Quantification of Tryptophan and Phenolic Metabolites

Dietary records were analyzed using the online food calculator ‘Nubel’ and information on standardized quantification of food products. Blood and urine human samples were used to measure creatinine and urea with standard laboratory techniques. In addition, human and mice samples were analyzed for a panel of metabolites, focusing on selected tryptophan and phenolic compounds. All analyses were based on single measurements. With respect to the tryptophan metabolites, we measured tryptophan, indoxyl sulfate, indoxyl glucuronide, indole-3-acetic acid, kynurenine, kynurenic acid and quinolinic acid. Phenolic compounds included p-cresyl sulfate, p-cresyl glucuronide, phenyl sulfate, phenyl glucuronide and phenylacetic acid. All metabolites were quantified using a dedicated liquid chromatography—tandem mass spectrometry (LC-MS/MS) method. Deuterated kynurenic acid was added as an internal standard for quantification, as described previously [24 (link)]. Before chromatography an aliquot of plasma was diluted in H₂O (1:1) and deproteinized with perchloric acid (final concentration 3.3% (v/v)). Next, samples were centrifuged at 12,000 x g for 3 min. Clear supernatant was injected into the LC-MS/MS system that consisted of an Accela HPLC system coupled to a TSQ Vantage triple quadropole mass spectrometer (Thermo Fischer Scientific, Breda, the Netherlands) equipped with a C18 HPLC column (Acquity UPLC HSS T3 1.8 μm; Waters, Milford, MA). The autosampler temperature was set at 8°C and the column temperature at 40°C. The flow rate was 350 μL/min. Eluent solvent A consisted of 10 mM NH4-acetate, solvent B was 5 mM NH4-acetate and 0.1% formic acid and solvent C was 100% methanol. Samples (10 μL) were injected three times. The first injection addressed the basic negative components, including indoxyl sulfate, p-cresyl sulfate, phenyl sulfate and phenylacetic acid, because these components are stable within the acidic environment for up to 16h. The second injection addressed the acidic negative components, including indoxyl glucuronide, quinolinic acid, p-cresyl glucuronide and phenyl glucuronide. The third injection addressed the acidic positive components tryptophan, indole-3-acetic acid, kynurenine, kynurenic acid and quinolinic acid. The elution gradient was as follows: for the first injection the gradient was 100% solvent A to 85% solvent C in 7 min, then for the second and third injection a gradient of 100% solvent B to 85% solvent C in 7 min. The effluent from the UPLC was passed directly into the electrospray ion source. Negative electrospray ionization (first two injections) achieved using a nitrogen sheath gas with ionization voltage at 2500 Volt. The positive (third injection) electrospray ionization was achieved using a nitrogen sheath gas with ionization voltage at 3500 Volt. The capillary temperature was set at 240°C. Detection of the components was based on isolation of the deprotonated (negative electrospray; [M-H]-) or protonated molecular ion (positive electrospray; [M+H]+) and subsequent MS/MS fragmentations and a selected reaction monitoring (SRM) were carried out. The UPLC-MS/MS operating conditions and SRM transitions used for parent compounds and ion products were optimized for each component and shown in Table 1.
With the 48h urinary collection of human subjects, we calculated the average daily urinary excretion of each metabolite, giving an estimate of its daily generation. Urinary collections were considered complete when urinary excretion of creatinine was within 2 standard deviations of the mean creatinine excretion for the geographical region of this study, derived from the INTERSALT study [25 (link)].