Assessment of Mitochondrial Function

As mitochondrial function changes as we age, it is important that we have tools at our disposal to measure accurately different aspects of mitochondrial function and follow the changes through the aging process.

There are a number of methods that can be employed to measure mitochondrial function, such as determination of oxidative enzyme activity, mitochondrial respiration, or mitochondrial ATP production rate (MAPR).

Muscle fibre type is also an indicator of the oxidative capacity of the muscle. For example, type I oxidative fibres, have a high oxidative capacity due to their high mitochondrial density, compared with that of glycolytic fibres (Jackman and Willis 1996). Glycolytic fibres, however, have a much lower mitochondrial density and hence oxidative capacity (Jackman and Willis 1996) (see Table 1). Therefore, histochemical staining of muscle sections to determine muscle fibre type can also assess the oxidative capacity of muscle. However this is not a very quantitative method in comparison to other methods discussed below.

! (*! 2.4.3.1 Mitochondrial preparation

Until recently, most of the research on mitochondria and mitochondrial dysfunction was conducted using mitochondrial tissue prepared from animals (Saks, Kapelko et al. 1989; Wibom, Lundin et al. 1990). This is because it was not always possible to obtain sufficiently large (>1 g of muscle) from human tissue to effectively measure mitochondrial function without some form of invasive surgery. To overcome the complications involved with open surgery biopsies, Rasmussen and colleagues (1997) developed a needle biopsy method to isolate mitochondria from only a small amount of skeletal muscle (25–100 mg) that can successfully be used to measure mitochondrial enzymatic activity and ATP synthesis (Rasmussen, Andersen et al. 1997) (see Chapter 3, Section 3.9.1)

2.4.3.2 Oxidative Enzymes

A number of studies have used the activities of various enzymes involved in oxidative metabolism as a measure of mitochondrial function (Blomstrand, Radegran et al. 1997; Wang, Williams et al. 1999; Carey, Williams et al. 2000). Enzymes measured in such studies include the Krebs cycle enzymes, citrate synthase (CS) (Blomstrand, Radegran et al. 1997; Wang, Williams et al. 1999; Carey, Williams et al. 2000), succinate dehydrogenase (SDH) (Blomstrand, Radegran et al. 1997), oxoglutarate dehydrogenase (OGDH) (Blomstrand, Radegran et al. 1997; Wang,

Williams et al. 1999), as well as !-hydroxyacyl-coA dehydrogenase (!-HAD) (Wang,

Williams et al. 1999), an enzyme involved in the !-oxidation of fatty acids.

The activity of ETC complexes (complexes I-IV) can also be measured spectrophotometrically/fluorometrically by isolating the respiratory enzyme of

! (+! interest in mitochondrial preparations and quantifying the activity of specific enzymes. For example, rotenone is a potent inhibitor of respiratory complex I (NADH dehydrogenase) and is often used to determine the activity of specific NADH oxidation reactions that occur through complex I (Chretien, Bourgeron et al. 1990; Wibom, Lundin et al. 1990).

Whilst these methods are useful in assessing mitochondrial oxidative capacity, they tend to be limited because they measure specific mitochondrial processes rather than overall mitochondrial function.

2.4.3.3 Oxygen Consumption

Other studies have used the determination of oxygen consumption as a measure of mitochondrial function (Chance and Williams 1955). This method assesses the rate of ADP-stimulated oxygen consumption either in whole tissues (Chance and Williams 1955) or in isolated mitochondria (Guerrero-Ontiveros and Wallimann 1998). The usefulness of this method is also limited because the electron transfer may not be completely coupled to the synthesis of ATP, as protons may leak back into the mitochondrial matrix across the inner membrane, rather than through ATP synthase. This process is termed ‘proton leak’ (Mitchell and Moyle 1967), and has been shown to occur in a number of tissues including skeletal muscle (Rolfe and Brand 1996).

The use of mitochondrial oxygen consumption as a measure of mitochondrial function may therefore be limited by the fact that not all the ATP produced is coupled to electron transport and oxygen consumption. This is quite likely in a number of

! (,! disease states, and may also be true in the elderly as inefficiencies and mtDNA changes begin to occur.

2.4.3.4 Mitochondrial ATP Production Rate (MAPR)

Other measures of aerobic metabolism have been based on the luminometric measurement of light emitted by a firefly luciferase reagent to measure ATP production (Lemasters and Hackenbrock 1973). The bioluminescent properties of firefly luciferase have been widely used in the measurement of ATP content in various tissues (Strehler and Totter 1952; Lemasters and Hackenbrock 1973; Lundin, Richardsson et al. 1976) including skeletal muscle (Wibom, Lundin et al. 1990; Wibom, Hultman et al. 1992; Wang, Williams et al. 1999) because the intensity of the light emitted (measured bioluminometrically at 560 nm) is proportional to the amount of ATP in the tissue (Wibom and Hultman 1990; Wibom, Lundin et al. 1990) (see Figure 3.9 in Chapter 3)

The MAPR method used by Wibom et al. (1992) is a reliable index of muscle oxidative capacity, as it is not concerned with oxygen consumption and thus proton leak, but rather the direct measurement of ATP production by the mitochondria. In addition, the use of different substrate combinations to measure MAPR provides a good reflection of ATP-production rates from different metabolic pathways representing carbohydrate, fat and amino acid metabolism (Wibom, Lundin et al. 1990).

When the different methods available for the assessment of mitochondrial function, and hence muscle oxidative capacity, are compared (oxidative enzyme activity, oxygen consumption as a measure of mitochondrial respiration, and MAPR),

! (-! it becomes clear that MAPR is the most reliable and effective method with the fewest drawbacks. It is for this reason that MAPR was chosen as the method for measurement of muscle oxidative capacity in two of the studies reported herein.