Biochemical Assays of Respiratory Chain Complex Activity

Publisher Summary

This chapter focuses on the biochemical assays of the respiratory chain (RC) complex activity. It presents the preparation of mitochondrial fractions from tissues and cultured cells for RC enzymology; the measurement of activity of the individual complexes I, II, III, IV, V, the mitochondrial matrix marker enzyme citrate synthase; and the combined activity of complexes II + III. RC enzyme activities are frequently expressed relative to its activity of citrate synthase. Such ratios are more robust than absolute activities because of the variability inherent in cell culture conditions, such as passage number and the degree of confluence, and the proliferation of mitochondria seen in tissues of many patients with mitochondrial disease. The effects of postmortem delay on RC enzymes from liver samples frozen at varying times after death were analyzed to assess the stability of RC enzyme activities postmortem. There can be considerable loss of RC enzyme activity postmortem, particularly in liver, but the observations suggest that muscle collected and frozen at -70°C within 6 h of death and liver within 2 h remain suitable for RC enzyme analysis. The chapter presents the effects of tissue pathology by comparing RC enzymes in tissues from patients without RC complexes I–IV defects with normal controls. The possibility of secondary decreases in enzyme activity and the broadening of reference ranges in the presence of tissue pathology should be considered in interpreting RC enzyme profiles.

Introduction

Mitochondrial disorders are estimated to affect at least 1 in 5000 of the population (Skladal et al., 2003). These are usually multisystem diseases; indeed, mitochondrial disease “can give rise to any symptom, in any tissue, at any age, and with any mode of inheritance” (Munnich and Rustin, 2001). Skeletal and cardiac muscle and the central nervous system are predominantly affected. Many mitochondrial disorders are caused by deficiencies of the activities of one or more of the enzyme complexes of the respiratory chain (RC), thus RC biochemistry is an important part of the diagnostic work up of a patient suspected of mitochondrial disease. This is especially true for children, where defects caused by mutations in mitochondrial DNA (mtDNA) are less common than in adults. RC enzyme deficiencies may be isolated, that is, affect only one of the enzyme complexes (Fig. 1A), or they may affect several, particularly those with mtDNA‐encoded subunits (Fig. 1B). Mutations in mitochondrial transfer RNA (tRNA) genes, and in nuclear genes encoding enzymes involved in regulation and metabolism of mitochondrial nucleoside pools, such as the mtDNA polymerase γ1 (POLG1) (Davidzon 2005, Naviaux 2004) and mitochondrial deoxyguanosine kinase (DUOGK) (Mandel 2001, Slama 2005) genes, can cause multiple RC enzyme defects. Accurate identification of such biochemical defects is desirable in order to direct efforts in identifying the molecular basis of the disease in a particular patient. It is therefore important to be able to measure individual RC complexes to enable correlation with both nuclear and mtDNA mutations, to direct screening of candidate genes (e.g., a biochemical complex I deficiency would direct one toward sequencing the mitochondrial MTND genes, or some of the many nuclear DNA‐encoded complex I subunits, while an isolated complex IV deficiency would more likely lead to investigation of genes involved in complex IV assembly such as SURF1), and to narrow down defects detected by other methods [e.g., cytochrome c oxidase (COX) histochemistry, substrate oxidation, adenosine triphosphate (ATP) synthesis].

There are many methods described to measure RC enzyme activities, and there is no one “correct” way. This chapter is written collaboratively by representatives from two laboratories that provide diagnostic services for RC enzymology using spectrophotometric assays. It is notable that although the precise methods differ slightly, the general approach in the two centers is remarkably similar. We find that linked assays are less useful in the diagnosis of RC deficiencies than the isolated activities. For example, the combined activity of complex II + III (succinate:cytochrome c reductase) is not very sensitive for the diagnosis of complex III defects, since the flux through these enzymes relies mostly on the complex II activity (Taylor et al., 1994). However, it can be useful in detecting complex II deficiency, and since it relies on endogenous coenzyme Q, complex II + III activity is useful in delineating coenzyme Q deficiency. Thus, if the isolated complexes II and III activities are normal, but the combined II + III activity is reduced, the RC abnormality may well be a defect in coenzyme Q synthesis or recycling.

Measurement of RC enzymes in cultured cells is useful to confirm a defect found in another tissue, although it is our experience, and that of others, that only about 50% of patients with an RC defect detected in skeletal muscle, liver or heart will express that defect in cultured fibroblasts (Faivre 2000, Niers 2003). In adults, the proportion of patients expressing a defect in fibroblasts is probably even less than this. Since fibroblasts, chorionic villus sampling (CVS) cells, and amniocytes have a common embryonic origin (Niers et al., 2003), expression of an enzyme defect in fibroblasts suggests that the deficiency will also be expressed in CVS and amniocytes, implying that enzyme‐based prenatal diagnosis may be possible in cases where a molecular defect has not been demonstrated. The ability to measure the RC complexes in cultured cells is also useful in cell biology studies to demonstrate the pathogenicity of a putative mtDNA mutation. Such studies may include correlation of mutant load with residual enzyme activity in cybrids (transmitochondrial cytoplasmic hybrids) or lack of phenotypic rescue (restoration of enzyme activity) in hybrids between RC enzyme‐deficient patient fibroblasts and mtDNA‐less cells (ρ⁰ cells).

Here, we describe the preparation of mitochondrial fractions from tissues and cultured cells for RC enzymology, and measurement of activity of the individual complexes I, II, III, IV, V, the mitochondrial matrix marker enzyme citrate synthase, and the combined activity of complexes II + III. Citrate synthase is not part of the RC, but RC enzyme activities are frequently expressed relative to its activity. Such ratios are more robust than absolute activities due to (1) the variability inherent in cell culture conditions such as passage number and the degree of confluence and (2) the proliferation of mitochondria seen in tissues of many patients with mitochondrial disease.