Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA028848

Local Sample ID:	Total Pool_3
Subject ID:	SU000581
Subject Type:	Animal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Genotype Strain:	C57BL/6
Gender:	Male
Animal Animal Supplier:	KO: bred and housed at the laboratory animal resource unit (LARU) of North Carolina A&T State University; WT: Charles River Laboratories
Animal Housing:	group cages of up to 5 mice per cage
Animal Light Cycle:	12:12h light:dark cycle
Animal Feed:	rat chow
Animal Water:	water ad libitum
Species Group:	Mammal

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Subject:

Subject ID:	SU000581
Subject Type:	Animal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Genotype Strain:	C57BL/6
Gender:	Male
Animal Animal Supplier:	KO: bred and housed at the laboratory animal resource unit (LARU) of North Carolina A&T State University; WT: Charles River Laboratories
Animal Housing:	group cages of up to 5 mice per cage
Animal Light Cycle:	12:12h light:dark cycle
Animal Feed:	rat chow
Animal Water:	water ad libitum
Species Group:	Mammal

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
Total Pool_3	SA028848	FL007032	-	Treatment
Total Pool_3	SA028848	FL007032	-	genotype
Total Pool_3	SA028848	FL007032	-	Week

Collection:

Collection ID:	CO000575
Collection Summary:	Urine samples were collected at 4 and 8 weeks post-STZ injection. Urine samples were obtained by bladder massage unto sterile petri dishes and stored at -80oC until used.
Sample Type:	Urine
Storage Conditions:	-80 C

Treatment:

Treatment ID:	TR000595
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:	SP000588
Sampleprep Summary:	Urine samples were thawed on ice for 30–60 min and vortexed on a multi-tube vortexer for 2 mins at 5,000 rpm, followed by centrifugation at room temperature at 16,000 rcf for 4 min. An aliquot of the supernatant was transferred to pre-labeled 1.5 mL low protein-binding microcentrifuge tube to make the individual study sample (40 µL). Another aliquot of supernatant (11 µL) was combined with those from all other urine 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 5 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 urine analysis. Acetonitrile containing the internal standard Tryptophan-d5 (120 µL; 0.0167 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 new, pre-labeled autosampler vials and 3 uL was injected into SYNAPT G2-Si.

Sampleprep ID:

SP000588

Sampleprep Summary:

Urine samples were thawed on ice for 30–60 min and vortexed on a multi-tube vortexer for 2 mins at 5,000 rpm, followed by centrifugation at room temperature at 16,000 rcf for 4 min. An aliquot of the supernatant was transferred to pre-labeled 1.5 mL low protein-binding microcentrifuge tube to make the individual study sample (40 µL). Another aliquot of supernatant (11 µL) was combined with those from all other urine 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 5 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 urine analysis. Acetonitrile containing the internal standard Tryptophan-d5 (120 µL; 0.0167 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 new, pre-labeled autosampler vials and 3 uL was injected into SYNAPT G2-Si.

Combined analysis:

Analysis ID	AN000858	AN000859
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	normalized ion counts	normalized ion counts

Chromatography:

Chromatography ID:	CH000611
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:	MS000759
Analysis ID:	AN000858
Instrument Name:	Waters Synapt G2 Si QTOF
Instrument Type:	QTOF
MS Type:	ESI
Ion Mode:	POSITIVE

MS ID:	MS000760
Analysis ID:	AN000859
Instrument Name:	Waters Synapt G2 Si QTOF
Instrument Type:	QTOF
MS Type:	ESI
Ion Mode:	NEGATIVE