Urinary metabolic profiles were measured by 1H NMR spectroscopy using the protocols described by Dona et al. (41) and Beckonert et al. (42). Briefly, 630 μl of urine sample was combined with 70 μl of phosphate buffer solution (pH 7.4, 100% D2O) containing 1 mM of the internal standard 3-trimethylsilyl-1-[2,2,3,3-2H4] propionate (TSP). Samples were then vortexed and spun (12,000g) for 10 min at 4°C before transfer to 5-mm NMR tubes. A pooled urine quality control sample was prepared by combining 5 μl of each individual sample of the study and was used to monitor variability of the analytical platform. One-dimensional 600-MHz 1H NMR spectra were acquired on a Bruker NMR spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany), equipped with a SampleJet system and a cooling rack of refrigerated tubes at 6°C. 1H NMR spectra acquisition was achieved using a standard one-dimensional solvent suppression pulse sequence (relaxation delay, 90° pulse, 4-μs delay, 90° pulse, mixing time, 90° pulse, acquire free induction decay). For each sample, 32 transients were collected in 64,000 frequency domain points with a spectral window set to 20 ppm (parts per million). A relaxation delay of 4 s, a mixing time of 10 ms, an acquisition time of 2.73 s, and 0.3-Hz line broadening were used. Spectra were referenced to the TSP resonance at δ 0.0. Spectral phasing and baseline correction were automatically performed using Topspin 3.2 (Bruker Biospin GmbH, Rheinstetten, Germany). The resulting raw NMR spectra were digitized, aligned, and normalized using the Imperial Metabolic Profiling and Chemometrics Toolbox (https://github.com/csmsoftware/IMPaCTS) in MATLAB (version 2018a, MathWorks Inc.). Briefly, after digitization of the spectra, redundant peaks (TSP, H2O, and urea) were removed and the resulting spectra were manually aligned to reference peaks using recursive segment-wise peak alignment (43). The aligned spectra were normalized using probabilistic quotient normalization (44). This approach adjusts the metabolite concentrations for differences in sample dilution as a result of differences in liquid and food intakes between infants, offsetting any potential confounding effect from these factors (45).

A targeted amino acid and tryptophan metabolite assay was carried out using UPLC-MS for the plasma samples using a Waters Acquity UPLC coupled to a Xevo TQ-XS mass spectrometer following the method published by Gray et al. (46). For the plasma samples, data were acquired and processed using Waters MassLynx (version 4.2) followed by multivariate analysis using SIMCA (version 15, Sartorius Stedim Biotech).

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