Quantichrom diur 100

ELISA sandwich assay was used to quantify the albumin concentration in the culture media collected throughout the experiments (dynamic biochips and static multi-well plates). The assays were performed using a human albumin ELISA Quantitation Set (E80-129, Bethyl Laboratories, Montgomery, TX, USA), following the manufacturer instructions.
For urea quantification in the culture medium, a colorimetric method was used (urea assay kit, QuantiChrom DIUR-100, BioAssay Systems, Hayward, CA, USA). The kit contains a chromogenic reagent that forms a colored complex specifically with urea. For both assays, the results were obtained with a Spectafluor Plus microplate reader (TECAN, Männedorf, Switzerland) set to a wavelength of 450 and 520 nm for albumin and urea, respectively.

Breath and urine samples were collected at baseline and isotopic plateau and were analyzed for ¹³CO₂ enrichment by continuous-flow isotope ratio mass spectrometry and L-[1-¹³C]phenylalanine enrichment by liquid chromatography-tandem mass spectrometry, respectively, as previously described [29 (link)]. Steady-state CO₂ production (VCO₂) was measured for 20 min after the fifth test drink by indirect calorimetry as previously described [29 (link)]. Urinary urea and creatinine excretion was determined by enzymatic assay according to manufacturer’s instructions (BioAssay Systems, QuantiChrom DIUR-100, DICT-500, Hayward, CA). Amino acid deamination and oxidization is reflected by an increase in urinary urea production [15 (link)] and urinary creatinine excretion is related to FFM [31 (link)]. Thus, the ratio of urea/creatinine was used herein as an estimate of total amino acid oxidation.