The tissue was weighed while frozen. After thawing, fetal brain (1:10, w/v) and placenta (1:10, w/v) were homogenized by sonication in ultrapure water (placental homogenate was further diluted 1:10, v/v, for KYNA determination). Maternal (1:4, v/v for kynurenine, 1:10, v/v for KYNA) and fetal (1:5, v/v for kynurenine, 1:20 v/v for KYNA) plasma were diluted in ultrapure water. Twenty-five μL of 25% perchloric acid were added to 100 μL of the tissue preparation, and precipitated proteins were removed by centrifugation (16,000 × g, 15 min). For determination ex vivo, the eluate was diluted 1:2 (v/v) in 6% perchloric acid and centrifuged (16,000 × g, 5 min).
For kynurenine and KYNA determination, 20 μL of each resulting supernatant were injected onto a 3 μm C18 reverse phase HPLC column (150 mm × 4 mm; Dr. Maisch GmbH, Ammerbuch, Germany), using a mobile phase containing 50 mM sodium acetate and 3% acetonitrile (pH adjusted to 6.2 with glacial acetic acid) at a flow rate of 0.5 mL/min. Zinc acetate (0.5 M; not pH adjusted), was delivered post column by a peristaltic pump (Dionex AXP, Thermo Fisher, Waltham, MA, USA) at a flow rate of 0.1 mL/min. In the eluate, analytes were detected fluorimetrically (kynurenine: excitation: 365 nm, emission: 480 nm; KYNA: excitation: 344 nm, emission: 398 nm; S200a fluorescence detector; Perkin Elmer, Waltham, MA, USA).
For kynurenine and KYNA determination, 20 μL of each resulting supernatant were injected onto a 3 μm C18 reverse phase HPLC column (150 mm × 4 mm; Dr. Maisch GmbH, Ammerbuch, Germany), using a mobile phase containing 50 mM sodium acetate and 3% acetonitrile (pH adjusted to 6.2 with glacial acetic acid) at a flow rate of 0.5 mL/min. Zinc acetate (0.5 M; not pH adjusted), was delivered post column by a peristaltic pump (Dionex AXP, Thermo Fisher, Waltham, MA, USA) at a flow rate of 0.1 mL/min. In the eluate, analytes were detected fluorimetrically (kynurenine: excitation: 365 nm, emission: 480 nm; KYNA: excitation: 344 nm, emission: 398 nm; S200a fluorescence detector; Perkin Elmer, Waltham, MA, USA).