Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA290633

Local Sample ID:	20220107_Cowley3_Blank_C18pos_2
Subject ID:	SU002865
Subject Type:	Mammal
Subject Species:	Rattus norvegicus
Taxonomy ID:	10116

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Subject:

Subject ID:	SU002865
Subject Type:	Mammal
Subject Species:	Rattus norvegicus
Taxonomy ID:	10116

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
20220107_Cowley3_Blank_C18pos_2	SA290633	FL035781	n/a	Treatment
20220107_Cowley3_Blank_C18pos_2	SA290633	FL035781	n/a	Source

Collection:

Collection ID:	CO002858
Collection Summary:	Plasma was collected through an arterial and renal venous catheter throughout the study (200 µL of arterial and renal venous blood were sampled at the day 7, 14, and 21). Overnight urine (18 hours) from the day before the blood draw was collected on ice. The kidneys were collected either at 14 days of HS (HS14) or 21 days of HS (HS21). The kidneys of only LS fed SD rats were also collected for comparison. The collected kidneys (n=5 for each group for metabolomics) were dissected to cortex and outer medulla and snap frozen with liquid nitrogen. Plasma, urine and tissue were stored in -80°C until further analysis.
Sample Type:	Arterial plasma; Venous plasma
Storage Conditions:	-80?

Treatment:

Treatment ID:	TR002874
Treatment Summary:	Rats (n=7, 10-11 weeks of age) were performed renal blood flow (RBF) probe implantation and femoral arterial catheterization5. Briefly, rats were anesthetized with isoflurane and arterial catheter was inserted. Following an abdominal incision, RBF probe was implanted on left renal artery and the cable was exposed at nape of the neck via the subcutaneous route. In addition to the RBF probe implantation, renal venous catheter was inserted through the femoral vein and placed in the left renal vein and secured to the luminal wall with 10-0 nylon. RBF and BP via arterial line were measured by conscious freely moving rats and recorded on average of every minute for 24 h/day. After 7-10 days of recovery period, 200 µL of arterial and renal venous blood were sampled and that blood was replaced from donor rats before and following 7, 14 and 21 days after the switch in diet from 0.4% (LS) to 4.0% (HS) salt diet (Dyets Inc, Bethlehem, PA). Overnight urine (18 hours) from the day before the blood draw was collected on ice. The kidneys were collected either at 14 days of HS (HS14) or 21 days of HS (HS21). The kidneys of only LS fed SD rats were also collected for comparison. The collected kidneys (n=5 for each group for metabolomics and mRNAseq analysis) were dissected to cortex and outer medulla and snap frozen with liquid nitrogen. Plasma, urine and tissue were stored in -80°C until further analysis.

Treatment ID:

TR002874

Treatment Summary:

Rats (n=7, 10-11 weeks of age) were performed renal blood flow (RBF) probe implantation and femoral arterial catheterization5. Briefly, rats were anesthetized with isoflurane and arterial catheter was inserted. Following an abdominal incision, RBF probe was implanted on left renal artery and the cable was exposed at nape of the neck via the subcutaneous route. In addition to the RBF probe implantation, renal venous catheter was inserted through the femoral vein and placed in the left renal vein and secured to the luminal wall with 10-0 nylon. RBF and BP via arterial line were measured by conscious freely moving rats and recorded on average of every minute for 24 h/day. After 7-10 days of recovery period, 200 µL of arterial and renal venous blood were sampled and that blood was replaced from donor rats before and following 7, 14 and 21 days after the switch in diet from 0.4% (LS) to 4.0% (HS) salt diet (Dyets Inc, Bethlehem, PA). Overnight urine (18 hours) from the day before the blood draw was collected on ice. The kidneys were collected either at 14 days of HS (HS14) or 21 days of HS (HS21). The kidneys of only LS fed SD rats were also collected for comparison. The collected kidneys (n=5 for each group for metabolomics and mRNAseq analysis) were dissected to cortex and outer medulla and snap frozen with liquid nitrogen. Plasma, urine and tissue were stored in -80°C until further analysis.

Sample Preparation:

Sampleprep ID:	SP002871
Sampleprep Summary:	Plasma/Urine Metabolite Extraction. Metabolites were extracted from 20 µL of plasma and 20 µL of urine from each SD rat in the study according to standard operating procedures in the Mass Spectrometry and Protein Chemistry Service at The Jackson Laboratory34. Metabolites were extracted using 500 µL of an ice cold 2:2:1 methanol:acetonitrile:water (MeOH:ACN:H2O) buffer; the sample was part of the water fraction. Caffeine, 1-napthylamine, and 9-anthracene carboxylic acid were all added at 0.5 ng/ µL in the extraction buffer as internal standards. Each sample was then vortexed for 30 seconds on the highest setting, subject to one minute of mixing with the Tissue Lyser II in pre-chilled cassettes, and then sonicated at 30 Hz for 5 minutes of 30 seconds on 30 seconds off in an ice water bath. Samples were then placed in the -20°C freezer overnight (16 hours) for extraction. Following the extraction, samples were centrifuged at 21,000 x g at 4°C and supernatant from each metabolite extract was equally divided into five 2 mL microcentrifuge tubes. Each sample supernatant was divided into five equal volume aliquots, one for each of the four modes and the rest to create equal representation pools of all samples, one for each mode. Each aliquot was then dried down using a vacuum centrifuge for storage at -80°C until further use. Tissue Metabolite Extraction. Metabolites were extracted from 20 mg of kidney cortex and medulla from each SD rat in the study according to standard operating procedures in the Mass Spectrometry and Protein Chemistry Service at The Jackson Laboratory34 as described for the plasma and urine samples with slight modification. Metabolites were extracted using 1000 µL of an ice cold 2:2:1 methanol:acetonitrile:water (MeOH:ACN:H2O) buffer containing internal standards as above per 20 mg of sample to ensure the extraction equivalents were normalized. Each sample had a 5 mm stainless steel bead added, then were pulverized in extraction buffer for two minutes usingTissue Lyser II. Samples were then placed in the -20°C freezer overnight (16 hours) for extraction and the supernatant was collected as with the urine/plasma samples. Each sample supernatant was divided into five equal volume aliquots, one for each of the four modes and the rest to create equal representation pools of all samples, one for each mode. Each aliquot was then dried down using a vacuum centrifuge for storage at -80°C until further use.

Sampleprep ID:

SP002871

Sampleprep Summary:

Plasma/Urine Metabolite Extraction. Metabolites were extracted from 20 µL of plasma and 20 µL of urine from each SD rat in the study according to standard operating procedures in the Mass Spectrometry and Protein Chemistry Service at The Jackson Laboratory34. Metabolites were extracted using 500 µL of an ice cold 2:2:1 methanol:acetonitrile:water (MeOH:ACN:H2O) buffer; the sample was part of the water fraction. Caffeine, 1-napthylamine, and 9-anthracene carboxylic acid were all added at 0.5 ng/ µL in the extraction buffer as internal standards. Each sample was then vortexed for 30 seconds on the highest setting, subject to one minute of mixing with the Tissue Lyser II in pre-chilled cassettes, and then sonicated at 30 Hz for 5 minutes of 30 seconds on 30 seconds off in an ice water bath. Samples were then placed in the -20°C freezer overnight (16 hours) for extraction. Following the extraction, samples were centrifuged at 21,000 x g at 4°C and supernatant from each metabolite extract was equally divided into five 2 mL microcentrifuge tubes. Each sample supernatant was divided into five equal volume aliquots, one for each of the four modes and the rest to create equal representation pools of all samples, one for each mode. Each aliquot was then dried down using a vacuum centrifuge for storage at -80°C until further use. Tissue Metabolite Extraction. Metabolites were extracted from 20 mg of kidney cortex and medulla from each SD rat in the study according to standard operating procedures in the Mass Spectrometry and Protein Chemistry Service at The Jackson Laboratory34 as described for the plasma and urine samples with slight modification. Metabolites were extracted using 1000 µL of an ice cold 2:2:1 methanol:acetonitrile:water (MeOH:ACN:H2O) buffer containing internal standards as above per 20 mg of sample to ensure the extraction equivalents were normalized. Each sample had a 5 mm stainless steel bead added, then were pulverized in extraction buffer for two minutes usingTissue Lyser II. Samples were then placed in the -20°C freezer overnight (16 hours) for extraction and the supernatant was collected as with the urine/plasma samples. Each sample supernatant was divided into five equal volume aliquots, one for each of the four modes and the rest to create equal representation pools of all samples, one for each mode. Each aliquot was then dried down using a vacuum centrifuge for storage at -80°C until further use.

Combined analysis:

Analysis ID	AN004475	AN004476	AN004477	AN004478
Analysis type	MS	MS	MS	MS
Chromatography type	Reversed phase	Reversed phase	HILIC	HILIC
Chromatography system	Thermo Vanquish	Thermo Vanquish	Thermo Vanquish	Thermo Vanquish
Column	Agilent InfinityLab Poroshell 120 EC-C18 (2.1 x 50 mm; 2.7-Micron)	Agilent InfinityLab Poroshell 120 HILIC-Z (2.1 x 50 mm; 2.7 micron; #689775-924)	Agilent InfinityLab Poroshell 120 HILIC-Z (2.1 x 50 mm; 2.7 micron; #689775-924)	Agilent InfiinityLab Poroshell 120 HILIC-Z (2.1 x 50 mm; 2.7 micron; #689775-924)
MS Type	ESI	ESI	ESI	ESI
MS instrument type	Orbitrap	Orbitrap	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap	Thermo Q Exactive Orbitrap	Thermo Q Exactive Orbitrap	Thermo Q Exactive Orbitrap
Ion Mode	POSITIVE	NEGATIVE	POSITIVE	NEGATIVE
Units	Area	Area	Area	Area

Chromatography:

Chromatography ID:	CH003360
Chromatography Summary:	This chromatography method was utilized for all C18 positive polarity runs in this study.
Instrument Name:	Thermo Vanquish
Column Name:	Agilent InfinityLab Poroshell 120 EC-C18 (2.1 x 50 mm; 2.7-Micron)
Column Temperature:	25C
Flow Gradient:	0-1 minutes at 98% A1/2% B1, 1-13 minutes from 98% A1/2% B1 to 10% A1/90% B1, 13-15 minutes at 10% A1/90% B1, 15-16 minutes from 10% A1/90% B1 to 98% A1/2% B1, and was re-equilibrated from 16-25 minutes at 98% A1/2% B1
Flow Rate:	0.1 mL/minute
Internal Standard:	Caffeine, 1-napthylamine, and 9-anthracene carboxylic acid were all added at 0.5 ng/ µL in the extraction buffer as internal standards
Solvent A:	100% water, 0.2% acetic acid
Solvent B:	100% acetonitrile, 0.2% acetic acid
Chromatography Type:	Reversed phase

Chromatography ID:	CH003361
Chromatography Summary:	This chromatography method was utilized for all C18 negative polarity runs in this study.
Instrument Name:	Thermo Vanquish
Column Name:	Agilent InfinityLab Poroshell 120 HILIC-Z (2.1 x 50 mm; 2.7 micron; #689775-924)
Column Temperature:	25C
Flow Gradient:	0-1 minutes at 2% A/98% B, 1-11 minutes from 2% A/98% B to 30% A/70% B, 11-12 minutes from 30% A/70% B to 40% A/60% B, 12-16 minutes from 40% A/60% B to 95% A/5% B, was held at 95% A/5% B from 16-18 minutes, 18-20 minutes from 95% A/5% B to 2% A/98% B, and was re-equilibrated from 20-25 minutes at 2% A/98% B
Flow Rate:	0.1 mL/minute
Internal Standard:	Caffeine, 1-napthylamine, and 9-anthracene carboxylic acid were all added at 0.5 ng/ µL in the extraction buffer as internal standards
Solvent A:	100% water, 0.2% acetic acid
Solvent B:	100% acetonitrile, 0.2% acetic acid
Chromatography Type:	Reversed phase

Chromatography ID:	CH003362
Chromatography Summary:	This chromatography method was utilized for all HILIC positive polarity runs in this study.
Instrument Name:	Thermo Vanquish
Column Name:	Agilent InfinityLab Poroshell 120 HILIC-Z (2.1 x 50 mm; 2.7 micron; #689775-924)
Column Temperature:	25C
Flow Gradient:	0-1 minutes at 2% A/98% B, 1-11 minutes from 2% A/98% B to 30% A/70% B, 11-12 minutes from 30% A/70% B to 40% A/60% B, 12-16 minutes from 40% A/60% B to 95% A/5% B, was held at 95% A/5% B from 16-18 minutes, 18-20 minutes from 95% A/5% B to 2% A/98% B, and was re-equilibrated from 20-25 minutes at 2% A/98% B
Flow Rate:	0.1 mL/minute
Internal Standard:	Caffeine, 1-napthylamine, and 9-anthracene carboxylic acid were all added at 0.5 ng/ µL in the extraction buffer as internal standards
Solvent A:	10 mM ammonium formate in H2O with 0.1% formic acid (Solvent A2)
Solvent B:	90% ACN with 10 mM ammonium formate in H2O with 0.1% formic acid (Solvent B2)
Chromatography Type:	HILIC

Chromatography ID:	CH003363
Chromatography Summary:	This chromatography method was utilized for all HILIC negative polarity runs in this study.
Instrument Name:	Thermo Vanquish
Column Name:	Agilent InfiinityLab Poroshell 120 HILIC-Z (2.1 x 50 mm; 2.7 micron; #689775-924)
Column Temperature:	25C
Flow Gradient:	0-1 minutes at 2% A/98% B, 1-11 minutes from 2% A/98% B to 30% A/70% B, 11-12 minutes from 30% A/70% B to 40% A/60% B, 12-16 minutes from 40% A/60% B to 95% A/5% B, was held at 95% A/5% B from 16-18 minutes, 18-20 minutes from 95% A/5% B to 2% A/98% B, and was re-equilibrated from 20-25 minutes at 2% A/98% B
Flow Rate:	0.1 mL/minute
Internal Standard:	Caffeine, 1-napthylamine, and 9-anthracene carboxylic acid were all added at 0.5 ng/ µL in the extraction buffer as internal standards
Solvent A:	10 mM ammonium acetate in H2O, pH 9.0 with 0.1% AffinityLab Deactivator Inhibitor (Agilent, #5191-3940; Solvent A3)
Solvent B:	85% ACN with 10 mM ammonium acetate in H2O with 0.1% AffinityLab Deactivator Inhibitor (Solvent B3)
Chromatography Type:	HILIC

MS:

MS ID:	MS004222
Analysis ID:	AN004475
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	C18 positive plasma data: The tandem mass spectrometry RAW data files (consisting of MS1 and MS2 spectra collected) were analyzed using Thermo Compound Discoverer (v3.2.0.421). The MS1 and MS2 data was searched against the Thermo mzCloud database, ChemSpider database, Metabolika Pathways, and mzLogic predicted composition in the Compound Discoverer workflow.
Ion Mode:	POSITIVE

MS ID:	MS004223
Analysis ID:	AN004476
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	C18 negative plasma data: The tandem mass spectrometry RAW data files (consisting of MS1 and MS2 spectra collected) were analyzed using Thermo Compound Discoverer (v3.2.0.421). The MS1 and MS2 data was searched against the Thermo mzCloud database, ChemSpider database, Metabolika Pathways, and mzLogic predicted composition in the Compound Discoverer workflow.
Ion Mode:	NEGATIVE

MS ID:	MS004224
Analysis ID:	AN004477
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	HILIC positive plasma data: The tandem mass spectrometry RAW data files (consisting of MS1 and MS2 spectra collected) were analyzed using Thermo Compound Discoverer (v3.2.0.421). The MS1 and MS2 data was searched against the Thermo mzCloud database, ChemSpider database, Metabolika Pathways, and mzLogic predicted composition in the Compound Discoverer workflow.
Ion Mode:	POSITIVE

MS ID:	MS004225
Analysis ID:	AN004478
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	HILIC negative plasma data: The tandem mass spectrometry RAW data files (consisting of MS1 and MS2 spectra collected) were analyzed using Thermo Compound Discoverer (v3.2.0.421). The MS1 and MS2 data was searched against the Thermo mzCloud database, ChemSpider database, Metabolika Pathways, and mzLogic predicted composition in the Compound Discoverer workflow.
Ion Mode:	NEGATIVE