Elsevier

Mitochondrion

Volume 11, Issue 6, November 2011, Pages 893-904
Mitochondrion

Optimization of respiratory chain enzymatic assays in muscle for the diagnosis of mitochondrial disorders

https://doi.org/10.1016/j.mito.2011.07.006Get rights and content

Abstract

The diagnosis of mitochondrial disorders is difficult due to clinical and genetic heterogeneity. Measurements of mitochondrial respiratory chain (RC) enzyme activities are essential for both clinical diagnoses and many basic research questions. Current protocols for RC analysis are not standardized, and so are prone to inter-laboratory variability, and also to biochemical interferences that lead to analytical discrepancies. Moreover, knowledge of the analytical performances of these assays, which is essential to draw meaningful conclusions from the results, is lacking. To understand this variability and to propose possible solutions, we systematically investigated the effect of different homogenization protocols and chemical conditions on RC assays using muscle homogenates. We developed optimized protocols and a novel complex III method with improved sensitivity, precision, and linearity. These methods can be reliably performed on minute muscle samples with a single-wavelength spectrophotometer. Moreover, we measured the variability of the proposed homogenization protocol and we provide a systematic evaluation of each assay's specificity, precision, and linearity. These data will be useful for quality control in both clinical and research laboratories.

Introduction

Mitochondrial disorders are a heterogeneous group of inherited metabolic diseases characterized by impaired function of the mitochondrial respiratory chain (RC). They can be caused by a large variety of mutations in either the mitochondrial or nuclear DNA, and can potentially affect every tissue in the organism. However, tissues with high metabolic rates such as the nervous system, skeletal muscles, and the heart are usually most severely affected (DiMauro and Schon, 2003).

Altogether, mitochondrial disorders are relatively common, with an estimated prevalence of 9.18 cases per 100,000 adults (Schaefer et al., 2008). Diagnosis is still a difficult task, due to the large number of nuclear genes involved and the heteroplasmy of mitochondrial DNA mutations in different tissues. Skeletal muscle is considered the most suitable tissue for the diagnosis of these disorders due to its availability and high metabolic rate. Morphological analyses by specific histochemical reactions and biochemical measures of RC enzyme activities are crucial for clinical diagnosis, clinical research, and many of the basic questions in cell biology.

Activities of RC complexes (I–IV) are assayed spectrophotometrically and the results are normalized to the total muscle protein content or to the activity of mitochondrial matrix enzyme citrate synthase. Despite the importance of biochemical measurements, there is still no consensus on the optimal conditions for these assays, nor a quality assurance scheme (Thorburn et al., 2004). Although most published protocols appear similar in principle, they have not been standardized and employ different muscle homogenization procedures, as well as different reaction conditions (Barrientos et al., 2009, Kirby et al., 2007, Rustin et al., 1994, Trounce et al., 1996). The lack of a uniform methodology has led to striking inconsistencies, as demonstrated by a recent multicenter study that compared the results of RC assays on the same muscle homogenate performed by several diagnostic laboratories specialized in mitochondrial disorders. There was considerable divergence of the results among different laboratories, with differences exceeding one order of magnitude (Gellerich et al., 2004). This issue has also been investigated by a French network of diagnostic laboratories for mitochondrial disorders, leading to the development of standardized assays that led to greater consistency (Medja et al., 2009). However, the performance of some assays (CIII, CI + III, and CII + III) was still unsatisfactory due to unreliability and the difficulty in obtaining linear kinetics and a systematic evaluation of their sensitivity, reliability, and linearity was not performed. Moreover, although the use of double-wavelength spectrophotometry has been suggested to be necessary (Trounce et al., 1996) or at least preferable to single-wavelength spectrophotometry for mitochondrial RC assays (Gellerich et al., 2004) no comparative study was performed between these two techniques.

The aim of our study was to measure the impact of different conditions on RC assays, both at the homogenization phase, and at the analytical phase (i.e. impact of different chemical conditions: and impact of single- vs double-wavelength spectrophotometry), in order to develop simple and reliable protocols for the standardized analysis of RC enzyme activities.

Section snippets

Sample preparation

In order to have a sufficient quantity of the same muscle tissue and to spare limited human samples, we used bovine quadriceps muscle obtained from a freshly slaughtered ox for the development of the protocols. Use of human muscle tissue from controls was limited to the validation of the assays and definition of preliminary reference values, as specified. The muscle was cut in fragments, flash-frozen in liquid nitrogen, and stored at − 80 °C. For each experiment, small amounts of frozen muscle

The type of homogenization buffer affects the quantity and quality of supernatant's proteins

We analyzed the effect of different homogenization buffers on the protein composition of the supernatants obtained after centrifugation of the muscle extracts homogenized in different buffers.

Homogenization of muscle in ionic buffers with relatively high osmolarity (ChP and KCl) resulted in higher total muscle protein levels compared to either sugar-based buffers or low-osmolarity potassium phosphate buffer (Fig. 1A; p < 0.0001). This difference was explained by solubilization of myosin in the

Discussion

At difference with other inborn errors of metabolism, residual enzymatic activities can be considerable in mitochondrial diseases (Trijbels et al., 1993, Thorburn et al., 2004). Therefore, to be clinically useful, mitochondrial RC enzymatic assays must be sufficiently sensitive to detect partial loss of function and sufficiently specific and precise to limit false positive results. Moreover, these methods should be sensitive enough to be performed in small amounts of tissue, especially in case

Conclusions

Different homogenization protocols and analytical conditions have a dramatic impact on the results of respiratory chain spectrophotometric assays used for the diagnosis of mitochondrial diseases and significantly affect their sensitivity and reliability. We presented protocols with optimized analytical performances that allow for the assay of electron transport complexes in mitochondria from muscle homogenates with a commercial single-wavelength spectrophotometer. Use of the detergent Tween 20

Acknowledgments

This work has been supported by a donation from the Stevanato Group SPA, in memory of its founder Giovanni Stevanato, from Telethon Italy grant GGP09207 and a grant from Fondazione Cariparo. We thank the Neuromuscular bank of Padova—Telethon Genetic Biobank Network (project no. GTB07001) supported by the Italian Telethon grants and Eurobiobank for providing us with human muscle specimens. This research is part of a project of the Telethon founded Italian Collaborative Network on Mitochondrial

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