Biomedical Engineering Reference
In-Depth Information
Chapter 16
Measurement of Mitochondrial ROS Production
Anatoly A. Starkov
Abstract
The significance of reactive oxygen species (ROS) as aggravating or primary factors in numerous pathologies
is widely recognized, with mitochondria being considered the major intracellular source of ROS. It is not
yet possible to routinely measure mitochondrial ROS in animals or cultured cells with a reasonable
degree of certainty. However, at the level of isolated mitochondria, one can easily monitor and quantify
the rate of ROS production, identify major sites of ROS production, and compare the rates of ROS pro-
duction in mitochondria isolated from normal and diseased tissue. In this chapter, we describe in detail
the most recent and reliable method to measure mitochondrial ROS as the rate of H
2
O
2
emission. This
method may be employed with minimal modifications to measure H
2
O
2
production by mitochondria
isolated from various tissues and under a wide variety of experimental conditions.
Key words:
Reactive oxygen species, Mitochondria, Amplex, Oxidative stress
1. Introduction
The significance of reactive oxygen species (ROS) as aggravating
or primary factors in numerous pathologies, ischemia, excitotox-
icity, neurodegenerative diseases, and senescence is widely recog-
nized and extensively reviewed elsewhere (
1-11
). More recent
data strongly suggest that ROS, and specifically mitochondria-
generated ROS, are involved in physiological signaling cascades
regulating various cellular and organ functions (
3, 12-14
), with
H
2
O
2
being a chief messenger molecule (
15
).
Mitochondria are believed to be the major intracellular source
of ROS. Several decades of research have firmly established that
ROS production is inherent to mitochondrial oxidative metabo-
lism. This literature has been extensively reviewed by us and oth-
ers elsewhere (
8, 12, 16-23
).
Whereas the primary ROS species produced in mitochondria
is thought to be superoxide (
17, 24
), by its chemical nature it is
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