JPET #161612
A silybin-phospholipids complex prevents mitochondrial dysfunction in a rodent model of non-alcoholic steatohepatitis
Gaetano Serviddio, Francesco Bellanti, Anna Maria Giudetti, Gabriele Vincenzo Gnoni, Antonio Petrella, Rosanna Tamborra, Antonino Davide Romano, Tiziana Rollo, Gianluigi Vendemiale and Emanuele Altomare
Supplemental information:
Measurement of mitochondrial H2O2 production. The rate of mitochondrial peroxide generation was measured according to a modification of the method by Barja (Barja, G., 1999). In this method the generation of hydrogen peroxide is measured following the reaction between H2O2 and Amplex Red® catalyzed by peroxidase, which forms a H2O2-Amplex Red® dimer that exhibits fluorescence at 590 nm when stimulated at 530 nm. The formation of the dimer is followed kinetically. Determination of peroxide production in mitochondria requires incubation of isolated mitochondria with 5mM glutamate and 2.5mM malate as complex I-linked substrate or with 10mM succinate and 2µM rotenone as complex II-linked substrates. For each type of substrates, the measurements was performed in triplicate. An internal standard was used with hydrogen peroxide at the following final concentration: 0, 1.0, 1.5, and 2.0 nmol/ml. The working solution of hydrogen peroxide should be prepared from the commercial stock solution of hydrogen peroxide (33,3% w/v) as follows: first, a 1/103 dilution in HEPES-phosphate buffer is performed and the precise H2O2 concentration is measured using its absorbance at 230 nm [(H2O2) = 72,4 mM-1 x cm-1]; subsequently, a 1/105 dilution is performed as the working solution to be used for the standard tubes.
Mitochondrial HNE- and MDA-protein adducts. Protein oxidative damage in liver mitochondria was evaluated by HNE- and MDA-protein adducts as previously reported (10). Briefly, tissue fluorescent adducts formed between peroxidation derived aldehydes (HNE and MDA) and liver mitochondria proteins were monitored by spectrofluorimetry as described by Tsuchida et al. (11): fluorescence emission was at 460 nm, excitation was at 390nm for MDA-adducts and at 355nm for HNE-adducts.
Evaluation of FOF1ATPase activity. FOF1ATPase activity was measured following ATP hydrolysis with an ATP-regenerating system coupled to NADPH oxidation, as described previously (8). In brief, FOF1ATPase hydrolytic activity was determined spectrophotometrically at 660 nm reference wavelength, in association with ATP hydrolysis. The reaction was carried out at 37°C, in 2 ml reaction medium containing a buffer solution (50 mM Tris-HCl pH 8.0, 50 mM KCl, 20 mM MgCl2, and BSA 5 mg/ml) and pyruvate kinase/l-lactate dehydrogenase 4U/ml, 1µM Antimycin A and 1µM CCCP. After the addition of mitochondria (40 µg of proteins), the reaction was initiated by adding 1.5 mM phosphoenolpyruvate and 50 µM NADH. 5 min later, oxidation of NADH was monitored at 660 nm by adding 350 µM ATP as a measure of total ATPase activity (ε=1.87 mM-1 cm-1). The same assay was performed with addiction of 1.5 µg/ml Oligomycin. Specific ATPase activity was calculated as the difference in total absorbance and absorbance in the presence of Oligomycin.