Summary of Study ST003672

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 PR002277. The data can be accessed directly via it's Project DOI: 10.21228/M89G0H 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	ST003672
Study Title	Advanced Lipidomics Using UHPLC-ESI-QTOF-MS/MS Reveals Novel Lipids in Hibernating Syrian Hamsters
Study Summary	Mammalian hibernation offers a unique model for exploring neuroprotective mechanisms relevant to neurodegenerative diseases. In this study, we employed untargeted lipidomics with iterative tandem mass spectrometry (MS/MS) to profile the brain lipidome of Syrian hamsters across different hibernation stages: late torpor, arousal, and euthermia (control). Previously, a lipid species identified as methyl-PA(16:0/0:0) showed a significant increase during torpor, but its precise structure was unresolved due to technological constraints. Leveraging iterative MS/MS and advanced lipid annotation tools (LipidAnnotator and MS-DIAL), we accurately annotated 377 lipid species, including the re-identification of methyl-PA(16:0/0:0) as methylated lysophosphatidic acid (PMeOH 16:0/0:0). This reannotation led to the discovery of two additional lipids during torpor: PMeOH 18:0/0:0 and PMeOH 18:1/0:0. Verification involved manual inspection of MS/MS spectra and Kendrick Mass Defect plots. The lipid alterations observed during torpor suggest biochemical adaptations to maintain membrane fluidity and protect against oxidative stress under hypothermic conditions. Elevated levels of PMeOH lipids and their lyso-forms may play roles in cell survival signalling. Additionally, a decrease in phosphatidic acid species and an increase in diacylglycerol species imply a metabolic shift favouring diacylglycerol production, potentially activating protein kinase C signalling pathways. The increased levels of monogalactosyl diglyceride lipids during torpor suggest a role in neuroprotection by enhancing oligodendrocyte function and myelination. Our comprehensive lipidomic profiling provides detailed insights into lipid dynamics associated with hibernation and underscores the potential of advanced MS/MS methodologies in lipidomics for developing therapeutic strategies against neurodegenerative diseases.
Institute	Universidad CEU San Pablo
Department	Centro de Metabolómica y Bioanálisis (CEMBIO)
Last Name	González
First Name	Carolina
Address	km 0, Universidad CEU-San Pablo Urbanización Montepríncipe. M-501
Email	carolina.gonzalezriano@ceu.es
Phone	646251045
Submit Date	2025-01-17
Raw Data Available	Yes
Raw Data File Type(s)	mzXML
Analysis Type Detail	LC-MS
Release Date	2025-02-05
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR002277
Project DOI:	doi: 10.21228/M89G0H
Project Title:	Advanced Lipidomics Using UHPLC-ESI-QTOF-MS/MS Reveals Novel Lipids in Hibernating Syrian Hamsters
Project Summary:	Mammalian hibernation offers a unique model for exploring neuroprotective mechanisms relevant to neurodegenerative diseases. In this study, we employed untargeted lipidomics with iterative tandem mass spectrometry (MS/MS) to profile the brain lipidome of Syrian hamsters across different hibernation stages: late torpor, arousal, and euthermia (control). Previously, a lipid species identified as methyl-PA(16:0/0:0) showed a significant increase during torpor, but its precise structure was unresolved due to technological constraints. Leveraging iterative MS/MS and advanced lipid annotation tools (LipidAnnotator and MS-DIAL), we accurately annotated 377 lipid species, including the re-identification of methyl-PA(16:0/0:0) as methylated lysophosphatidic acid (PMeOH 16:0/0:0). This reannotation led to the discovery of two additional lipids during torpor: PMeOH 18:0/0:0 and PMeOH 18:1/0:0. Verification involved manual inspection of MS/MS spectra and Kendrick Mass Defect plots. The lipid alterations observed during torpor suggest biochemical adaptations to maintain membrane fluidity and protect against oxidative stress under hypothermic conditions. Elevated levels of PMeOH lipids and their lyso-forms may play roles in cell survival signalling. Additionally, a decrease in phosphatidic acid species and an increase in diacylglycerol species imply a metabolic shift favouring diacylglycerol production, potentially activating protein kinase C signalling pathways. The increased levels of monogalactosyl diglyceride lipids during torpor suggest a role in neuroprotection by enhancing oligodendrocyte function and myelination. Our comprehensive lipidomic profiling provides detailed insights into lipid dynamics associated with hibernation and underscores the potential of advanced MS/MS methodologies in lipidomics for developing therapeutic strategies against neurodegenerative diseases.
Institute:	Universidad CEU San Pablo
Department:	Centro de MEtabolómica y Bioanálisis (CEMBIO)
Last Name:	Gonzalez-Riano
First Name:	Carolina
Address:	Facultad de Farmacia, Universidad CEU San Pablo, Campus Monteprincipe, Boadilla del Monte, Boadilla del Monte, Madrid, 28668, Spain
Email:	car.gonzalez@ceindo.ceu.es
Phone:	00 34 91 3724753
Funding Source:	This work was supported by Grant PID2021-122490NB-I00 funded by MCIN/AEI/10.13039/501100011033 and by ERDF A way of making Europe (CGR and CB) and COST Action 19105-EpiLipidNET (CGR).

Subject:

Subject ID:	SU003804
Subject Type:	Mammal
Subject Species:	Mesocricetus auratus
Taxonomy ID:	10036
Age Or Age Range:	3 month old Syrian hamsters (M. auratus)
Gender:	Male
Animal Animal Supplier:	Janvier Laboratories (Le Genest-Saint-Isle, France)

Factors:

Subject type: Mammal; Subject species: Mesocricetus auratus (Factor headings shown in green)

mb_sample_id	local_sample_id	Sample source	Hibernation stage
SA401707	A12	brain	Arousal
SA401708	A3	brain	Arousal
SA401709	A1	brain	Arousal
SA401710	A6	brain	Arousal
SA401711	A11	brain	Arousal
SA401721	C1	brain	control
SA401722	C2	brain	control
SA401723	C3	brain	control
SA401724	C4	brain	control
SA401725	C5	brain	control
SA401712	QC1	brain	Quality Control
SA401713	QC2	brain	Quality Control
SA401714	QC3	brain	Quality Control
SA401715	QC4	brain	Quality Control
SA401716	T4	brain	Torpor
SA401717	T5	brain	Torpor
SA401718	T10	brain	Torpor
SA401719	T11	brain	Torpor
SA401720	T2	brain	Torpor

Showing results 1 to 19 of 19

Showing results 1 to 19 of 19

Collection:

Collection ID:	CO003797
Collection Summary:	All experimental procedures were carried out at the animal facility of San Pablo CEU University of Madrid (SVACEU. USP, registration no. ES 28022 0000015) and were approved by the Institutional Animal Experiment Ethics Committee. A total of 15 male, 3-month-old Syrian hamsters (M. auratus) were purchased from Janvier Laboratories (Le Genest-Saint-Isle, France). The animals had free access to food and water and were kept at 23 °C with an 8 h/16 h light/dark cycle for a 4 to 6-week acclimatization period in our animal facility. As previously described, to obtain arousal and torpor experimental groups, some animals were transferred to a specialized chamber that allowed for the control of temperature and photoperiod, two essential factors that affect hibernation. This chamber (developed by Tiselius) was designed with six individual cages to induce hibernation, based on previous studies. The chamber enabled the gradual reduction of temperature (via LM35 sensors), controlled illumination (adjustable RGB LEDs for intensity and color), and monitoring of the hamsters by measuring general locomotor activity with a passive infrared sensor mounted on top of each cage. All recorded data were saved on a notebook computer to distinguish between torpor and arousal phases during the hibernation cycle using the software program Fastwinter 1.9 (developed by Tiselius s.l.). The time at which animals exhibited periods of inactivity lasting 24 hours was considered the starting point for torpor. The status of the animals was confirmed by body temperature measurements (via an infrared thermometer). Since torpor bout durations are irregular at the beginning of hibernation, we classified animals as torpid only when they had completed three full bouts of torpor before being sacrificed. All animals were euthanized by decapitation. Brains were then removed and immediately transferred to a recipient containing liquid nitrogen to freeze the tissues. Aroused animals were awakened by gentle handling with a thermal blanket, a process taking 45−50 minutes for the hamsters to reach at least 35 °C (measured multiple times with an infrared thermometer pointed at the hamster’s head). Syrian hamsters were thus compared at three stages: control or euthermic (n = 5), torpor (n = 5), and arousal (n = 5).
Sample Type:	Brain

Treatment:

Treatment ID:	TR003813
Treatment Summary:	All experimental procedures were carried out at the animal facility of San Pablo CEU University of Madrid (SVACEU. USP, registration no. ES 28022 0000015) and were approved by the Institutional Animal Experiment Ethics Committee. A total of 15 male, 3-month-old Syrian hamsters (M. auratus) were purchased from Janvier Laboratories (Le Genest-Saint-Isle, France). The animals had free access to food and water and were kept at 23 °C with an 8 h/16 h light/dark cycle for a 4 to 6-week acclimatization period in our animal facility. As previously described, to obtain arousal and torpor experimental groups, some animals were transferred to a specialized chamber that allowed for the control of temperature and photoperiod, two essential factors that affect hibernation. This chamber (developed by Tiselius) was designed with six individual cages to induce hibernation, based on previous studies. The chamber enabled the gradual reduction of temperature (via LM35 sensors), controlled illumination (adjustable RGB LEDs for intensity and color), and monitoring of the hamsters by measuring general locomotor activity with a passive infrared sensor mounted on top of each cage. All recorded data were saved on a notebook computer to distinguish between torpor and arousal phases during the hibernation cycle using the software program Fastwinter 1.9 (developed by Tiselius s.l.). The time at which animals exhibited periods of inactivity lasting 24 hours was considered the starting point for torpor. The status of the animals was confirmed by body temperature measurements (via an infrared thermometer). Since torpor bout durations are irregular at the beginning of hibernation, we classified animals as torpid only when they had completed three full bouts of torpor before being sacrificed. All animals were euthanized by decapitation. Brains were then removed and immediately transferred to a recipient containing liquid nitrogen to freeze the tissues. Aroused animals were awakened by gentle handling with a thermal blanket, a process taking 45−50 minutes for the hamsters to reach at least 35 °C (measured multiple times with an infrared thermometer pointed at the hamster’s head). Syrian hamsters were thus compared at three stages: control or euthermic (n = 5), torpor (n = 5), and arousal (n = 5).

Treatment ID:

TR003813

Treatment Summary:

All experimental procedures were carried out at the animal facility of San Pablo CEU University of Madrid (SVACEU. USP, registration no. ES 28022 0000015) and were approved by the Institutional Animal Experiment Ethics Committee. A total of 15 male, 3-month-old Syrian hamsters (M. auratus) were purchased from Janvier Laboratories (Le Genest-Saint-Isle, France). The animals had free access to food and water and were kept at 23 °C with an 8 h/16 h light/dark cycle for a 4 to 6-week acclimatization period in our animal facility. As previously described, to obtain arousal and torpor experimental groups, some animals were transferred to a specialized chamber that allowed for the control of temperature and photoperiod, two essential factors that affect hibernation. This chamber (developed by Tiselius) was designed with six individual cages to induce hibernation, based on previous studies. The chamber enabled the gradual reduction of temperature (via LM35 sensors), controlled illumination (adjustable RGB LEDs for intensity and color), and monitoring of the hamsters by measuring general locomotor activity with a passive infrared sensor mounted on top of each cage. All recorded data were saved on a notebook computer to distinguish between torpor and arousal phases during the hibernation cycle using the software program Fastwinter 1.9 (developed by Tiselius s.l.). The time at which animals exhibited periods of inactivity lasting 24 hours was considered the starting point for torpor. The status of the animals was confirmed by body temperature measurements (via an infrared thermometer). Since torpor bout durations are irregular at the beginning of hibernation, we classified animals as torpid only when they had completed three full bouts of torpor before being sacrificed. All animals were euthanized by decapitation. Brains were then removed and immediately transferred to a recipient containing liquid nitrogen to freeze the tissues. Aroused animals were awakened by gentle handling with a thermal blanket, a process taking 45−50 minutes for the hamsters to reach at least 35 °C (measured multiple times with an infrared thermometer pointed at the hamster’s head). Syrian hamsters were thus compared at three stages: control or euthermic (n = 5), torpor (n = 5), and arousal (n = 5).

Sample Preparation:

Sampleprep ID:	SP003811
Sampleprep Summary:	Following removal of the brains (n = 15), the right hemispheres were isolated, immediately snap-frozen in liquid nitrogen, and then kept at −80 °C to prevent any post-mortem metabolic activity. Subsequent sample preparation for liquid chromatography−mass spectrometry (LC−MS) took place at CEMBIO (Madrid, Spain) (Gonzalez-Riano et al., 2019). The extraction procedure was originally developed and validated at CEMBIO for a multi-platform analysis of lung tissue (Naz et al., 2013), and later extended for use with hippocampal samples (Gonzalez-Riano et al., 2017, 2021). In this study, an entire right hemisphere (approximately 300 mg) was employed to minimize variability linked to brain region selection. To generate the homogenate, cold (−20 °C) methanol/water (1:1, v/v) was added at a 1:10 ratio of tissue to solvent. Tissue disruption was achieved using a Tissue-Lyser LT homogenizer (Qiagen, Germany), ensuring efficient metabolite extraction. Subsequently, 100 μL of the homogenate was vortexed with 320 μL of methanol for two minutes, followed by the addition of 80 μL of MTBE to extract nonpolar compounds. The vials were then sealed immediately and agitated for one hour at room temperature. Centrifugation of the samples was performed at 4000g for 20 minutes at 20 °C. For LC−MS measurements, 90 μL of the supernatant was placed into an ultra-high-performance liquid chromatography−mass spectrometry (UHPLC−MS) vial with an insert and directly injected into the instrument. Quality control (QC) samples were prepared by pooling equal aliquots of homogenate from each sample, followed by the same processing and analytical procedures. All samples, including QCs, were randomized before any treatment and subsequent analysis.

Sampleprep ID:

SP003811

Sampleprep Summary:

Following removal of the brains (n = 15), the right hemispheres were isolated, immediately snap-frozen in liquid nitrogen, and then kept at −80 °C to prevent any post-mortem metabolic activity. Subsequent sample preparation for liquid chromatography−mass spectrometry (LC−MS) took place at CEMBIO (Madrid, Spain) (Gonzalez-Riano et al., 2019). The extraction procedure was originally developed and validated at CEMBIO for a multi-platform analysis of lung tissue (Naz et al., 2013), and later extended for use with hippocampal samples (Gonzalez-Riano et al., 2017, 2021). In this study, an entire right hemisphere (approximately 300 mg) was employed to minimize variability linked to brain region selection. To generate the homogenate, cold (−20 °C) methanol/water (1:1, v/v) was added at a 1:10 ratio of tissue to solvent. Tissue disruption was achieved using a Tissue-Lyser LT homogenizer (Qiagen, Germany), ensuring efficient metabolite extraction. Subsequently, 100 μL of the homogenate was vortexed with 320 μL of methanol for two minutes, followed by the addition of 80 μL of MTBE to extract nonpolar compounds. The vials were then sealed immediately and agitated for one hour at room temperature. Centrifugation of the samples was performed at 4000g for 20 minutes at 20 °C. For LC−MS measurements, 90 μL of the supernatant was placed into an ultra-high-performance liquid chromatography−mass spectrometry (UHPLC−MS) vial with an insert and directly injected into the instrument. Quality control (QC) samples were prepared by pooling equal aliquots of homogenate from each sample, followed by the same processing and analytical procedures. All samples, including QCs, were randomized before any treatment and subsequent analysis.

Combined analysis:

Analysis ID	AN006029	AN006030
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Agilent 1290 Infinity II	Agilent 1290 Infinity II
Column	Agilent InfinityLab Poroshell 120 EC-C18 (100 x 3mm,2.7um)	Agilent InfinityLab Poroshell 120 EC-C18 (100 x 3mm,2.7um)
MS Type	ESI	ESI
MS instrument type	QTOF	QTOF
MS instrument name	Agilent 6545 QTOF	Agilent 6545 QTOF
Ion Mode	POSITIVE	NEGATIVE
Units	Peak area	Peak area

Chromatography:

Chromatography ID:	CH004582
Chromatography Summary:	The Agilent 1290 Infinity II Multisampler system was employed with a multiwash option, and 1 µL of extracted samples was injected. The multisampler temperature was maintained at 15 °C to ensure the stability of compounds and prevent lipid precipitation. For chromatographic separation, an Agilent InfinityLab Poroshell 120 ECC18 (3.0 × 100 mm, 2.7 µm) (Agilent Technologies) column and a compatible guard column (Agilent InfinityLab Poroshell 120 ECC18, 3.0 × 5 mm, 2.7 µm) were employed and held at 50 °C. The chromatography gradient was initiated at 70% of B at 0–1 min, increased to 86% at 3.5–10 min, and reached 100% B at 11–17 min. The initial conditions were restored by minute 17, followed by a 2-minute re-equilibration, resulting in a total running time of 19 min. The mobile phases for positive and negative ionization modes comprised (A) 10 mM ammonium acetate, 0.2 mM ammonium fluoride in a 9:1 water/methanol ratio and (B) 10 mM ammonium acetate, 0.2 mM ammonium fluoride in a 2:3:5 acetonitrile/methanol/isopropanol ratio. The flow rate was maintained at 0.6 mL/min. The multisampler's multiwash strategy involved a methanol:isopropanol (50:50, v/v) mixture with a 15-second wash time and an aqueous:organic phases (30:70, v/v) mixture to achieve the initial conditions.
Instrument Name:	Agilent 1290 Infinity II
Column Name:	Agilent InfinityLab Poroshell 120 EC-C18 (100 x 3mm,2.7um)
Column Temperature:	50 °C
Flow Gradient:	Started at 70% of B at 0 –1 min, 86% B at 3.5 –10 min, and 100% B at 11–17 min
Flow Rate:	0.6 mL/min
Solvent A:	90% water/10% methanol; 10 mM ammonium acetate; 0.2 mM ammonium fluoride
Solvent B:	20% acetonitrile/30% methanol/50% isopropanol; 10 mM ammonium acetate; 0.2 mM ammonium fluoride
Chromatography Type:	Reversed phase

MS:

MS ID:	MS005740
Analysis ID:	AN006029
Instrument Name:	Agilent 6545 QTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	150 V fragmentor, 65 V skimmer, 3500 V capillary voltage, 750 V octopole radio frequency voltage, 10 L/min nebulizer gas flow, 200 °C gas temperature, 50 psi nebulizer gas pressure, 12 L/min sheath gas flow, and 300 °C sheath gas temperature. Data were collected in positive and negative ESI modes in separate runs, operated in full scan mode from 40 to 1700 m/z with a scan rate of 3 spectra/s. A solution consisting of two reference mass compounds was infused throughout the whole analysis: purine (C5H4N4) at m/z 121.0509 and HP-0921 (C18H18O6N3P3F24) at m/z 922.0098. These masses were continuously infused into the system through an Agilent 1260 Iso Pump at a 1 mL/min (split ratio 1:100) to provide a constant mass correction.
Ion Mode:	POSITIVE

MS ID:	MS005741
Analysis ID:	AN006030
Instrument Name:	Agilent 6545 QTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	150 V fragmentor, 65 V skimmer, 3500 V capillary voltage, 750 V octopole radio frequency voltage, 10 L/min nebulizer gas flow, 200 °C gas temperature, 50 psi nebulizer gas pressure, 12 L/min sheath gas flow, and 300 °C sheath gas temperature. Data were collected in positive and negative ESI modes in separate runs, operated in full scan mode from 40 to 1700 m/z with a scan rate of 3 spectra/s. A solution consisting of two reference mass compounds was infused throughout the whole analysis: purine (C5H4N4) at m/z 119.0363 and HP-0921 (C18H18O6N3P3F24) at m/z 980.0163 (HP-0921 + acetate). These masses were continuously infused into the system through an Agilent 1260 Iso Pump at a 1 mL/min (split ratio 1:100) to provide a constant mass correction.
Ion Mode:	NEGATIVE