Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA221046

Local Sample ID:	spleen_44066
Subject ID:	SU002384
Subject Type:	Mammal
Subject Species:	Rattus norvegicus
Taxonomy ID:	10116

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Subject:

Subject ID:	SU002384
Subject Type:	Mammal
Subject Species:	Rattus norvegicus
Taxonomy ID:	10116

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
spleen_44066	SA221046	FL027480	non-stress	Experimental factor

Collection:

Collection ID:	CO002377
Collection Summary:	Individual urine samples were collected in ice-cooled vessels containing 1% sodium azide (0.1 ml) for 2 h using a metabolic cage at 0, 24, and 48 h post-stress, respectively, and immediately frozen at -80°C. Animals were sacrificed by exsanguination under isoflurane anesthesia at 48 h post-stress. The blood sample was divided into two aliquots, one serum for biochemical analysis and the other heparinized plasma for NMR spectroscopic analysis. After weighing, brain, kidney, liver, lung, and spleen tissue were excised in duplicate: one being fixed in 10% formalin for histopathological examination, the other immediately snap-frozen in liquid nitrogen for tissue extraction. These samples were stored at -80°C until used.
Collection Protocol Filename:	Protocols.pdf
Sample Type:	serum, urine, brain, kidney, liver, lung, spleen

Treatment:

Treatment ID:	TR002396
Treatment Summary:	According to the National Institutes of Health Guide for the Care and Use of Laboratory Animals, all animals involved in this study were cared for, and protocols were reviewed and approved by the Anhui Laboratory Animal Care Committee. The specific pathogen-free (SPF) seven-week-old male Sprague Dawley (SD) rats (weight 233 ± 5 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd and used in this study. The environmental conditions were set at 21-26°C with a relative humidity of 50 ± 10% and a 12/12 h light/dark cycle. Food and tap water were provided ad libitum, and body weights were recorded daily. After one week of acclimatization, rats were randomly assigned to the groups of non-stressed control (n = 6) or the stressed (n = 7). For restraint stress, rats were individually placed in a ventilated plastic tube restrainer for 120 minutes, using a previously modified method. According to the general protocol, control rats were left undistributed in a home cage and allowed to contact each other without food and water.

Treatment ID:

TR002396

Treatment Summary:

According to the National Institutes of Health Guide for the Care and Use of Laboratory Animals, all animals involved in this study were cared for, and protocols were reviewed and approved by the Anhui Laboratory Animal Care Committee. The specific pathogen-free (SPF) seven-week-old male Sprague Dawley (SD) rats (weight 233 ± 5 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd and used in this study. The environmental conditions were set at 21-26°C with a relative humidity of 50 ± 10% and a 12/12 h light/dark cycle. Food and tap water were provided ad libitum, and body weights were recorded daily. After one week of acclimatization, rats were randomly assigned to the groups of non-stressed control (n = 6) or the stressed (n = 7). For restraint stress, rats were individually placed in a ventilated plastic tube restrainer for 120 minutes, using a previously modified method. According to the general protocol, control rats were left undistributed in a home cage and allowed to contact each other without food and water.

Sample Preparation:

Sampleprep ID:	SP002390
Sampleprep Summary:	Samples of plasma (255 μl) were mixed with 255 μl of phosphate D2O buffer solution (NaH2PO4 and K2HPO4, 60 mM, pH 7.4). After centrifugation at 10000 × g at 4°C for 10 min to remove the precipitates, the supernatants were transferred to 5 mm NMR tubes and analyzed by NMR. Samples of urine (455 μl) were mixed with 55 μl of D2O buffer solution (NaH2PO4 and K2HPO4, 1.5 M, including 0.1% TSP (sodium 3-(trimethylsilyl) propionate-2,2,3,3-d4), pH 7.4) to minimize any gross variation in the pH of the urine samples. The mixture was left to stand for 10 min and centrifuged at 10000 × g at 4°C for 10 min to remove the precipitates. The supernatants were transferred to 5 mm NMR tubes and analyzed by NMR. The polar metabolites in the rat tissue were extracted according to the protocol established in our previous work. In brief, pre-weighed brain, kidney, liver, lung, or spleen samples (100 mg) were homogenized in 400 μl of CH3OH and 85 μl of H2O at 4°C. The homogenates were transferred into a 2.5-ml tube, combined with 400 μl of CHCl3 and 200 μl of H2O, and then kept in a vortex for 60 s. After 10-min partitioning on ice, the samples were centrifuged for 5 min (10000 × g, 4°C). The upper supernatants were transferred into 1.5-ml tubes and lyophilized to remove CH3OH and H2O. The extracts were reconstituted in 0.5 ml D2O containing 1 mM TSP, then transferred into 5-mm NMR tubes and analyzed by NMR spectroscopy.

Sampleprep ID:

SP002390

Sampleprep Summary:

Samples of plasma (255 μl) were mixed with 255 μl of phosphate D2O buffer solution (NaH2PO4 and K2HPO4, 60 mM, pH 7.4). After centrifugation at 10000 × g at 4°C for 10 min to remove the precipitates, the supernatants were transferred to 5 mm NMR tubes and analyzed by NMR. Samples of urine (455 μl) were mixed with 55 μl of D2O buffer solution (NaH2PO4 and K2HPO4, 1.5 M, including 0.1% TSP (sodium 3-(trimethylsilyl) propionate-2,2,3,3-d4), pH 7.4) to minimize any gross variation in the pH of the urine samples. The mixture was left to stand for 10 min and centrifuged at 10000 × g at 4°C for 10 min to remove the precipitates. The supernatants were transferred to 5 mm NMR tubes and analyzed by NMR. The polar metabolites in the rat tissue were extracted according to the protocol established in our previous work. In brief, pre-weighed brain, kidney, liver, lung, or spleen samples (100 mg) were homogenized in 400 μl of CH3OH and 85 μl of H2O at 4°C. The homogenates were transferred into a 2.5-ml tube, combined with 400 μl of CHCl3 and 200 μl of H2O, and then kept in a vortex for 60 s. After 10-min partitioning on ice, the samples were centrifuged for 5 min (10000 × g, 4°C). The upper supernatants were transferred into 1.5-ml tubes and lyophilized to remove CH3OH and H2O. The extracts were reconstituted in 0.5 ml D2O containing 1 mM TSP, then transferred into 5-mm NMR tubes and analyzed by NMR spectroscopy.

Analysis:

MB Sample ID:	SA221046
Analysis ID:	AN003754
Analysis Type:	NMR
Results File:	ST002298_AN003754_Results.txt
Units:	Peak area

NMR:

NMR ID:	NM000253
Analysis ID:	AN003754
Instrument Name:	Varian 500 MHz spectrometer/Bruker-AV600 spectrometer
Instrument Type:	FT-NMR
NMR Experiment Type:	1D-1H
Spectrometer Frequency:	500 MHz/600 MHz