Ultimate 3000 vwd

Methionine, hypotaurine, and taurine were derivatized with ο-phthalaldehyde/2-mercaptoethanol and quantified using an HPLC with a fluorescence detector (FLD-3100; Thermo Scientific; excitation 338 nm and emission 425 nm) [43 (link),45 (link)]. They were separated with a Hector T-C18 column (3 µm × 4.6 mm × 100 mm; Chiral Technology Korea, Daejeon, Korea). The examination of both SAM and SAH concentrations were determined by HPLC separation with a UV detector (UltiMate™ 3000 VWD; Thermo Scientific; 254 nm). Homocysteine, cysteine, and GSH were analyzed by the SBD-F derivatization method [46 (link)] using an HPLC with a fluorescence detector (FLD-3100; Thermo Scientific; excitation 385 nm and emission 515 nm). The chromatographic separation was achieved with a Hector M-C18 column (3 µm × 4.6 mm × 150 mm: Chiral Technology Korea).

Liver lysate was obtained by homogenization of liver tissue with four-fold volume of buffer (150 mM NaCl, pH 7.4). The liver lysate was mixed with 1 M perchloric acid containing 2 mM EDTA or methanol, and the centrifuged (10,000× g, 10 min, 4 °C) supernatant was used for the determination of SAM/SAH/cysteine/GSH or methionine/taurine, respectively. The levels of cysteine and GSH were determined using HPLC with a fluorescence detector (excitation at 385 nm and emission at 515 nm; FLD-3100, Thermo Scientific). They were separated with a Hector M-C18 column (3 µm × 4.6 mm × 150 mm; Chiral Technology Korea, Daejeon, Korea) and fluorescence detector using the SBD-F derivation method. HPLC with Kromasil column (3 µm × 4.6 mm × 250 mm: Kromasil, Bohus, Sweden) and 254 nm UV detector (UltiMate™ 3000 VWD; Thermo Scientific) was used to analyze hepatic SAM and SAH concentration. Methione and taurine were derivatized using o-phthalaldehyde/2-mercaptoethanol and measured using an HPLC system with a fluorescence detector. They were separated using a Hector T-C18 column (3 µm × 4.6 mm × 100 mm; Chiral Technology Korea, Daejeon, Korea).