Respiratory chain enzyme analysis in muscle and liver

doi:10.1016/j.mito.2004.07.003

Mitochondrion

Volume 4, Issues 5–6, September 2004, Pages 363-375

https://doi.org/10.1016/j.mito.2004.07.003 Get rights and content

Abstract

Respiratory Chain (RC) enzyme analysis remains the mainstay for diagnosis of children suspected of having a RC disorder. A previous international workshop suggested a set of criteria for the ideal approach to diagnosis but concluded that probably no single centre fulfilled all these criteria. Major practical issues relate to the range of tissues tested, whether frozen tissue biopsies can be used reliably, assay methods, difficulty in defining realistic reference ranges, and the lack of an external quality assurance scheme. We discuss these issues and describe our experience over the last decade with assaying RC enzymes in over 600 skeletal muscle and 300 liver biopsies from patients, a range of different controls (other known inborn errors, end-stage liver disease, post-mortem samples) and single donated normal muscle and liver samples assayed on more than 100 occasions over 5- to 10-year periods. Our experience is that ‘sick’ tissues have wider ‘normal’ ranges than ‘healthy’ tissues. Caution is therefore needed to ensure that secondary RC defects are not misdiagnosed as primary RC defects. We describe diagnostic criteria that integrate the results of RC enzyme assays with clinical, histological, metabolic and molecular investigations to determine whether the overall diagnostic certainty is possible, probable or definite.

Section snippets

‘Atypical’ patients are the rule in pediatrics

Most of the best known mitochondrial disorders were defined by adult physicians specialising in neurology or ophthalmology. These include conditions such as Leber Hereditary Optic Neuropathy (LHON), Mitochondrial Encephalomyopathy, Lactic Acidosis and Stroke-like disorder (MELAS), Myoclonic Epilepsy with Ragged Red Fibers (MERRF), Neurogenic weakness, Ataxia and Retinitis Pigmentosa (NARP), Mitochondrial Neuro-GastroIntestinal Encephalomyopathy (MNGIE), Chronic Progressive External

The ideal approach to respiratory chain enzyme diagnosis

Prior to a workshop on Clinical and Enzyme Diagnosis of RC Disorders, 15 major international mitochondrial centers completed a questionnaire on the criteria for defining the ideal mitochondrial diagnostic centre. As described elsewhere (Thorburn and Smeitink, 2001), there was broad agreement on a number of criteria, which included that such a center would: be part of a complete (clinical and laboratory) diagnostic program, clinically investigate all suspected patients before invasive procedures

The Melbourne experience: history and demographics

Enzyme diagnosis of RC disorders in Melbourne was initiated in the early 1980s by Dr Garry Brown, now at Oxford University. Initial studies were an adjunct to the main focus of the laboratory on pyruvate dehydrogenase deficiency, and involved assay of complexes I+III, II+III, IV and V in crude fibroblast mitochondrial preparations. A major expansion of the RC work was begun in the early 1990s, with introduction of the newer assays for individual enzyme complexes (Table 2) and skeletal muscle

Alternative approaches to developing reference ranges

Most centers define normal ranges for RC enzymes using enzyme ratios, but it is often not practical to match patients and controls exactly for all parameters that could potentially affect RC enzymes such as age, biopsy site, type of anesthesia, fresh or frozen samples and tissue pathology. We have attempted to address this problem by comparing the extent of variation in activities and ratios for RC complexes I, II, II+III, III and IV in five different types of sample groups: (i) ‘normal’

Reference ranges and diagnostic criteria

Reference ranges quoted by other international centers vary widely depending on the methods used, the type of normal controls, and the method of expressing activity or enzyme ratios. For example, some centers quote normal ranges based on mean±2 SD giving lower limits for normal skeletal muscle complex I activity of 24% (Morris et al., 1996), 25% (Zheng et al., 1990), 28% (Pitkanen et al., 1996) and 51% (Birch-Machin et al., 1994). Others quote observed ranges with lower limits for complex I or

Acknowledgements

This work was supported by a Senior Research Fellowship (DT) and grants from the National Health and Medical Research Council (NHMRC) of Australia as well as grants from the Muscular Dystrophy Association (MDA) of USA.

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Respiratory chain enzyme analysis in muscle and liver

Abstract

Section snippets

‘Atypical’ patients are the rule in pediatrics

The ideal approach to respiratory chain enzyme diagnosis

The Melbourne experience: history and demographics

Alternative approaches to developing reference ranges

Reference ranges and diagnostic criteria

Acknowledgements

Am. J. Pathol.

Biochem. Med. Metab. Biol.

Clin. Chim. Acta.

Lancet

J. Biol. Chem.

Lancet Neurol.

Biochim. Biophys. Acta

Lethal infantile mitochondrial disease with isolated complex I deficiency in fibroblasts but with combined complex I and IV deficiencies in muscle

Neurology

Diagnostic criteria for respiratory chain disorders in adults and children