA167962	S-FMD016_014	AL-Serum
SA167963	S-FMD014_012	AL-Serum
SA167964	S-FMD012_010	AL-Serum
SA167965	S-FMD013_011	AL-Serum
SA167966	S-FMD011_009	AL-Serum
SA167997	L-FMD036_085	FMD-Liver
SA167998	L-FMD024_073	FMD-Liver
SA167999	L-FMD025_074	FMD-Liver
SA168000	L-FMD026_075	FMD-Liver
SA168001	L-FMD023_072	FMD-Liver
SA168002	L-FMD022_071	FMD-Liver
SA168003	L-FMD035_084	FMD-Liver
SA168004	L-FMD020_069	FMD-Liver
SA168005	L-FMD021_070	FMD-Liver
SA168006	L-FMD027_076	FMD-Liver
SA168007	L-FMD019_068	FMD-Liver
SA168008	L-FMD034_083	FMD-Liver
SA168009	L-FMD028_077	FMD-Liver
SA168010	L-FMD032_081	FMD-Liver
SA168011	L-FMD033_082	FMD-Liver
SA168012	L-FMD031_080	FMD-Liver
SA168013	L-FMD030_079	FMD-Liver
SA168014	L-FMD029_078	FMD-Liver
SA168015	S-FMD026_023	FMD-Serum
SA168016	S-FMD028_025	FMD-Serum
SA168017	S-FMD025_022	FMD-Serum
SA168018	S-FMD027_024	FMD-Serum
SA168019	S-FMD021_018	FMD-Serum
SA168020	S-FMD029_026	FMD-Serum
SA168021	S-FMD022_019	FMD-Serum
SA168022	S-FMD023_020	FMD-Serum
SA168023	S-FMD024_021	FMD-Serum
SA168024	S-FMD033_030	FMD-Serum
SA168025	S-FMD036_033	FMD-Serum
SA168026	S-FMD020_017	FMD-Serum
SA168027	S-FMD019_016	FMD-Serum
SA168028	S-FMD035_032	FMD-Serum
SA168029	S-FMD034_031	FMD-Serum
SA168030	S-FMD031_028	FMD-Serum
SA168031	S-FMD032_029	FMD-Serum
SA168032	S-FMD030_027	FMD-Serum
SA168033	L-FMD111_196	HCR-Liver
SA168034	L-FMD105_190	HCR-Liver
SA168035	L-FMD104_189	HCR-Liver

Showing page 1 of 2 Results: 1 2 Next Showing results 1 to 100 of 196

Collection:

Collection ID:	CO001879
Collection Summary:	Mice were euthanized and serum was collected thereafter. Liver was perfused in PBS, frozen in LN2 and pulverized thereafter.
Sample Type:	Blood (serum)

Treatment:

Treatment ID:	TR001899
Treatment Summary:	6, 5-8 All mice were identified with FMD followed by a 3 digit number SD - day SD - night FMD - fed iFMD - fed FMD - fast iFMD - fast FMD - refed iFMD - refed

Sample Preparation:

Sampleprep ID:	SP001892
Sampleprep Summary:	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. Sample preparation of blood plasma or serum samples for GCTOF analysis Purpose: This SOP describes sample extraction and sample preparation for primary metabolism profiling by gas chromatography/time-of-flight mass spectrometry (GCTOF). References: Fiehn O, Kind T (2006) Metabolite profiling in blood plasma. In: Metabolomics: Methods and Protocols. Weckwerth W (ed.), Humana Press, Totowa NJ. Fiehn, O. Metabolomics by gas chromatography - mass spectrometry: combined targeted and untargeted profiling. 2016. Curr. Protoc. Mol. Biol. 114:30.4.1-30.4.32. doi: 10.1002/0471142727.mb3004s114. Starting material: Plasma/serum: 30 µL sample volume or aliquot Equipment: Centrifuge Eppendorf 5415 D Calibrated pipettes 1-200µL 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) Nitrogen line with Pasteur pipette Chemicals and consumables: Product Manufacturer & Part Number Eppendorf tubes 1.5 mL, uncolored Eppendorf 022363204 Crushed ice UC Davis Water, LC/MS Grade Fisher Optima W6-4 Acetonitrile, LC/MS Grade Fisher Optima A955-4 Isopropanol, LC/MS Grade Fisher A461-4 pH paper 5-10 Millipore Sigma 1095330001 Bioreclamation human plasma (disodium EDTA) Bioreclamation HMPLEDTA Sample Preparation: Preparation of extraction 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 at room temperature (or in the refrigerator at 4˚C) and vortex 10 sec at low speed to homogenize. Aliquot 30 μL of plasma sample into a 1.5 mL Eppendorf tube. Keep all samples on ice. Add 1 mL 3:3:2 (v/v/v) ACN:IPA:H2O extraction solvent (prechilled in a -20°C freezer). Vortex the sample for 10 sec. Shake for 5 min at 4°C using the Orbital Mixing Chilling/Heating Plate. Continue to keep all extracted samples on ice. Centrifuge samples for 2 min at 14000 rcf. Aliquot two 450 μL portions of the supernatant into 1.5 mL Eppendorf tubes (one for analysis and one as a backup sample). Transfer 100 μL of the remaining supernatant from each sample to a 2, 15, or 50 mL tube for pools, depending on number of samples in the study. Evaporate one 450 μL aliquot of the sample in the Labconco Centrivap cold trap concentrator to complete dryness. Proceed with cleanup or store tubes at -20°C until cleanup. Pooling Transfer multiple 475 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. Centrifuge pool samples for 2 min at 14000 rcf. Remove 450 µL supernatant to new 1.5 mL Eppendorf tube. Evaporate to complete dryness in the Labconco Centrivap cold trap concentrator. Proceed with cleanup or store tubes at -20°C until cleanup. Cleanup Resuspend the dried aliquot with 500 μL 50:50 (v/v) ACN:H2O (degassed as given above) and vortex for about 10 sec. Centrifuge for 2 min at 14000 rcf. Remove 475 μL supernatant to a new 1.5 mL Eppendorf tube. Evaporate the transferred supernatant to complete dryness in the Labconco Centrivap cold trap concentrator. Submit to derivatization (see SOP “Derivatization of GC Samples & Standards”) or store at -20°C until ready for analysis. Quality assurance For every 50 samples, perform one method blank negative control extraction by applying the total procedure (i.e. all materials and plastic ware) without biological sample. If no combined pool was made from the extracted samples, use one commercial plasma/serum pool sample per 10 authentic subject samples as control instead. 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.

Sampleprep ID:

SP001892

Sampleprep Summary:

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. Sample preparation of blood plasma or serum samples for GCTOF analysis Purpose: This SOP describes sample extraction and sample preparation for primary metabolism profiling by gas chromatography/time-of-flight mass spectrometry (GCTOF). References: Fiehn O, Kind T (2006) Metabolite profiling in blood plasma. In: Metabolomics: Methods and Protocols. Weckwerth W (ed.), Humana Press, Totowa NJ. Fiehn, O. Metabolomics by gas chromatography - mass spectrometry: combined targeted and untargeted profiling. 2016. Curr. Protoc. Mol. Biol. 114:30.4.1-30.4.32. doi: 10.1002/0471142727.mb3004s114. Starting material: Plasma/serum: 30 µL sample volume or aliquot Equipment: Centrifuge Eppendorf 5415 D Calibrated pipettes 1-200µL 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) Nitrogen line with Pasteur pipette Chemicals and consumables: Product Manufacturer & Part Number Eppendorf tubes 1.5 mL, uncolored Eppendorf 022363204 Crushed ice UC Davis Water, LC/MS Grade Fisher Optima W6-4 Acetonitrile, LC/MS Grade Fisher Optima A955-4 Isopropanol, LC/MS Grade Fisher A461-4 pH paper 5-10 Millipore Sigma 1095330001 Bioreclamation human plasma (disodium EDTA) Bioreclamation HMPLEDTA Sample Preparation: Preparation of extraction 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 at room temperature (or in the refrigerator at 4˚C) and vortex 10 sec at low speed to homogenize. Aliquot 30 μL of plasma sample into a 1.5 mL Eppendorf tube. Keep all samples on ice. Add 1 mL 3:3:2 (v/v/v) ACN:IPA:H2O extraction solvent (prechilled in a -20°C freezer). Vortex the sample for 10 sec. Shake for 5 min at 4°C using the Orbital Mixing Chilling/Heating Plate. Continue to keep all extracted samples on ice. Centrifuge samples for 2 min at 14000 rcf. Aliquot two 450 μL portions of the supernatant into 1.5 mL Eppendorf tubes (one for analysis and one as a backup sample). Transfer 100 μL of the remaining supernatant from each sample to a 2, 15, or 50 mL tube for pools, depending on number of samples in the study. Evaporate one 450 μL aliquot of the sample in the Labconco Centrivap cold trap concentrator to complete dryness. Proceed with cleanup or store tubes at -20°C until cleanup. Pooling Transfer multiple 475 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. Centrifuge pool samples for 2 min at 14000 rcf. Remove 450 µL supernatant to new 1.5 mL Eppendorf tube. Evaporate to complete dryness in the Labconco Centrivap cold trap concentrator. Proceed with cleanup or store tubes at -20°C until cleanup. Cleanup Resuspend the dried aliquot with 500 μL 50:50 (v/v) ACN:H2O (degassed as given above) and vortex for about 10 sec. Centrifuge for 2 min at 14000 rcf. Remove 475 μL supernatant to a new 1.5 mL Eppendorf tube. Evaporate the transferred supernatant to complete dryness in the Labconco Centrivap cold trap concentrator. Submit to derivatization (see SOP “Derivatization of GC Samples & Standards”) or store at -20°C until ready for analysis. Quality assurance For every 50 samples, perform one method blank negative control extraction by applying the total procedure (i.e. all materials and plastic ware) without biological sample. If no combined pool was made from the extracted samples, use one commercial plasma/serum pool sample per 10 authentic subject samples as control instead. 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.

Combined analysis:

Analysis ID	AN002932
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:	CH002173
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:	MS002723
Analysis ID:	AN002932
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