Taurine Quantification in Plasma and Muscle

Taurine was measured in the plasma and muscle using reverse phase high performance liquid chromatography (HPLC) as previously described [20 (link)]. In brief, plasma samples were precipitated by the addition of 20 times by weight of 5% trichloroacetic acid (TCA). Frozen muscle was crushed using a mortar and pestle under liquid nitrogen and homogenised in 100 times 5% TCA. Zebrafish tails were collected from four independent replicates (12 tails for each experimental condition). The sample for each replicate was split into two, to allow technical replication, and the mean of the technical replicates was used in the subsequent analysis. Samples were homogenised in 200 µL 5% TCA. After centrifugation, supernatants were removed and stored at −80 °C before analysis. Analytes were separated using HPLC with fluorescent detection, with pre-column derivatisation with o-phthalaldehyde (OPA) and 2-mercaptoethanol (2ME). OPA reacts rapidly with amino acids and sulfhydryl groups to yield intensely fluorescent derivatives, and 2ME, a reducing agent, prevents the OPA reagent from oxidising. An internal standard, O-phospho-DL-serine, dissolved in 5% TCA was added. Sodium borate was used to adjust the pH to 9. Samples were placed in an autosampler, which was maintained at 4 °C. Samples were mixed on a sample loop with a derivatising solution containing 20 mM OPA and 60 mM 2ME in 100 mM sodium borate, pH 10, for 30 s before injection onto the column. Separation was achieved with a C18 column (4 µm, 4.6 × 100 mm, Agilent, Santa Clara, CA, USA) using an Agilent 1260 Infinity HPLC system. Mobile phase A consisted of 50 mM potassium phosphate buffer, methanol, and tetrahydrofuran (94:3:3). Mobile phase B consisted of 90% methanol, with a gradient increase in B from 0 to 100%. Fluorescence was set at 360 nm and 455 nm for excitation and emission, respectively. The protein content of the muscle and zebrafish samples were quantified by solubilising the pellet in 0.5M sodium hydroxide, before incubation at 80 °C for 15 min. Once fully dissolved, protein concentrations of supernatants were quantified using a Bradford protein assay (Bio-Rad Australia, South Granville, NSW, Australia).