2.12. Thiol- and Amino Acid Concentrations

Free amino acids and thiol compounds were extracted from 30 mg kidney tissue with 0.3 mL of 0.1 mol/L HCl in an ultrasonic ice-bath for 10 min. The resulting homogenates were centrifuged twice for 10 min at 4 °C and 16,400× g to remove cell debris. Total glutathione was quantified by reducing disulfides with DTT followed by thiol derivatization with the fluorescent dye monobromobimane (Thiolyte, Calbiochem, Darmstadt, Germany). For quantification of GSSG, free thiols were first blocked by NEM followed by DTT reduction and monobromobimane derivatization. GSH equivalents were calculated by subtracting GSSG from total glutathione levels. Derivatization was performed as described in Wirtz et al. [30]. For ultra performance liquid chromatography-fluorescence detector (UPLC-FLR) analysis an Acquity H-class UPLC system coupled to an Acquity FLR detector (Waters, Milford, CT, USA) was used. Separation was carried out using a binary gradient of buffer A (100 mmol/L potassium acetate, pH 5.3) and solvent B (acetonitrile) with the following gradient: 0 min 2.3% buffer B; 0.99 min 2.3%, 1 min 70%, 1.45 min 70%, and re-equilibration to 2.3% B in 1.05 min at a flow rate of 0.85 mL min⁻¹. The column (Acquity BEH Shield RP18 column, 50 mm x 2.1 mm, 1.7 µm, Waters, Milford, USA) was maintained at 45 °C and sample temperature was kept constant at 14 °C. Monobromobimane conjugates were detected by fluorescence at 480 nm after excitation at 380 nm. Data acquisition and processing was performed with the Empower3 software suite (Waters, Milford, CT, USA).

Determination of proteinogenic amino acid levels was done as described in Weger et al. [31]. Beta-Alanine content was analyzed after specific labeling with the fluorescence dye AccQ-Tag™ (Waters, Milford, CT, USA) according to the manufacturer’s protocol using an Acquity I-class UPLC system coupled to a VION Ion Mobility Separation QTof (Waters, Milford, CT, USA). Separation was carried out using a Cortecs C18 column (100 mm × 2.1 mm, 1.6 µm, Waters) at 40 °C. The mobile UPLC phase consisted of binary gradients of ACN with 0.1% formic acid (B) and 0.1% aqueous formic acid (A), flowing at 0.5 mL min⁻¹. Analytes were initially eluted with 98% A and A was decreased linearly to 35% over 6 min. After this, the column was washed with 85% B for 2 min and re-equilibrated under the initial conditions for 2 min. Measurements were performed with an ESI source operated in positive mode (1.00 kV capillary voltage; source temperature 120 °C, desolvation temperature 500 °C; observed m/z 260.103 Da; observed CCS value 161.84). Unifi software (version 1.9.3, Waters, Milford, CT, USA) was used to control the instrument and to acquire and process the MS data.