Figure 4. PDC-mediated H₂O₂ production: Effects of NADH/NAD and mitochondrial membrane potential.

(A-G) Data were generated using PDC from porcine heart. (A) Representative H₂O₂ production experiment. Black arrows indicate the addition of pyruvate (1 mM). (B) H₂O₂ production in the absence and presence of 25 U/ml SOD. (C) H₂O₂ production in the absence and presence of 100 U/ml catalase. For these experiments, HRP was reduced to 100 mU/ml in order to allow catalase to outcompete HRP for any potential H₂O₂. (D-E) H₂O₂ production in response to increasing concentrations of pyruvate in the absence (D) or presence (E) of NAD⁺. K_m and V_max values (with respect to H₂O₂ production) were calculated using Prism statistical software. *Different from - NAD⁺ condition. (F) Representative H₂O₂ production (black trace) and NADH production (grey trace) experiment in response to 1 mM pyruvate (50 μM NAD⁺ was present for all assays). (G) Quantified flux derivative from experiment depicted in panel F. JH₂O₂ and JNADH were plotted together (pmol/sec). (H) Permeabilized fibers were prepared from RG of C57BL/6NJ mice and pre-treated with 0 or 100 nM CDNB. JH₂O₂ emission was assessed in response to succinate (10 mM) and pyruvate (1 mM) in the absence and presence of FCCP (5 μM). (I) Permeabilized fibers were prepared from RG of Sprague Dawley rats and pre-treated with varying concentrations of CDNB. JH₂O₂ emission was assessed in response to pyruvate (1 mM) in the absence (State 4) and presence (State 3) of maximal ADP (2 mM). Data are mean ± SEM, n = 3-4/group (A-G), n = 6-8/group (H-I).