Ehrlich s reagent

Protein was extracted from MLR media by addition of one half-volume 30% trichloroacetic acid. Precipitated proteins were pelleted out and supernatants were plated on a 96-well plate alongside an L-kynurenine (Sigma) standard curve (1,200 μM−75 μM, two-fold serial dilutions). An equal volume of freshly prepared Ehrlich's Reagent (1% 4-(Dimethylamino)benzaldehyde [Sigma] in glacial acetic acid) was added to each well, and absorbance at OD490 was read on an ELISA plate reader (SpectraMax 340pc, Molecular Devices, San Jose, CA, USA) using SoftMax software. Kynurenine concentrations (μM) were calculated against a linear regression curve.

The measurement of Uδ-ALA was performed according to the method described by Wada et al. [32 (link)]. This method depends on the development of reddish colour when chloroform is added to urine containing high amount of δ-ALA, while faint yellow or faint red colour represents normal amounts of δ-ALA in urine. In brief, we added 2 ml of 20% acetic acid solution to a tube containing 2 ml of urine followed by loading 8 ml of n-butanol to the mixture and homogenised it well using the vortex shaker (Vortex-Genie®, Scientific Industries, USA). Two test tubes were then used; each tube contained 0.5 ml of the mixture aqueous part, one was used as a blank after adding 1.5 ml of sodium phosphate buffer only, while the other (sample tube) was loaded with 1.5 ml of sodium phosphate buffer containing ethyl acetoacetate. Following ten minutes of incubation of both tubes in boiling water, they were allowed to cool down. Then, 2 ml of Ehrlich's reagent (Sigma-Aldrich® Company, Germany) was mixed. The samples were allowed to settle down for another ten minutes; after that, 4 ml of chloroform was added and mixed well with the vortex shaker. After 5 to 30 min, the absorbance of the chloroform phase was read at 556 nm by the spectrophotometer (Biotech-UV2601®-UK) against the blank. The concentration in urine was shown as μmol/l.

TSB activity was measured according to Last et al. [39 (link)]. All steps were performed at 4 °C, unless indicated otherwise. Plant extracts were prepared by grinding 1 g of leaves from five-leaf stage rice seedlings into a paste with a prechilled mortar and pestle containing 1.5 mL of 0.1 M K-phosphate (pH 8.2), 0.3 g quartz sand, and 0.1 g polyvinylpolypyrrolidone. Homogenates were then centrifuged twice at 12,000 g for 15 min and the supernatants were used as the enzyme extracts. The 1 mL reaction solution was prepared containing 60 mM L-serine, 0.2 mM indole, 80 mM potassium phosphate (pH8.2), 10 μg pyridoxal phosphate, and 0.4 mL plant extract with gentle agitation at 30 °C. The reaction was stopped by the addition of 0.1 mL of 0.2 M sodium hydroxide after 90 min. The residual indole was extracted into 4 mL of toluene by gentle vortexing (vigorous agitation created a permanent emulsion). After centrifugation for 15 min at 1500 g, 0.5 mL of the toluene layer was added to 2 mL ethanol and 1 mL Ehrlich’s Reagent (Sigma). The color could develop for 30 min at room temperature and the absorbance of the product was measured spectrophotometrically at 540 nm.