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MB Sample ID: SA098970
| Local Sample ID: | CKD004 |
| 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 |
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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:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| CKD004 | SA098970 | FL014029 | CKD | Group |
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. |
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 |
