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MB Sample ID: SA098977
Local Sample ID: | Con007 |
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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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. |