Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA028825

Local Sample ID:	516-8
Subject ID:	SU000580
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:	SU000580
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
516-8	SA028825	FL007029	STZ	Treatment
516-8	SA028825	FL007029	Meprin bKO	Genotype
516-8	SA028825	FL007029	8	Week

Collection:

Collection ID:	CO000574
Collection Summary:	Blood samples were collected at 4 and 8 weeks post-STZ injection. Blood was obtained by tail nicking at week 4 and cardiac puncture at week 8, collected into heparin tubes, centrifuged to obtain plasma, and stored at -80oC until used.
Sample Type:	Blood
Storage Conditions:	-80 C
Additives:	Heparin
Blood Serum Or Plasma:	Plasma

Treatment:

Treatment ID:	TR000594
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:	SP000587
Sampleprep Summary:	Plasma samples were thawed on ice for 30–60 min and vortexed on a multi-tube vortexer for 4 min 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 plasma 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 6 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 plasma analysis. Methanol containing the internal standard Tryptophan-d5 (320 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 (290 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 h and lyophilized for 18 h. Acetonitrile:Water (125 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:

SP000587

Sampleprep Summary:

Plasma samples were thawed on ice for 30–60 min and vortexed on a multi-tube vortexer for 4 min 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 plasma 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 6 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 plasma analysis. Methanol containing the internal standard Tryptophan-d5 (320 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 (290 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 h and lyophilized for 18 h. Acetonitrile:Water (125 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	AN000856	AN000857
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	Waters Synapt-G2-Si
Ion Mode	POSITIVE	NEGATIVE
Units	normalized ion counts	normalized ion counts

Chromatography:

Chromatography ID:	CH000610
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:	MS000757
Analysis ID:	AN000856
Instrument Name:	Waters Synapt-G2-Si
Instrument Type:	QTOF
MS Type:	ESI
Ion Mode:	POSITIVE

MS ID:	MS000758
Analysis ID:	AN000857
Instrument Name:	Waters Synapt-G2-Si
Instrument Type:	QTOF
MS Type:	ESI
Ion Mode:	NEGATIVE