ROS production can be measured with different techniques including chemiluminescence, electron spin
Handbook of Models for Human Aging
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resonance (ESR), spectrophotometry, and fluorometry. These techniques have been used to estimate ROS levels in submitochondrial particles, mitochondria and whole cells. Any appropriate method for estimating ROS production must be highly sensitive since the amount of ROS produced by intact mitochondria in the absence of inhibitors is small. In many physiological comparisons (e.g., related to aging) it is essential to be able to measure basal ROS production without inhibitors. Chemilumies-cent methods are highly sensitive (Chance and Gao, 1994), but unfortunately have low chemical specificity. This is why they are seldom used to assay mitochondrial ROS production. On the other hand, spin-trap ESR techniques can have enough sensitivity and specificity, and their main advantage is their capacity to detect free radicals directly. These methods have been employed to measure ROS levels in brain mitochondria (Dykens,
1994), heart submitochondrial particles (Giulivi et al.,
1995), cultured vascular cells (Inoguchi et al., 2000), or liver mitoplasts (Han et al., 2001). However, ESR techniques require expensive equipment not frequently present in biochemistry laboratories, and in many occasions need the use of spin trap intermediates.
The two more classical methods of estimating free radical production in mitochondria use spectrophotome-try and fluorometry. The measurement of O2 production by spectrophotometry in mitochondrial preparations has been commonly performed by kinetic assays of superoxide dismutase-sensitive epinephrine reduction to adrenochrome or reduction of acetylated or succinylated cytochrome c (Boveris et al., 1976; Takeshige and Minakami, 1979; Lass and Sohal, 2000). They have been also applied to submitochondrial particles or to isolated complexes (Takeshige and Minakami, 1979; Cadenas et al., 1977). However, the detection of ROS production in functional mitochondria needs methods which measure H2O2 rather than O2 . Chance and co-workers developed a method for measuring the rate of mitochondrial H2O2 production (Boveris and Chance, 1973). They used double wavelength spectrophotometry to follow the enzymesubstrate complex between H2O2 and cytochrome c peroxidase. However cytochrome c peroxidase is not commercially available, and it must be prepared from yeast or other sources, which complicates routine assays (Prat et al., 1991). Although substitution of cytochrome c peroxidase by horseradish is possible (Turrens et al., 1985), it further decreases the intrinsically low sensitivity of spectrophotometric techniques.
Fluorometric techniques have the advantage that they are more sensitive. One of the earliest fluorometric methods developed for evaluating H2O2 production (Loschen et al., 1971) was scopoletin (6-methyl-7-hydroxy-1,2-benzopyrone), and sometimes it is still employed (Mattiasson, 2004). During the assay, fluorescent scopoletin is oxidized by H2O2 to a nonfluorescent substance in the presence of horseradish peroxidase. This assay is specific, but it has the inconvenience that it is a negative method because it is based on the disappearance of fluorescence. It requires the use of graded quantities of scopoletin in order to get the best range for measurements (Loschen et al., 1971).
There are other fluorescent probes available for the measurement of ROS production in mitochondria: diacetyldichlorfluorescin, p-hydroxyphenylacetate, homo-vanillic acid (4-hydroxy-3-methoxy-phenylacetic acid) and Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine), all of them specific for H2O2 due to the presence of horseradish peroxidase. Nowadays, the last two probes are the ones most frequently used. The general principle is similar for all these substances (Tarpey et al., 2004): in the presence of H2O2, hydrogen donors (AH2) are oxidized by horseradish-peroxidase (HRP) generating a fluorescent compound:
In the assay described here, homovanillic acid is used since it is less expensive than Amplex Red and does not spontaneously generate fluorescence in the absence of H2O2. Amplex Red can have interferences with NADH or reduced glutathione (Votyakova and Reynolds, 2004; Towne et al., 2004). However, Amplex Red is being frequently used (Zhou et al., 1997) and can produce interesting results (Muller et al., 2004) when properly handled. Homovanillic has been used to specifically detect H2O2 production in isolated mitochondria from a wide variety of tissues and animal species (Drew et al., 2003; Gredilla et al., 2001, Hagopian et al., 2005). The method, which is described in detail below, is useful to (1) quantify basal ROS production, (2) localize the main sites of oxygen radical generation in the respiratory chain, (3) determine the free radical leak (see below), and (4) study the effect of different compounds on mitochondrial ROS production.
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