Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA237806

Local Sample ID:	CG_720
Subject ID:	SU002471
Subject Type:	Mammal
Subject Species:	Rattus norvegicus
Taxonomy ID:	10116

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Subject:

Subject ID:	SU002471
Subject Type:	Mammal
Subject Species:	Rattus norvegicus
Taxonomy ID:	10116

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
CG_720	SA237806	FL029549	MHG	Treatment

Collection:

Collection ID:	CO002464
Collection Summary:	Metabolite extraction was carried out as previously described (96). Briefly, ~20-30 mg of kidney cortex was homogenized under cryogenic conditions in cryomill tubes containing beads for homogenization (Precellys bead-mill with a Cryolys attachment, Bertin Technologies, France) and 600 mL of 3:1 methanol:water (v/v) containing 0.5 nmol 13C6-sorbitol and 5 nmol 13C5,15N-valine as internal standards. Homogenates (480 mL) were subsequently vortexed in fresh Eppendorf tubes containing 120 ml chloroform. The resultant extracts were centrifuged to pellet cell debris and precipitated protein. The supernatant was used for subsequent analysis. In addition, an aliquot from each sample was pooled and re-aliquoted to generate pooled biological quality controls (PBQC). Samples and PBQCs were evaporated dry by speed vacuum centrifugation and then derivatized online using the Shimadzu AOC6000 autosampler robot with methoxyamine hydrochloride (30 mg/mL in pyridine) and N, O - bis (trimethylsilyl) trifluoroacetamide [BSTFA] + 1% chlorotrimethylsilane [TMCS] (both Thermo Fisher Scientific, Waltham, USA). Samples were left for 1 h before 1 µL was injected onto the GC column using a hot needle technique. Split (1:10) injections were performed for each sample.
Sample Type:	Kidney

Treatment:

Treatment ID:	TR002483
Treatment Summary:	Male Sprague Dawley rats were housed in groups of three rats per cage in a temperature-controlled environment, with a 12 h light/dark cycle and ad libitum access to food and water. Experimental diabetes was induced in six week old male Sprague Dawley rats (200-250 g, n = 35) by i.v. injection of streptozotocin (55 mg/kg, sodium citrate buffer pH 4.5) following an overnight fast, as previously described (80). One group of rats received citrate buffer vehicle (0.42% in sterile saline, pH 4.5) as a non-diabetic control with normal blood glucose (NG) (n = 16). One week following STZ treatment, diabetic rats were further assigned to two groups: standard insulin therapy (n = 17 rats), resulting in severe hyperglycemia (SHG) and intensive insulin therapy (n = 19 rats), resulting in moderate hyperglycemia (MHG) using a single daily insulin injection (long-lasting Humulin NPH; Eli Lilly, Indianapolis, USA) to titrate blood glucose levels to >28 mM (1-2 units, s.c. per day) and ∼20 mM (6-7 units, s.c. per day) as required, respectively. Blood glucose and body weight were monitored weekly. Blood glucose was measured using a handheld glucometer (Accutrend; Boehringer Manheim Biochemica, Manheim, Germany) during the study time course. After the completion of the study, plasma glucose was measured using a colorimetric glucose assay kit from Cayman Chemical Company (Ann Arbor, MI, USA), performed according to the manufacturer’s instructions. Hemoglobin A1c (HbA1c) was determined by a Cobas Integra 400 autoanalyzer (Roche Diagnostics Corporation, USA). Plasma C-peptide was determined using a commercially available ELISA kit (Alpco, Salem, NH, USA) according to the manufacturer’s instructions. In the final week of the study, rats were placed individually into metabolic cages (Iffa Credo, L’Arbresele, France) for 24 hours to collect urine.

Treatment ID:

TR002483

Treatment Summary:

Male Sprague Dawley rats were housed in groups of three rats per cage in a temperature-controlled environment, with a 12 h light/dark cycle and ad libitum access to food and water. Experimental diabetes was induced in six week old male Sprague Dawley rats (200-250 g, n = 35) by i.v. injection of streptozotocin (55 mg/kg, sodium citrate buffer pH 4.5) following an overnight fast, as previously described (80). One group of rats received citrate buffer vehicle (0.42% in sterile saline, pH 4.5) as a non-diabetic control with normal blood glucose (NG) (n = 16). One week following STZ treatment, diabetic rats were further assigned to two groups: standard insulin therapy (n = 17 rats), resulting in severe hyperglycemia (SHG) and intensive insulin therapy (n = 19 rats), resulting in moderate hyperglycemia (MHG) using a single daily insulin injection (long-lasting Humulin NPH; Eli Lilly, Indianapolis, USA) to titrate blood glucose levels to >28 mM (1-2 units, s.c. per day) and ∼20 mM (6-7 units, s.c. per day) as required, respectively. Blood glucose and body weight were monitored weekly. Blood glucose was measured using a handheld glucometer (Accutrend; Boehringer Manheim Biochemica, Manheim, Germany) during the study time course. After the completion of the study, plasma glucose was measured using a colorimetric glucose assay kit from Cayman Chemical Company (Ann Arbor, MI, USA), performed according to the manufacturer’s instructions. Hemoglobin A1c (HbA1c) was determined by a Cobas Integra 400 autoanalyzer (Roche Diagnostics Corporation, USA). Plasma C-peptide was determined using a commercially available ELISA kit (Alpco, Salem, NH, USA) according to the manufacturer’s instructions. In the final week of the study, rats were placed individually into metabolic cages (Iffa Credo, L’Arbresele, France) for 24 hours to collect urine.

Sample Preparation:

Sampleprep ID:	SP002477
Sampleprep Summary:	The GC-MS system consisted of an AOC6000 autosampler, a 2030 Shimadzu gas chromatograph and a TQ8040 quadrupole mass spectrometer (Shimadzu, Japan), which was tuned according to the manufacturer’s recommendations using tris-(perfluorobutyl)-amine (CF43). GC-MS was performed on a 30 m Agilent DB-5 column with 1 µm film thickness and 0.25 mm internal diameter. The injection temperature (Inlet) and the MS transfer line were both set at 280°C and the ion source adjusted to 200°C. Helium was used as the carrier gas at a flow rate of 1 mL/min and argon gas was used as the collision cell gas to generate the multiple reaction monitoring (MRM) product ion. The analysis was performed under the following temperature program; start at injection 100°C, a hold for 4 minutes followed by a 10°C min-1 oven temperature ramp to 320°C followed by a final hold off of 11 minutes. Approximately 520 quantifying MRM targets were collected using Shimadzu Smart Database along with qualifier for each target, which covers about 350 endogenous metabolites and multiple 13C labelled internal standards. Both chromatograms and MRMs were evaluated using the Shimadzu GCMS browser and LabSolutions Insight software.

Sampleprep ID:

SP002477

Sampleprep Summary:

The GC-MS system consisted of an AOC6000 autosampler, a 2030 Shimadzu gas chromatograph and a TQ8040 quadrupole mass spectrometer (Shimadzu, Japan), which was tuned according to the manufacturer’s recommendations using tris-(perfluorobutyl)-amine (CF43). GC-MS was performed on a 30 m Agilent DB-5 column with 1 µm film thickness and 0.25 mm internal diameter. The injection temperature (Inlet) and the MS transfer line were both set at 280°C and the ion source adjusted to 200°C. Helium was used as the carrier gas at a flow rate of 1 mL/min and argon gas was used as the collision cell gas to generate the multiple reaction monitoring (MRM) product ion. The analysis was performed under the following temperature program; start at injection 100°C, a hold for 4 minutes followed by a 10°C min-1 oven temperature ramp to 320°C followed by a final hold off of 11 minutes. Approximately 520 quantifying MRM targets were collected using Shimadzu Smart Database along with qualifier for each target, which covers about 350 endogenous metabolites and multiple 13C labelled internal standards. Both chromatograms and MRMs were evaluated using the Shimadzu GCMS browser and LabSolutions Insight software.

Combined analysis:

Analysis ID	AN003881
Analysis type	MS
Chromatography type	GC
Chromatography system	Shimadzu 2030
Column	Agilent DB5-MS (30m x 0.25mm, 0.25um)
MS Type	ESI
MS instrument type	Triple quadrupole
MS instrument name	Shimazu TQ8040
Ion Mode	UNSPECIFIED
Units	Peak area

Chromatography:

Chromatography ID:	CH002875
Instrument Name:	Shimadzu 2030
Column Name:	Agilent DB5-MS (30m x 0.25mm, 0.25um)
Chromatography Type:	GC

MS:

MS ID:	MS003621
Analysis ID:	AN003881
Instrument Name:	Shimazu TQ8040
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	The GC-MS system consisted of an AOC6000 autosampler, a 2030 Shimadzu gas chromatograph and a TQ8040 quadrupole mass spectrometer (Shimadzu, Japan), which was tuned according to the manufacturer’s recommendations using tris-(perfluorobutyl)-amine (CF43). GC-MS was performed on a 30 m Agilent DB-5 column with 1 µm film thickness and 0.25 mm internal diameter. The injection temperature (Inlet) and the MS transfer line were both set at 280°C and the ion source adjusted to 200°C. Helium was used as the carrier gas at a flow rate of 1 mL/min and argon gas was used as the collision cell gas to generate the multiple reaction monitoring (MRM) product ion. The analysis was performed under the following temperature program; start at injection 100°C, a hold for 4 minutes followed by a 10°C min-1 oven temperature ramp to 320°C followed by a final hold off of 11 minutes. Approximately 520 quantifying MRM targets were collected using Shimadzu Smart Database along with qualifier for each target, which covers about 350 endogenous metabolites and multiple 13C labelled internal standards. Both chromatograms and MRMs were evaluated using the Shimadzu GCMS browser and LabSolutions Insight software.
Ion Mode:	UNSPECIFIED