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Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases
08:57

Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases

Published on: February 24, 2018

Moderate dependence of ROS formation on DeltaPsim in isolated brain mitochondria supported by NADH-linked substrates

Laszlo Tretter1, Vera Adam-Vizi

  • 1Department of Medical Biochemistry, Semmelweis University, P.O. Box 262, Budapest H-1444, Hungary.

Neurochemical Research
|August 26, 2006
PubMed

Insights

Mitochondrial membrane potential (DeltaPsim) influences hydrogen peroxide (H2O2) generation, but only under highly polarized conditions. Most reactive oxygen species (ROS) production is independent of DeltaPsim when using NADH-linked substrates.

Area of Science:

  • Mitochondrial Physiology
  • Biochemistry
  • Cellular Respiration

Background:

  • Mitochondria generate reactive oxygen species (ROS) during cellular respiration.
  • The relationship between mitochondrial membrane potential (DeltaPsim) and ROS production is complex and substrate-dependent.
  • Previous studies suggested a strong link between DeltaPsim and ROS formation, particularly with succinate-supported respiration.

Purpose of the Study:

  • To investigate the dependence of hydrogen peroxide (H2O2) generation on mitochondrial membrane potential (DeltaPsim) in guinea-pig brain mitochondria.
  • To determine the role of bovine serum albumin (BSA) in modulating mitochondrial polarization and ROS production.
  • To differentiate DeltaPsim-dependent and independent pathways of ROS generation.

Main Methods:

  • Isolated guinea-pig brain mitochondria were used, respiring on glutamate plus malate (NADH-linked substrates).
  • Mitochondrial membrane potential (DeltaPsim) was assessed, and hydrogen peroxide (H2O2) formation was measured.
  • Experiments were conducted in the presence and absence of bovine serum albumin (BSA) and with the uncoupler FCCP and rotenone.

Main Results:

  • In the absence of BSA, FCCP-induced depolarization did not affect H2O2 formation.
  • BSA addition hyperpolarized mitochondria and increased H2O2 production.
  • FCCP-induced depolarization in the presence of BSA reduced H2O2 formation only to the level seen without BSA, indicating a DeltaPsim-independent component.
  • Rotenone stimulated H2O2 formation irrespective of BSA presence or DeltaPsim.

Conclusions:

  • Mitochondrial H2O2 formation from NADH-linked substrates is sensitive to DeltaPsim changes only when mitochondria are highly polarized.
  • A significant portion (approximately 60%) of ROS generation in this system is independent of DeltaPsim.
  • This contrasts with findings for succinate-supported mitochondria where ROS formation is highly sensitive to DeltaPsim due to reverse electron flow.

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