Liver H2S Production Quantification

H₂S production in liver tissue was evaluated following the lead sulfide method as previously described, with modifications [29 (link)]. Briefly, 100 mg of liver tissue were homogenized in 1.2 mL PBS and their protein levels measured with the BCA Assay Kit (Thermo Fisher, Waltham, MA, USA). For total H₂S production measurement, 200 μg of protein were incubated at 37 °C in the presence of 10 mM Cys (Sigma-Aldrich, St. Louis, MO, USA) and 20 μM Pyridoxal 5′-phosphate (PLP) (Sigma-Aldrich, St. Louis, MO, USA) on 96-well plates covered with a lead acetate membrane. In the case of enzymatic H₂S production, 15 mM EDTA solution was added to the previous reaction mix to chelate Fe²⁺, a metal used in the non-enzymatic production, according to Yang et al. [30 (link)]. Incubations were performed after 2 h, until dots of lead sulfide were detected but not saturated. In order to prepare the membranes, Whatman n° 2 paper was soaked in 20 mM lead acetate (Sigma-Aldrich, St. Louis, MO, USA) and vacuum dried. Dots were densitometered (BioRad Densitometer G-800, Hercules, CA, USA) for quantification. A standard curve from 0 to 1000 mM NaHS was performed for each membrane.