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. Author manuscript; available in PMC: 2014 Oct 11.
Published in final edited form as: Free Radic Biol Med. 2013 Apr 11;0:298–309. doi: 10.1016/j.freeradbiomed.2013.04.006

Table 2.

Rates of H2O2 production from sites IF (plus any other sites that respond to NADH reduction state), IIIQo, and IIF predicted from the reduction states of NAD(P)H and cytochrome b566 and the inhibition by malonate in rat skeletal muscle mitochondria with various substrate combinations.

Substrate Site IF
Site IIIQo
Site IIF corrected H2O2 (pmol · min−1 · mg protein−1)
NAD(P)H (% reduced) H2O2 predicted (pmol · min−1 · mg protein−1) Cytochrome b566 (% reduced) H2O2 predicted (pmol · min−1 · mg protein−1)
15 μM palmitoylcarnitine 15.1±0.7 (6) 11.9±1.4 26.1±2.4 (6) 7.8±5.4 15.5±7.5
15 μM palmitoylcarnitine + 2 mM carnitine 79.9±0.7 (6) 32.9±2.8 42.2±3.0 (6) 39.5±14.5 39.6±21.0
15 μM palmitoylcarnitine + 5 mM malate 65.7±1.4 (6) 21.3±1.8 47.5±2.3 (6) 66.2±18.0
15 μM palmitoylcarnitine + 0.5 mM malonate 13.8±0.8 (5) 11.8±1.4 16.6±1.0 (4) 2.7±4.9
15 μM palmitoylcarnitine + 2 mM carnitine + 0.5 mM malonate 79.3±1.1 (6) 32.2±2.8 43.8±2.4 (6) 46.2±14.4

The reduction states of NAD(P) and cytochrome b566 were measured under the conditions of Figs. 1 and 2. The rates of H2O2 production from sites IF and IIIQo were predicted from the calibration curves in Fig. 2A and B (insets). To calculate the corrected rates from site IIF, the measured decreases in H2O2 production rate caused by addition of malonate in Fig. 1B were corrected for the changes in rate from sites IF and IIIQo predicted from the changes in reduction state of the endogenous reporters ± malonate. Values are means ± SEM (n independent experiments). The SEM of the predicted rates of H2O2 production from sites IF and IIIQo were calculated by propagation of calibration and measurement errors using the equations in [19]. The SEM of the corrected rate from site IIF was calculated as SEM = √(SEMa2 + SEMb2 + SEMc2), where SEMa, SEMb, and SEMc represent the errors of the predicted rates from sites IF and IIIQo before and after malonate addition and the SEM of the rate from site IIF assessed as the malonate-sensitive rate in Fig. 1B.