Summary of Study ST001361

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000931. The data can be accessed directly via it's Project DOI: 10.21228/M8BT4S This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

Study ID	ST001361
Study Title	Serum tryptophan metabolomics in CKD
Study Type	MS quantitative analysis
Study Summary	Serum was processed using a targeted metabolomics platform for quantifying tryptophan metabolites as a number of these metabolites are well establish uremic toxins.
Institute	University of Florida
Last Name	Ryan
First Name	Terence
Address	1864 Stadium Rd, FLG 114, Gainesville, FL, 32611, USA
Email	ryant@ufl.edu
Phone	352-294-1700
Submit Date	2020-04-02
Num Groups	2
Total Subjects	16
Num Males	8
Num Females	8
Raw Data Available	Yes
Analysis Type Detail	LC-MS
Release Date	2020-12-31
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR000931
Project DOI:	doi: 10.21228/M8BT4S
Project Title:	Tryptophan metabolomics in CKD serum
Project Type:	MS quantitative analysis
Project Summary:	Targeted tryptophan metabolomics were performed in mouse serum collected from mice with and without chronic kidney disease
Institute:	University of Florida
Department:	Applied Physiolog and Kinesiology
Last Name:	Ryan
First Name:	Terence
Address:	1864 Stadium Rd, FLG 114, Gainesville, FL, 32611, USA
Email:	ryant@ufl.edu
Phone:	352-294-1700
Funding Source:	NIH/NHLBIR01-HL149704 and R01-HL148597

Subject:

Subject ID:	SU001435
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Genotype Strain:	C57BL6J
Age Or Age Range:	18-20 weeks
Weight Or Weight Range:	20-30g
Gender:	Male and female
Animal Animal Supplier:	Jackson Labs
Animal Housing:	5/cage
Animal Light Cycle:	12h
Animal Feed:	Ad libitum. Control mice received custom casein-diet. Chronic kidney disease was induced by supplementing casein-based diet with 0.15% adenine
Animal Water:	Ad libitum

Factors:

Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id	local_sample_id	Group
SA098963	CKD003	CKD
SA098964	CKD002	CKD
SA098965	CKD005	CKD
SA098966	CKD007	CKD
SA098967	CKD008	CKD
SA098968	CKD001	CKD
SA098969	CKD006	CKD
SA098970	CKD004	CKD
SA098971	Con004	Control
SA098972	Con003	Control
SA098973	Con002	Control
SA098974	Con005	Control
SA098975	Con006	Control
SA098976	Con008	Control
SA098977	Con007	Control
SA098978	Con001	Control
SA098979	C2	N/A
SA098980	C3	N/A
SA098981	C7	N/A
SA098982	QC1	N/A
SA098983	QC2	N/A
SA098984	C1	N/A
SA098985	C6	N/A
SA098986	C4	N/A
SA098987	C5	N/A
SA098988	QC3	N/A

Showing results 1 to 26 of 26

Showing results 1 to 26 of 26

Collection:

Collection ID:	CO001430
Collection Summary:	Serum was collected ketamine/xylazine anesthesia, from a 1mm tail snip, allowed to clot for 20 minutes at room temperature and centrifuged at 4000xG for 10 min. Serum was collected from stored at -80C until analysis.
Sample Type:	Blood (serum)
Storage Conditions:	-80℃

Treatment:

Treatment ID:	TR001450
Treatment Summary:	We utilized an established adenine-diet model to induce CKD in mice. Mice were assigned to a casein-based chow diet for 7 days, followed by induction of renal tubular injury by supplementing the diet with 0.2% adenine for 7 days, and were subsequently maintained on a 0.15% adenine diet for 7 more weeks. CKD mice were then placed back on control casein diet for 2 weeks to prior to euthanasia and terminal experiments. Control mice received casein diet for the duration of the study.
Animal Anesthesia:	Ketamine/Xylazine
Animal Endp Euthanasia:	Ketamine/Xylazine

Sample Preparation:

Sampleprep ID:	SP001443
Sampleprep Summary:	Twenty-five microliters of each mouse serum was spiked with 5 µL internal standards (IS) solution consisted of tryptophan ¹³C₁₁, serotonin D4, kynurenine D4, kynurenic Acid D5, xanthurenic acid D4, anthranilic acid ¹³C₆, indoxyl sulfate ¹³C₆, p-cresol sulfate D7 and 3-indole-acetate D7. Metabolite extraction was done by protein precipitation using 200 µl of 8:1:1 Acetonitrile: Methanol: Acetone with 0.1% formic acid. Further protein precipitation was allowed by incubating the samples at 4°C for 20 min. Samples were placed in an ultrasonic bath for 10 min and then centrifuged at 20 000 xg for 5min at 4°C to pellet the protein. 190 µl supernatant was transferred from each sample into clean tube and dried under a gentle stream of nitrogen at 30°C. The dried extracts were re-suspended with 25 µL water with 0.1% formic acid. Resuspension was allowed at 4°C for 10 -15 min then samples were centrifuged at 20000 xg for 5 min at 4°C. Supernatants were transferred into clean LC-vials for targeted LC-MS quantitation on a Thermo Q-Exactive Oribtrap mass spectrometer with Dionex UHPLC and autosampler.

Sampleprep ID:

SP001443

Sampleprep Summary:

Twenty-five microliters of each mouse serum was spiked with 5 µL internal standards (IS) solution consisted of tryptophan ¹³C₁₁, serotonin D4, kynurenine D4, kynurenic Acid D5, xanthurenic acid D4, anthranilic acid ¹³C₆, indoxyl sulfate ¹³C₆, p-cresol sulfate D7 and 3-indole-acetate D7. Metabolite extraction was done by protein precipitation using 200 µl of 8:1:1 Acetonitrile: Methanol: Acetone with 0.1% formic acid. Further protein precipitation was allowed by incubating the samples at 4°C for 20 min. Samples were placed in an ultrasonic bath for 10 min and then centrifuged at 20 000 xg for 5min at 4°C to pellet the protein. 190 µl supernatant was transferred from each sample into clean tube and dried under a gentle stream of nitrogen at 30°C. The dried extracts were re-suspended with 25 µL water with 0.1% formic acid. Resuspension was allowed at 4°C for 10 -15 min then samples were centrifuged at 20000 xg for 5 min at 4°C. Supernatants were transferred into clean LC-vials for targeted LC-MS quantitation on a Thermo Q-Exactive Oribtrap mass spectrometer with Dionex UHPLC and autosampler.

Combined analysis:

Analysis ID	AN002265
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Thermo Dionex
Column	ACE 5 C18-300 (100 x 2.1mm)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap
Ion Mode	POSITIVE
Units	ng/ml

Chromatography:

Chromatography ID:	CH001664
Chromatography Summary:	Twenty-five microliters of each mouse serum was spiked with 5 µL internal standards (IS) solution consisted of tryptophan ¹³C₁₁, serotonin D4, kynurenine D4, kynurenic Acid D5, xanthurenic acid D4, anthranilic acid ¹³C₆, indoxyl sulfate ¹³C₆, p-cresol sulfate D7 and 3-indole-acetate D7. Metabolite extraction was done by protein precipitation using 200 µl of 8:1:1 Acetonitrile: Methanol: Acetone with 0.1% formic acid. Further protein precipitation was allowed by incubating the samples at 4°C for 20 min. Samples were placed in an ultrasonic bath for 10 min and then centrifuged at 20 000 xg for 5min at 4°C to pellet the protein. 190 µl supernatant was transferred from each sample into clean tube and dried under a gentle stream of nitrogen at 30°C. The dried extracts were re-suspended with 25 µL water with 0.1% formic acid. Resuspension was allowed at 4°C for 10 -15 min then samples were centrifuged at 20000 xg for 5 min at 4°C. Supernatants were transferred into clean LC-vials for targeted LC-MS quantitation on a Thermo Q-Exactive Oribtrap mass spectrometer with Dionex UHPLC and autosampler. All samples were analyzed in positive and negative heated electrospray ionization for all with a mass resolution of 35,000 at m/z 200 as separate injections. Tryptophan, serotonin, kynurenine, kynurenic acid, xanthurenic acid and anthranilic acid were quantified in the positive ionization while indoxyl sulfate, p-cresol sulfate and 3-indole-acetate were analyzed in negative ionization. Separation was achieved on an ACE 18-PFP 100 x 2.1 mm, 2 µm column using a gradient with mobile phase A as 0.1% formic acid in water and mobile phase B as acetonitrile. The flow rate was 350 µL/min with a column temperature of 25°C and injection volume of 2 µL. Run time was 20.5 min. A 9-point calibration curve and QC samples were prepared for targeted quantitation of tryptophan, serotonin, kynurenine, kynurenic acid, xanthurenic acid, anthranilic acid, indoxyl sulfate, p-cresol sulfate and 3-indole-acetate. 20 µL of each calibrator and QCs were supplemented with 5 µl indoxyl sulfate. Peak areas of each analyte and corresponding internal standard in the calibrator, QCs and samples were integrated using Xcalibur 4.0. A calibration curve was generated by plotting nominal concentration of the analyte in the calibrators versus peak area ratio of analyte and IS. QCs and samples were quantitated against the calibration curve.
Instrument Name:	Thermo Dionex
Column Name:	ACE 5 C18-300 (100 x 2.1mm)
Column Temperature:	25C
Flow Rate:	350ul/min
Solvent A:	100% water; 0.1% formic acid
Solvent B:	100% acetonitrile
Chromatography Type:	Reversed phase

MS:

MS ID:	MS002109
Analysis ID:	AN002265
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Tryptophan, serotonin, kynurenine, kynurenic acid, xanthurenic acid and anthranilic acid were quantified in the positive ionization while indoxyl sulfate, p-cresol sulfate and 3-indole-acetate were analyzed in negative ionization. Separation was achieved on an ACE 18-PFP 100 x 2.1 mm, 2 µm column using a gradient with mobile phase A as 0.1% formic acid in water and mobile phase B as acetonitrile. The flow rate was 350 µL/min with a column temperature of 25°C and injection volume of 2 µL. Run time was 20.5 min. A 9-point calibration curve and QC samples were prepared for targeted quantitation of tryptophan, serotonin, kynurenine, kynurenic acid, xanthurenic acid, anthranilic acid, indoxyl sulfate, p-cresol sulfate and 3-indole-acetate. 20 µL of each calibrator and QCs were supplemented with 5 µl indoxyl sulfate. Peak areas of each analyte and corresponding internal standard in the calibrator, QCs and samples were integrated using Xcalibur 4.0. A calibration curve was generated by plotting nominal concentration of the analyte in the calibrators versus peak area ratio of analyte
Ion Mode:	POSITIVE