Laboratory Diagnosis in Neurology, 1 Ed.

Mitochondrial Diseases

Definition of Mitochondrial Diseases

The term “mitochondrial diseases” does not apply to all disorders of metabolism in mitochondria: it refers only to impairments of the energy metabolism, particularly those of the electron transport chain (respiratory chain), and in part also to disturbances of the immediate pyruvate metabolism and the citrate cycle.


The estimated prevalence of mitochondrial diseases is 15 per 100 000 head of population.

Physiology and Etiology

Mitochondria are cellular organelles surrounded by a double membrane; inside these organelles, diverse metabolic processes take place (Fig. 17.1):

• Dehydrogenation and decarboxylation of pyruvate to acetyl CoA.

• The subsequent citrate cycle.

• Oxidative phosphorylation in the electron transport chain.

• β-Oxidation of fatty acids.

• Parts of the urea cycle.

The electron transport chain, in which the reoxidation of coenzymes is coupled to the synthesis of adenosine triphosphate (ATP)—the central energy source of the cell—consists of five enzyme complexes:

• Complex I: ubiquinone reductase.

• Complex II: succinate dehydrogenase.

• Complex III: cytochrome c oxidoreductase.

• Complex IV: cytochrome c oxidase (COX).

• Complex V: ATP synthase.

Mitochondria are inherited maternally and contain several copies of their own mitochondrial DNA (mtDNA). The mitochondrial DNA consists of a circular double strand of 16 569 base pairs; it codes for 13 polypeptides of the electron transport chain (mit genes) and also for two ribosomal RNAs and 22 transfer RNAs (syn genes). The majority of mitochondrial proteins, however, are encoded by nuclear DNA (nDNA) and are imported into the mitochondria after synthesis. Gene products of nDNA are also essential for maintaining and replicating the mtDNA. Normally, an organism contains only one mtDNA species. When a mutation occurs, a cell often contains both mutant mtDNA and wildtype mtDNA (heteroplasmy), with both being randomly distributed during cell division. As a result, one cell may receive only mutant DNA and another only wild-type DNA (segregation). Cell functions are only affected when the proportion of mutant mtDNA within a cell exceeds a critical threshold level. This explains, in part, the different manifestations of mtDNA mutations within a family.


Fig. 17.1 Mitochondrial metabolism.

PDH, pyruvate dehydrogenase Electron transport chain (respiratory chain):

Complex I

—ubiquinone reductase

Complex II

—succinate CO oxidoreductase

Complex III

—cytochrome c oxidoreductase

Complex IV

—cytochrome c oxidase (COX)

Complex V

—ATP synthase


—coenzyme Q

Cyt c

—cytochrome c

Table 17.2 Symptoms of electron transport chain defects



Central nervous system

Epilepsy, stroke-like episodes, lethargy, coma, psychosis, retardation, spasticity, dystonia, ataxia, paraparesis, myoclonus, cranial nerve defects, strabismus, nystagmus, central hypotension, cortical blindness, sensorineural loss of hearing

Peripheral nervous system

Sensorimotor axonal neuropathy


Hypotension, weakness, stress intolerance, ptosis


Retinitis pigmentosa, optic atrophy, cataract


Cardiomyopathy (usually hypertrophic), arrhythmia, impaired conduction


Liver failure (especially in infancy)


Exocrine pancreatic insufficiency

Gastrointestinal tract

Vomiting, diarrhea, villous atrophy, intestinal pseudo-obstruction


Proximal tubulopathy with aminoaciduria, focal segmental glomerulosclerosis

Endocrine system

Diabetes mellitus, diabetes insipidus, short stature, hypothalamic hypogonadism, hypothyroidism, hypoparathyroidism, adrenal dysfunction, primary ovarian dysfunction

Bone marrow

Anemia, leukopenia, pancytopenia


Pigmentation anomalies, hypertrichosis, alopecia, hair follicle anomalies

Clinical Features

Defects in the mitochondrial electron transport chain may manifest in any organ and at any age during life. They may show very variable patterns of inheritance, and they may be chronic or rapidly progressive. Table 17.2 presents an overview of possible symptoms of an electron transport chain defect. The cardinal symptoms of pyruvate dehydrogenase (PDH) deficiency include developmental delay, epilepsy, ataxia, and progressive encephalopathy.

If there are two symptoms that cannot be explained otherwise, and particularly if they belong to different organ systems, the possibility of a mitochondrial disorder should be considered.

However, mitochondrial disease can also manifest as pure myopathy. Typical constellations of clinical features have been grouped together and designated as syndromes (Table 17.3). Whereas mtDNA-coded defects causing typical syndromes are found more often in adults, mutations of nuclear genes causing variable symptoms predominate in children (Table 17.3). There is no 1:1 relationship between clinical manifestations and the underlying dysfunctions: Leigh's syndrome, for example, can be caused by a variety of defects, whereas on the other hand a particular enzyme defect can result in very different clinical features (see Tables 17.3 and 17.4) (Smeitink et al., 2001; Chinnery and Schon, 2003; DiMauro and Schon, 2003; Schmiedel et al., 2003).


Diagnosis of a mitochondrial disease requires global consideration of all symptoms and signs (Bernier et al., 2002).

Organ diagnosis. Clinical suspicion will prompt on the one hand a clinical diagnostic process appropriate to the organ in question: e. g., fundoscopy, ECG, EMG, relevant laboratory tests (e. g., blood count, transaminases, HbA1 c, hormones), or cranial MRI—the MRI is especially important because it may yield pathognomonic findings. On the other hand are the specific laboratory tests.

Plasma metabolites. Irrespective of the etiology (e. g., hypoxia, mitochondrial disease), every dysfunction in the electron transport chain causes a rise in the following (see also Fig. 17.1):

• Pyruvate (normal value: < 100 μmol/L).

• Lactate (normal value: < 1.8 mmol/L).

• Alanine (normal value: < 500 μmol/L).

Laboratories should calibrate their procedures according to the above values.

The cardinal biochemical finding of a mitochondrial disease is a rise in lactic acid in the plasma.

In lactemia, there is lactic acidosis in the blood. Even if the pH is balanced, an increased

• Anion gap (= [Na+] – ([Cl] + ([HCO3–]); normal value: < 16 mmol/L)

may indicate an increase in lactic acid. The metabolites mentioned are also elevated in cases of pyruvate dehydrogenase (PDH) deficiency. Only in the presence of electron transport chain defects are there increases in:


Table 17.4 Genetic classification of mitochondrial disorders

Genetic defect

Clinical manifestations

Mutations in mitochondrial DNA


Sporadic major deletions and duplications

• Chronic progressive external ophthalmoplegia (CPEO)

• Kearns-Sayre syndrome (KSS)

• Pearson's syndrome

• Diabetes mellitus and deafness (DAD)

• Myopathy

Point mutation of genes for structural proteins (mit mutations)—maternal inheritance

• Leber's hereditary optic atrophy (LHON)

• Myopathy

• Neuropathy with ataxia and retinitis pigmentosa (NARP)

• Leigh's syndrome (subacute necrotizing encephalopathy, SNE)

Point mutations of genes for transfer RNA (syn mutations)—maternal inheritance

• Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS)

• Myoclonic epilepsy with ragged red fibers (MERRF)

• Chronic progressive external ophthalmoplegia (CPEO)

• Myopathy

• Cardiomyopathy

• Encephalomyopathy

• Diabetes mellitus and deafness (DAD)

• Nonsyndromic deafness

Point mutations of genes for ribosomal RNA (syn mutations)—maternal inheritance

• Aminoglycoside-induced and nonsyndromic deafness

Mutations in nuclear DNA


Mutations in genes coding for structural proteins of the electron transport chain

• Autosomal recessive

– Leigh's syndrome: complex I: NDUFS 2, 4, 7, 8 or NDUFV1; complex II: flavoprotein

– macrocephaly, leukodystrophy, and myoclonic epilepsy: NDUFV1

– hypertrophic cardiomyopathy and encephalomyopathy: NDUFS 2

• Autosomal dominant: optic atrophy and ataxia:.avoprotein

Impaired assembly of electron transport chain complexes—autosomal recessive

• Leigh's syndrome: complex IV: SURF1 COX10

• Cardioencephalomyopathy: complex IV: SCO2

• Neonatal hepatopathy and encephalopathy: complex IV: SCO1

• Proximal tubulopathy, hepatopathy, and encephalopathy: complex III: Bcs1 p

Impaired mitochondrial protein import—X-linked recessive

• Deafness dystonia syndrome: deafness dystonia protein

Multiple mtDNA deletions due to nDNA mutation—autosomal dominant or recessive

• Autosomal dominant progressive external ophthalmoplegia: adenine nucleotide translocator 1 (ANT1)

• DNA polymerase gamma (POLG) or Twinkle helicase (C 10orf2)

• Myoneurogenic gastrointestinal encephalopathy (MNGIE): thymidine phophorylase (TP)

Depletion of mtDNA due to nDNA mutation—autosomal recessive

• Myopathy: thymidine kinase 2 (TK2)

• Hepatocerebral syndrome: deoxyguanosine kinase (DGUOK)

• Alpers’ syndrome: polymerase gamma (POLG)

Secondary dysfunctions of electron transport chain

• Friedreich's ataxia: frataxin

• Hereditary spastic paraplegia (SPG7): paraplegin

mtDNA, mitochondrial DNA; nDNA, nuclear DNA; NDUFS, NADH-ubiquinone oxidoreductase Fe-S protein; NDUFV, NADH-ubiquinone oxidoreductase flavoprotein; SURF, surfeit gene cluster; COX, cytochrome c oxidase; SCO, synthesis of cytochrome c oxidase; C 10orf2, chromosome 10 open reading frame 2; Bcs1 p, stabilizing protein for cytochrome bc 1 complex (complex III).

• Lactate/pyruvate ratio (normal value: < 20).

• 3-Hydroxybutyrate/acetoacetate ratio (normal value: < 3).

There are just a few mitochondrial disorders (e. g., Leber's hereditary optic neuropathy) in which the increase in lactic acid regularly fails to occur.

As to the determination of metabolites, the following apply:

• Lactate is measured by most routine laboratories. Values obtained in plasma by the enzymatic method and those obtained in whole blood by amperometric determination (lactate-sensitive electrodes) are comparable.

• Blood gas analysis and determination of electrolytes can also be done in routine laboratories.

• Pyruvate is determined enzymatically in plasma in specialized laboratories. The test usually yields little additional information and is susceptible to error.

• Alanine concentration is frequently of diagnostic value. It is measured by a specialized laboratory during chromatographic analysis of amino acids in EDTA-blood.

• The ketone bodies acetoacetate and 3-hydroxybutyrate are determined by spectrometry or chromatography. The findings are only of secondary importance.

Pitfalls in Blood Analysis

Care needs to be taken during blood collection and transport that the concentrations of the above metabolites are not secondarily falsified (Chariot et al., 1994; Carragher et al., 2003):

image Blood collection: Making a fist for more than 20 seconds clearly increases the concentration of these metabolites, and so does hyperventilation caused by pain. Contrary to commonly held opinion, brief venous stasis provides more reliable values than repeated punctures.

image Glycolysis and hemolysis: The concentration of metabolites increases if intracellular glycolysis continues after blood collection. Hemolysis leads to release of lactate dehydrogenase (LDH) and changes the lactate/pyruvate ratio in the sample. To measure lactate levels, it is sufficient to stop glycolysis with sodium fluoride. To measure pyruvate, the blood should be rapidly deproteinized; this is commonly done by mixing whole blood 1:1 (or 1:2) with 10% perchloric acid.

image Transport: For lactate measurement, the sample should ideally be cooled, but brief transportation at room temperature does no harm. For pyruvate measurement, the sample should be transported on ice and centrifuged within a short time. EDTA-blood for alanine determination should reach the laboratory within 24 hours; otherwise the plasma should be centrifuged and frozen. The same applies for determination of individual ketone bodies.

image In some cases, demonstration of elevated lactate levels requires repeated pre- and postprandial testing. Sometimes it is helpful to determine lactate (and pyruvate) after mild ergometer exercise (30 W over 15 minutes) or after glucose loading (2 g/kg body weight, max. 50 g). The latter test should only be done if postprandial lactate values are not already markedly increased! In both tests, a lactic acid increase of more than 20% or to above 2.1 mmol/L is considered pathological.

Urine metabolites. Urinary lactate is less dependent than plasma lactate on acute influences such as hyperventilation or muscle activity. Lactate is elevated when tubular reabsorption is impaired. The lactate concentration is presented relative to the creatinine concentration; it should be determined in at least three spontaneous urine samples. For preservation during shipment, 2–3 drops of chloroform should be added to each sample. Further indications are provided by measuring the mitochondrial metabolites presented as organic acids (e. g., 3-OH-butyrate, succinate, fumarate, ethylmalonic acid, 2-oxoglutaric acid) by gas chromatography–mass spectroscopy in individual urine samples.

CNS metabolites. Measuring lactate (and alanine) levels in the CSF can be useful in more cases than just where there are central nervous system symptoms. CSF values depend only slightly on acute influences and may be elevated even if blood values are normal. Elevated CSF lactate is a clear indication of a mitochondrial disease with central nervous system manifestation (Finsterer, 2001). Lactate concentrations in brain tissue can be assessed by proton MR spectroscopy. Lactic acid peaks may be present, even in areas that appear normal on MRI (Bianchi et al., 2003). However, normal brain lactate levels do not completely rule out mitochondrial disease of the central nervous system.

Molecular genetic diagnosis. Analysis of mtDNA is carried out with blood cells (EDTA-blood, shipped at room temperature). It is useful not just when a mitochondrial syndrome resulting from changes in the mtDNA is suspected (Table 17.4). As a rule, screening for deletions and duplications is performed; the number of routinely examined mtDNA point mutations varies from laboratory to laboratory. Mutant mtDNA may be distributed differently in various tissues of the body (see above) and so, if genetic diagnosis of blood cells shows no abnormalities, mtDNA analysis of other tissues may detect a mutation. The same does not hold for nDNA mutation analysis, but this is still very limited in routine analysis.

Biopsy. Muscle biopsies are the main interest. Material should be obtained for light and electron microscopy as well as for biochemical and genetic analyses. When a mitochondrial disease is suspected, a biochemical diagnosis should be attempted irrespective of any myopathological findings. If a mitochondrial disorder has already been demonstrated genetically, biochemistry may help to classify it more precisely. The biopsy technique should be discussed with the laboratory doing the tests. It makes sense to select a muscle that is moderately affected clinically. Common sampling sites with known muscle structure (e. g., vastus lateralis muscle) are advantageous for histological assessment. The tissue may be obtained by needle biopsy or open biopsy; it is usually easier to obtain sufficient material by open biopsy.

Procedure for Muscle Biopsy

image The muscle biopsy tissue must not be fixed in formalin.

image For light microscopy, biopsy material the size of a coffee bean should be frozen in liquid nitrogen. Ideally, it is first embedded in a material suitable for frozen sections. Samples are transported on dry ice.

image For electron microscopy, biopsy material the size of a rice grain should be immediately fixed in cold 3–6% glutaraldehyde solution. It may be shipped in this solution but must not be frozen.

image To determine the electron transport chain enzymes or PDH, and for genetic analysis of muscle tissue, biopsy material at least the size of a pea should be immediately frozen in liquid nitrogen, stored at −80°C, and shipped on dry ice. Some laboratories ask for fixation in isopentane prior to freezing.

image For functional biochemical studies, the biopsy material should be chilled on ice and arrive at the laboratory within 2 hours. This test requires a relatively large amount of tissue (> 300 mg).

Muscle biopsy can provide the following information:

• Ragged red fibers may be found by light microscopy in sections stained with the Gomori trichrome reaction. This is diagnostically important if they constitute more than about 1% of the muscle fibers. Ragged red fibers are subsarcolemmal aggregates of mitochondria. They are usually not detected in Leber's hereditary optic neuropathy, in Leigh syndrome, or in the syndrome called neuropathy, ataxia, and retinitis pigmentosa (NARP). Absence of ragged red fibers does not rule out a mitochondrial disorder.

• Enzyme histochemical studies (COX, ATPase) and immunohistochemical studies (antibodies against polypeptides of the electron transport chain) are carried out under the light microscope. Depending on the defect, COX activity may be reduced or increased.

• Electron microscopy may reveal unusual numbers, sizes, or shapes of mitochondria or unusual mitochondrial inclusions even before abnormalities are found by light microscopy (Sladky, 2001).

• In the biochemical analysis of frozen tissue, the electron transport chain complexes, PDH, and citrate synthase (a mitochondrial marker enzyme) can be determined individually by spectrophotometry.

• Other disorders, such as transport defects and impaired ATP synthesis, may be detected in fresh muscle tissue using functional tests. For this purpose, fresh muscle homogenate is incubated with various radioactively labeled substrates and specific inhibitors of metabolic pathways. This permits radiometric assessment of the oxidation rates of pyruvate, malate, and succinate, and of the production rates of ATP and creatine phosphate. This test enables global evaluation of the mitochondrial energy metabolism and helps to localize the dysfunction. If fresh muscle is available in the laboratory, this study is performed prior to any enzyme determination (Trijbels et al., 1993; Janssen et al., 2003).

Pitfalls in Biochemical Tissue Analysis

Abnormalities in the biochemical analysis of tissue may be caused not only by primary but also by secondary disturbances of mitochondrial function (anoxia, diabetes mellitus, medications, and other metabolic disorders).

Some of the defects can be verified in fibroblasts. For fibroblast cultures, a skin biopsy should be placed in an appropriate culture medium and then shipped in the culture medium.