Ammonia test wako

A part of the cultivation medium was sampled and centrifuged at 5000×g for 10 min, then the supernatant obtained was used to determine the concentrations of ammonia, nitrite, and nitrate. The ammonia concentration was assayed spectrophotometrically by the indophenol blue method using the Ammonia Test Wako (Fujifilm Wako Pure Chemical Co., Osaka, Japan). Concentrations of nitrite and nitrate were measured spectrophotometrically by a diazo-coupling method³⁹ and a brucine method⁴⁰ , respectively. POD activity was determined by measuring the rate of nitrite production in the assay solution containing 20 mM Tris–HCl buffer (pH 8.0), 1.0 mM sodium ascorbate, and 1.0 mM pyruvic oxime. Pyruvic oxime was synthesized according to Quastel et al.^{41 (link)}. The protein concentration was measured using a BCA protein assay kit (Pierce, Rockford, IL) with bovine serum albumin as the standard, and the specific activity was calculated.

The hepatic G3P concentrations were determined using an enzymatic method³¹ . Hepatic amino acid concentrations were determined by liquid chromatography/mass spectrometry (LC/MS) (Acquity ultra performance liquid chromatography/Acquity TQD tandem-quadrupole mass spectrometer; Waters, Milford, MA, USA) after solid phase extraction and derivatization^{32 (link)} using the EZ:faast amino acid analysis kit (Phenomenex Ltd., Los Angeles, USA). Blood ammonia concentration was determined with Ammonia Test Wako (Wako Pure Chemicals, Osaka, Japan). In the perfusion experiments, concentrations of ammonia, urea, lactate and Pyr were quantified enzymatically^{33 –35} .

The concentration of albumin produced in the medium was measured by enzyme-linked immunosorbent assay (ELISA) using a Human Albumin ELISA Quantitation Set (E80-129; Bethyl). To measure the ammonia removal rate, the culture supernatant was collected after 24 h incubation in fresh medium (500 µL/well) containing 2 mM NH₄Cl (Fujifilm Wako Pure Chemical). The ammonia concentration was measured using a commercially available kit (Ammonia-Test Wako; Fujifilm Wako Pure Chemical). The removal rate was determined by calculating the amount of ammonia reduction. To measure cytochrome P450 activity, the culture supernatant was collected after 1 h incubation in fresh medium (300 μL/well) with 3.0 μM luciferin substrate (Luciferin-IPA; Promega, Madison, WI, USA). The luciferin produced by CYP3A4 enzymatic activity in hepatic cells was quantified using luciferase (Luciferin Detection Reagent; Promega), and the luminescence intensity was measured using a Glo-max Luminometer (Promega). These assays were performed in accordance with the manufacturer’s instructions.