Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA027990

Local Sample ID:	525
Subject ID:	SU000562
Subject Type:	mice
Subject Species:	Mus musculus
Taxonomy ID:	10090
Genotype Strain:	C57BL/6
Gender:	Male
Species Group:	Mammal

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Subject:

Subject ID:	SU000562
Subject Type:	mice
Subject Species:	Mus musculus
Taxonomy ID:	10090
Genotype Strain:	C57BL/6
Gender:	Male
Species Group:	Mammal

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
525	SA027990	FL006936	STZ	Treatment
525	SA027990	FL006936	wild-type	Genotype

Collection:

Collection ID:	CO000556
Collection Summary:	Kidneys were harvested at 8-weeks post STZ-injection. The mice were sacrificed by CO2 asphyxiation. The kidneys were excised, decapsulated and individually weighed, snap-frozen in liquid nitrogen, and stored at -80oC until used.
Sample Type:	Kidney Tissue
Storage Conditions:	-80C

Treatment:

Treatment ID:	TR000576
Treatment Summary:	Type 1 diabetes was induced at age 8 weeks. The mice were fasted for 6 hours prior to being injected with low dose STZ (50 mg/kg body weight) in sodium citrate buffer (10 mmol/L, pH 4.5) daily for 5 days following the protocols described by Tesch and Allen and recommended by the Animal Models of Diabetic Complications Consortium (AMDCC; amdcc.org)1. Control mice were injected with equivalent volumes of the sodium citrate buffer. Diabetes was confirmed by measuring fasting blood glucose levels using a standard glucose meter at 10 days post-STZ injection. Mice with a fasting glucose level ≥280 mg/dL were considered diabetic. STZ-injected mice with a fasting blood glucose of <280 mg/dL (15mmol/L) were culled and eliminated from the study.
Treatment Compound:	streptozotocin
Treatment Dose:	50 mg/kg body weight
Treatment Doseduration:	daily; 5 days
Treatment Vehicle:	sodium citrate
Animal Endp Tissue Coll List:	plasma, urine, kidney tissue

Sample Preparation:

Sampleprep ID:	SP000569
Sampleprep Summary:	Frozen kidney tissue samples on dry ice were transferred to pre-chilled, pre-labeled tubes, and their weights recorded. For every 1 mg of tissue sample, 2 µL of 50:50 Acetonitrile:Water was added to the tube. Ceramic beads (2.3 mm; ~15-20 prewashed & dried) were added to the tubes, and the samples were homogenized on the MagNA Lyser system using a 30 s pulse at 4,000 rpm. The samples were centrifuged at room temperature at 16,000 rcf for 4 min. An aliquot of the supernatant was transferred to pre-labeled 2.0 mL low protein-binding microcentrifuge tube to make the individual study sample (40 µL). Another aliquot of supernatant (18 µL) was combined with those from all other kidney tissue samples in a 1.5 mL low protein-binding microcentrifuge tube to make the total pool quality control sample. The total pool quality control sample was aliquoted (40 µL) to make 4 Total Pool Samples and 4 Column Equilibration samples. 132 µL of the total pool quality control sample was added to the total study sample pool to make a pool of kidney, urine, and plasma samples. The total study pool sample was used to prepare 9 Total Study Samples (40 µL) to aid in alignment of the three studies. Of these, 3 total study samples were included in the kidney tissue analysis. Acetonitrile containing the internal standard Tryptophan-d5 (460 µL; 0.0125 mg/ml) was added to all samples, and samples were vortexed on a multiple tube vortexer for 2 min at 5000 rpm followed by centrifugation at 16,000 rcf for 4 min. The supernatants (420 µL) were transferred into new, pre-labeled 2.0 mL low protein-binding microcentrifuge tubes and capped with disposable rubber stoppers. Samples were placed at -80 °C for 1.5 h and lyophilized for 18 h. Acetonitrile:Water (150 µL; 95:5) was added to each tube and vortex mixed for 10 min at 5000 rpm, followed by centrifugation at room temperature at 16,000 rcf for 4 min. The supernatants were transferred to pre-labeled autosampler vials and 3 µL was injected into SYNAPT G2 Si.

Sampleprep ID:

SP000569

Sampleprep Summary:

Frozen kidney tissue samples on dry ice were transferred to pre-chilled, pre-labeled tubes, and their weights recorded. For every 1 mg of tissue sample, 2 µL of 50:50 Acetonitrile:Water was added to the tube. Ceramic beads (2.3 mm; ~15-20 prewashed & dried) were added to the tubes, and the samples were homogenized on the MagNA Lyser system using a 30 s pulse at 4,000 rpm. The samples were centrifuged at room temperature at 16,000 rcf for 4 min. An aliquot of the supernatant was transferred to pre-labeled 2.0 mL low protein-binding microcentrifuge tube to make the individual study sample (40 µL). Another aliquot of supernatant (18 µL) was combined with those from all other kidney tissue samples in a 1.5 mL low protein-binding microcentrifuge tube to make the total pool quality control sample. The total pool quality control sample was aliquoted (40 µL) to make 4 Total Pool Samples and 4 Column Equilibration samples. 132 µL of the total pool quality control sample was added to the total study sample pool to make a pool of kidney, urine, and plasma samples. The total study pool sample was used to prepare 9 Total Study Samples (40 µL) to aid in alignment of the three studies. Of these, 3 total study samples were included in the kidney tissue analysis. Acetonitrile containing the internal standard Tryptophan-d5 (460 µL; 0.0125 mg/ml) was added to all samples, and samples were vortexed on a multiple tube vortexer for 2 min at 5000 rpm followed by centrifugation at 16,000 rcf for 4 min. The supernatants (420 µL) were transferred into new, pre-labeled 2.0 mL low protein-binding microcentrifuge tubes and capped with disposable rubber stoppers. Samples were placed at -80 °C for 1.5 h and lyophilized for 18 h. Acetonitrile:Water (150 µL; 95:5) was added to each tube and vortex mixed for 10 min at 5000 rpm, followed by centrifugation at room temperature at 16,000 rcf for 4 min. The supernatants were transferred to pre-labeled autosampler vials and 3 µL was injected into SYNAPT G2 Si.

Combined analysis:

Analysis ID	AN000820	AN000821
Analysis type	MS	MS
Chromatography type	HILIC	HILIC
Chromatography system	Waters Acquity I-Class	Waters Acquity I-Class
Column	Waters Acquity BEH Amide (150 x 2.1mm,1.7um)	Waters Acquity BEH Amide (150 x 2.1mm,1.7um)
MS Type	ESI	ESI
MS instrument type	QTOF	QTOF
MS instrument name	Waters Synapt G2 Si QTOF	Waters Synapt G2 Si QTOF
Ion Mode	POSITIVE	NEGATIVE
Units	m/z	m/z

Chromatography:

Chromatography ID:	CH000586
Chromatography Summary:	Hydrophilic Interaction Liquid Chromatography (HILIC) Gradient Seperation
Instrument Name:	Waters Acquity I-Class
Column Name:	Waters Acquity BEH Amide (150 x 2.1mm,1.7um)
Chromatography Type:	HILIC

MS:

MS ID:	MS000721
Analysis ID:	AN000820
Instrument Name:	Waters Synapt G2 Si QTOF
Instrument Type:	QTOF
MS Type:	ESI
Ion Mode:	POSITIVE

MS ID:	MS000722
Analysis ID:	AN000821
Instrument Name:	Waters Synapt G2 Si QTOF
Instrument Type:	QTOF
MS Type:	ESI
Ion Mode:	NEGATIVE