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

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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.