Rp hplc

The renal tissue was homogenized in Tris-HCl (0.01 M)-EDTA (0.001 M) buffer of pH 7.4 and centrifuged at 12,000× g for 30 min at 4 °C to obtain supernatant for subsequent biochemical assays [6 (link)]. The levels of ROS, NO, H₂O₂, NADPH oxidase, TBARS, carbonylated proteins, GSH, GSSG, SOD, CAT, GPx, GR, and GST in the renal tissue homogenates were measured following the established protocols mentioned earlier. Co-enzymes Q9 and Q10 in the cell lysate were separated and quantified using reverse phase-high performance liquid chromatographic (RP-HPLC) (Dionex, Germany) methods as described by Zhang and co-workers [34 (link)]. DNA fragmentation in the renal cells was measured using the diphenylamine reagent [11 (link)]. The extent of DNA oxidation in the renal cells was measured by quantifying 8-hydroxy-2′-deoxyguanosine (8-OHdG) using a RP-HPLC (Dionex, Idstein, Germany) method [11 (link)].

The levels of ROS, NO, H₂O₂, NADPH oxidase, co-enzyme Q9, co-enzyme Q10, SOD, CAT, G6PD, GR, GST, GPx, GSH, and GSSG in the kidneys of the mice receiving different treatments were measured following the established protocol mentioned earlier. The extent of DNA fragmentation in the renal cells was evaluated by the diphenylamine reaction, while DNA oxidation in the renal cells was measured by RP-HPLC (Dionex, Germany) analysis [49 (link)].

Donor solutions of FLUO, LY, GA, and EGCG were prepared and sterile-filtered in HBSS at pH 6.5 and pH 7.4 to perform transepithelial studies. Standard curves of GA and EGCG dissolved in HBSS at pH 6.5 and pH 7.4 were obtained by HPLC analysis. 200 µL samples withdrawn from the donor and acceptor compartments during transport experiments across cell monolayers were quantitatively analyzed using RP-HPLC (Thermo Fisher Scientific, Denmark). A C18 column (3.0 × 100 mm) and 0.5 mL/min flow rate were used. GA and EGCG were quantified with detection at 255 nm and 270 nm, respectively. FLUO and LY aliquots were instead analyzed by UV-vis spectrometry (Nanodrop One^C, Thermo Fisher Scientific, Denmark), recording their absorbance at 490 nm and 430 nm, respectively. The amount of each compound transported across the cell monolayers within a time interval of 8 h was calculated for both apical-to-basolateral (AB) and basolateral-to-apical (BA) directions. FLUO, LY, GA, and EGCG that remained entrapped within the cell monolayers, insert membranes and nanofibers were likewise quantified at the end of the permeability studies.