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Clinical Chemistry 52: 860-871, 2006. First published March 16, 2006; 10.1373/clinchem.2005.062414
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(Clinical Chemistry. 2006;52:860-871.)
© 2006 American Association for Clinical Chemistry, Inc.

Endocrinology and Metabolism

Measurement of the Energy-Generating Capacity of Human Muscle Mitochondria: Diagnostic Procedure and Application to Human Pathology

Antoon J.M. Janssen1, Frans J.M. Trijbels1, Rob C.A. Sengers1, Liesbeth T.M. Wintjes1, Wim Ruitenbeek1, Jan A.M. Smeitink1, Eva Morava1, Baziel G.M. van Engelen2, Lambert P. van den Heuvel1 and Richard J.T. Rodenburg1,a

1 Department of Pediatrics and Laboratory of Pediatrics and Neurology, the Nijmegen Centre for Mitochondrial Disorders (NCMD), and 2 Department of Neurology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.

aAddress correspondence to this author at: Laboratory of Pediatrics and Neurology, UMC St. Radboud, Geert Grooteplein zuid 10, 6525 GA Nijmegen, The Netherlands. Fax 31-24-3618900; e-mail R.Rodenburg{at}cukz.umcn.nl.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Diagnosis of mitochondrial disorders usually requires a muscle biopsy to examine mitochondrial function. We describe our diagnostic procedure and results for 29 patients with mitochondrial disorders.

Methods: Muscle biopsies were from 43 healthy individuals and 29 patients with defects in one of the oxidative phosphorylation (OXPHOS) complexes, the pyruvate dehydrogenase complex (PDHc), or the adenine nucleotide translocator (ANT). Homogenized muscle samples were used to determine the oxidation rates of radiolabeled pyruvate, malate, and succinate in the absence or presence of various acetyl Co-A donors and acceptors, as well as specific inhibitors of tricarboxylic acid cycle or OXPHOS enzymes. We determined the rate of ATP production from oxidation of pyruvate.

Results: Each defect in the energy-generating system produced a specific combination of substrate oxidation impairments. PDHc deficiencies decreased substrate oxidation reactions containing pyruvate. Defects in complexes I, III, and IV decreased oxidation of pyruvate plus malate, with normal to mildly diminished oxidation of pyruvate plus carnitine. In complex V defects, pyruvate oxidation improved by addition of carbonyl cyanide 3-chlorophenyl hydrazone, whereas other oxidation rates were decreased. In most patients, ATP production was decreased.

Conclusion: The proposed method can be successfully applied to the diagnosis of defects in PDHc, OXPHOS complexes, and ANT.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The human mitochondrion contains at least 100 different proteins directly involved in the mitochondrial energy-generating system (MEGS)1 (1). MEGS enzymes are localized in the mitochondrial matrix space [pyruvate dehydrogenase complex (PDHc) and tricarboxylic acid (TCA) cycle] and in the mitochondrial inner membrane [oxidative phosphorylation (OXPHOS) complex]. Oxidation of pyruvate or fatty acids yields acetyl-CoA, which is oxidized in the TCA cycle, yielding NADH and FADH2, which in turn are oxidized by the respiratory chain (RC) and complex V to yield ATP (2). Complete oxidation of 1 mole of pyruvate delivers 15 moles of ATP. The adenine nucleotide translocator (ANT) transports ATP out of the mitochondrion. Deficiencies have been found in all OXPHOS complexes, PDHc, the TCA cycle (3), and ANT. Pathogenic mutations have been identified in the nuclear-encoded structural genes for complex I, II, and III and PDHc (4)(5)(6)(7)(8)(9)(10)(11), and in the mitochondrial DNA (mtDNA) (12). Here we describe a diagnostic procedure to examine the MEGS in detail by measurement of substrate oxidation rates and ATP production rates in intact mitochondria from a muscle biopsy.

Although fibroblasts or lymphocytes can be used for measurement of MEGS capacity, a muscle biopsy is preferred because a deficiency in muscle tissue is not always seen in other cell types (2). The reverse situation is also possible: one of our patients with an mtDNA ND6 mutation and complex I deficiency in fibroblasts showed normal complex I activities in muscle and liver tissue (13). The MEGS capacity can be measured either by oxygen consumption assays in isolated mitochondria (14), permeabilized single muscle fibers (14), and in cultured fibroblasts (8), or by measuring 14CO2 production rates from oxidation of [1-14C]pyruvate and carboxyl-14C–labeled TCA cycle intermediates. We developed a unique set of incubations with 3 carboxyl-14C–labeled substrates, in combination with measurement of ATP production, in intact muscle mitochondria that gives maximum information about the MEGS capacity. Control values were obtained from muscle tissue of 43 healthy individuals. The results from muscle biopsies from 29 patients with deficiencies in PDHc and OXPHOS enzymes illustrate the rationale of our approach.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
materials
[1-14C]Sodium pyruvate (0.4–1.1 GBq/mmol) and L-[1,4(2,3)-14C]malate ([U-14C]malate; 1.5–2.3 GBq/mmol) were obtained from Amersham Life Sciences. [1,4-14C]Succinate (0.55–1.11 GBq/mmol) and carbonyl cyanide 3-chlorophenyl hydrazone (CCCP) were from ICN. Sodium pyruvate, acetyl-D,L-carnitine hydrochloride, L-malate, L-carnitine hydrochloride, creatine, sodium-m-arsenite, atractyloside, and p1,p5-di(adenosine-5') pentaphosphate were from Sigma. Succinate and malonate were from Fluka, and ADP was from Roche. All other chemicals were of the highest purity commercially available. Glass incubation vials (20 mL) with injection caps and rubber septa, hydroxide of hyamine 10-X (hyamine), and Insta Fluor were from Packard BioScience.

collection of muscle biopsies
Musculus semitendinosus samples were obtained after informed consent from 22 otherwise healthy individuals undergoing arthroscopic anterior cruciate ligament reconstruction using semitendinosus tendon; specimens were scraped from the semitendinosus tendon. Musculus quadriceps samples from 6 healthy individuals were obtained after informed consent by needle biopsy under local anesthesia of the skin with lidocaine. Because lidocaine can influence some OXPHOS enzymes (15)(16), a minimal dose of lidocaine was used and biopsies were taken at some distance from the incision. Control muscle samples [musculus quadriceps from 7 children (age range, 2–11 years) and 8 adults] were taken surgically from patients with minimal suspicion of a mitochondrial myopathy, definitively excluded in subsequent clinical examinations. Substrate oxidation rates, ATP production, and OXPHOS enzyme activities were within the values measured in healthy individuals. Fiber typing and enzyme histochemical studies revealed no differences between musculus semitendinosus and musculus quadriceps. Musculus quadriceps samples from patients (needle or open biopsies) were taken as described above. For all patients and controls, the muscle biopsy used for this study was the only biopsy that was taken.

homogenization of muscle tissue
After biopsy, muscle tissue was immediately put in ice-cold SETH buffer (0.25 mol/L sucrose, 2 mmol/L EDTA, 10 mmol/L Tris, 5 x 104 IU/L heparin, pH 7.4) and transported to the laboratory within 3 h. Fat and connective tissue were removed. Muscle tissue was minced with a Sorvall TC2 tissue chopper, homogenized in SETH buffer, and centrifuged at 600g (17). A portion of the 600g supernatant was used for measuring oxidation rates and ATP production rates. The remaining 600g supernatant was frozen in 100-µL aliquots in liquid nitrogen and kept at –80 °C for enzymatic measurements. Citrate synthase (CS) activity was measured according to Srere (18) with minor modifications. Protein concentrations were measured according to Lowry et al. (19) with minor modifications.

incubations
Incubations were performed in a shaking water-bath at 37 °C in 20-mL glass incubation vials closed with injection caps and rubber septa. The vials for measuring 14C-labeled substrate oxidations contained a small plastic tube with 0.2 mL of hyamine. The incubation time was 20 min. Incubation volume was 0.5 mL, containing 30 mmol/L potassium phosphate, 75 mmol/L potassium chloride, 8 mmol/L Tris, 1.6 mmol/L EDTA, 5 mmol/L MgCl2, 0.2 mmol/L p1,p5-di(adenosine-5') pentaphosphate (myo-adenylate kinase inhibitor), and where indicated, 2.0 mmol/L ADP, 1 mmol/L pyruvate, 1 mmol/L malate, 1 mmol/L succinate, (with or without) 8.3 kBq of [1-14C]pyruvate, (with or without) 8.3 kBq of [U-14C]malate, (with or without) 8.3 kBq of [1,4-14C]succinate, 5 mmol/L L-carnitine, 2 mmol/L acetyl-D,L-carnitine, 2 mmol/L sodium arsenite, 5 mmol/L malonate, 2 µmol/L CCCP, and 10 µmol/L atractyloside, pH 7.4. To regenerate ADP by creatine kinase in the 600g supernatant, 20 mmol/L creatine was added to all ADP-containing incubations. [1-14C]Pyruvate solution was made fresh, and the purity was checked by comparing the concentrations measured by radioactivity counting and an enzymatic assay with lactate dehydrogenase and NADH.

Oxidation rates of [U-14C]malate were measured in the presence of malonate [inhibitor of succinate dehydrogenase (SDH)] to prevent the oxidation of [2,3-14C]malate to proceed beyond 1 TCA cycle. The end product, [2,3-14C]succinate, is transported from the mitochondrion and does not interfere with the substrate oxidation reactions. ATP production was measured in incubations containing pyruvate, malate, creatine, and ADP, in both the absence and presence (blank reaction) of arsenite. Incubations were started with 50 µL of 600g supernatant and stopped by addition of 0.2 mL of 3 mol/L perchloric acid through the rubber septum via a hypodermic syringe. Incubations were kept on ice for 1 h to trap the 14CO2 in the hyamine. The hyamine was mixed with 5 mL of Insta Fluor and counted in a Wallac 1400 LSC. Incubations for ATP production measurements were kept on ice for 15 min and then centrifuged (5 min at 14 000g and 2 °C) in an Eppendorf 5402 centrifuge, after which 0.5 mL of the supernatant was neutralized with 0.6 mL of ice-cold 1 mol/L KHCO3. The mixtures were kept on ice for 15 min and frozen at –20 °C.

atp and phosphocreatine measurements
Samples were thawed, put on ice for 5 min, and centrifuged (2 min at 14 000g and 2 °C) in an Eppendorf 5402 centrifuge. ATP and phosphocreatine (CrP) were measured in the supernatant according to the method of Lamprecht et al. (20) with minor modifications.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The results of the biochemical examination of control muscles are given in Table 1 . The intraassay variation (CV) for substrate oxidation rates and ATP production was determined by assaying 4 different 600g supernatants prepared from the same muscle biopsy (Table 1 ). The CV for the CS activity measurement was 7.6%. Substrate oxidation rates and the ATP production were expressed on CS base. The influence of an acetyl-CoA trap on the oxidation rate of [1-14C]pyruvate, of an acetyl-CoA donor on the oxidation rate of [U-14C]malate, and of malonate on the oxidation rate of [U-14C]malate + pyruvate and ATP production is given in Table 2 .


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  		Table 1. Oxidation rates of [1-14C]pyruvate, [U-14C]malate, and [1,4-14C]succinate; ATP production rate from the oxidation of pyruvate + malate; incubation ratios; and intraassay variations in control muscle biopsies.1


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  		Table 2. Substrate oxidation experiments in control muscle biopsies testing the influence of malonate on the malate oxidation rate and ATP production rate, and the effect of cosubstrates on the malate and pyruvate oxidation rates.1

We tested the validity of the methods by examining muscles of 29 patients with deficiencies in either one of the OXPHOS complexes, PDHc, or ANT. In 24 patients, the genetic defect was established. An overview of these patients is given in Table 3 , and results of the biochemical examinations are shown in Table 4 .


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  		Table 3. Genetic and clinical data of the 29 patients examined in this study.5


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  		Table 3A. Continued.5


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  		Table 4. Substrate oxidation rates, ATP production rate, and incubation ratios in patients with an RC deficiency, PDHc deficiency, complex V deficiency, and ANT deficiency.1


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  		Table 4A. Continued


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Any disturbance of the MEGS, apart from complex II deficiency, will lead to a lower pyruvate oxidation rate and ATP production. We measured pyruvate oxidation in the presence of malate or carnitine, which was added to remove acetyl-CoA (Fig. 1 ) to prevent inhibition of PDHc by accumulation of its product. PDHc is regulated by the ATP/ADP, NADH/NAD+, and acetyl-CoA/CoA ratios (2). Therefore, a defect in the TCA cycle or RC will lead to a decreased oxidation rate for [1-14C]pyruvate + malate as the result of an increase in the acetyl-CoA/CoA or NADH/NAD+ ratio, respectively. A disturbance in complex V or ANT leads to a decreased oxidation rate for [1-14C]pyruvate + malate as the result of an increase in the NADH/NAD+ or ATP/ADP ratio, respectively. In incubation 2, in which acetyl-CoA is removed by carnitine acetyltransferase, 1 mole of pyruvate oxidized yields 1 mole of NADH, whereas incubation 1 yields 4 moles of NADH and 1 mole of FADH2 (Fig. 1 ). In case of an OXPHOS or ANT deficiency, the oxidation rate in incubation 2 will be less decreased than in incubation 1, giving an increased ratio of incubation 2 to incubation 1. A PDHc deficiency will lead to equally diminished oxidation rates in incubations 1 and 2, and therefore the ratio of incubation 2 to incubation 1 remains ~1. Incubation 3 is performed to determine the ADP stimulation factor (ratio of incubation 1 to incubation 3), a measure for the coupling state of oxidation and phosphorylation in mitochondria. In incubation 4, addition of CCCP makes the pyruvate oxidation independent from ADP, complex V, and ANT. A deficiency in complex V or ANT will lead to a higher oxidation rate in incubation 4 than in incubation 1 and therefore an increased incubation 4/incubation 1 ratio. We found normal PDHc activities in 52 patients with normal oxidation rates in incubation 2 and 99 patients with normal oxidation rates in incubation 4, all showing decreased oxidation rates in incubation 1 (data not shown), which indicates that a normal oxidation rate in incubation 2 or 4, combined with a decreased oxidation rate in incubation 1, excludes a PDHc deficiency.


Figure 1
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  		Figure 1. Schematic representation of the oxidation of [1-14C]pyruvate in the presence of ADP and with either malate or carnitine as acetyl-CoA acceptor.

Malate is converted into oxaloacetate, which subsequently traps acetyl-CoA by the CS-catalyzed formation of citrate. In the presence of carnitine, acetyl-CoA is converted into acetylcarnitine and Co-A (CoA-SH) by the mitochondrial enzyme carnitine-acetyltransferase, which is endogenously present in sufficient amounts in muscle tissue. In addition, the production of ATP from the oxidation of pyruvate is shown. CI, CII, CIII, CIV, and CV, complexes I, II, III, IV, and V, respectively.

Incubation 5 measures the pyruvate oxidation rate in the presence of atractyloside. In the absence of exogenous ADP (incubation 3), the oxidation of pyruvate is dependent on endogenous ADP inside the mitochondrion. Because ATP cannot be transported through the mitochondrial membrane, atractyloside will further inhibit this residual substrate oxidation, giving a high ATP/ADP ratio and feedback inhibition of PDHc. In case of an ANT deficiency, addition of atractyloside will have little or no effect on this oxidation rate. A decreased oxidation rate in incubation 1 and equal oxidation rates in incubations 3 and 5, which gives a decreased incubation 3/incubation 5 ratio, are indicative of an ANT deficiency. Oxidation of malate and succinate strongly depends on an acetylCoA donor. Without pyruvate, the oxidation rate of [U-14C]malate is only 6% of that with pyruvate (Table 2Up ). We measured malate and succinate oxidation rates in the presence of pyruvate or acetylcarnitine (Fig. 2 ).


Figure 2
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  		Figure 2. Schematic representation of the oxidation of [U-14C]malate in the presence of either pyruvate or acetylcarnitine as acetyl-CoA donor.

Inhibition sites of arsenite (//1) on PDHc and 2-ODHc and malonate (//2) on SDH are indicated. CoA-SH, coenzyme A.

Incubation 6 measures the TCA cycle activity except for SDH and fumarase. Disturbances in PDHc, TCA cycle (except SDH and fumarase), OXPHOS, and ANT will produce a decreased oxidation rate in incubation 6. In theory, during oxidation of 1 mole of pyruvate, 1 mole of malate is oxidized, yielding twice as much 14CO2 in incubation 6 as in incubation 1. However, the ratio of incubation 6 to incubation 1 is lower (Table 1Up ) because of partial transport of 2-oxoglutarate out of the mitochondrion (A. Janssen, unpublished observations), thereby decreasing the amount of 14CO2 formed. Malonate, added to incubations 6 and 7, prevents FADH2 production by SDH, producing, in theory, 13 instead of 15 moles of ATP from oxidation of 1 mole of pyruvate. We found that addition of malonate indeed lowered the amount of ATP produced to 87% of the amount produced in the absence of malonate (Table 2Up ), indicating that malonate specifically inhibits SDH in our incubations.

Incubation 7 yielded 2 moles of 14CO2 from oxidation of 1 mole of [U-14C]malate. Incubation 8 yielded only 1 mole of 14CO2 because 2-oxoglutarate dehydrogenase complex (2-ODHc) is inhibited by arsenite, giving a incubation 7/incubation 8 ratio of ~2 (Table 1Up ). In case of a 2-ODHc deficiency, this ratio will be close to 1 because the reaction will not proceed beyond the formation of 2-oxoglutarate (Fig. 1Up ). Incubations 7 and 8 are performed in the presence of acetylcarnitine, as acetyl-CoA donor, and are therefore independent on PDHc. By contrast, incubation 6 is dependent on PDHc. Therefore, a PDHc deficiency will lead to an increased ratio of incubation 7 to incubation 6.

Because [U-14C]malate oxidation rates are independent of SDH and fumarase (Fig. 1Up ), we included incubation 9, in which 14CO2 production from oxidation of succinate is measured. A decreased oxidation rate of [1,4-14C]succinate combined with moderately decreased or normal oxidation rates of [U-14C]malate is indicative of an SDH or fumarase deficiency. The ATP production and ATP/pyruvate ratio are a measure of the efficiency of the total MEGS. In theory, the maximum value for the ATP/pyruvate ratio is 15. In practice, we obtained a mean value of 9.8 (Table 1Up ), which is most likely attributable to export of 2-oxoglutarate and succinate from the mitochondria.

The majority of complex-I–deficient patients had decreased substrate oxidation rates and ATP production. The ratio of incubation 2 to incubation 1 was increased in 11 of 12 patients. In patients 4, 5, and 8, the ratio of incubation 4 to incubation 1 was increased, suggesting that CCCP inhibits the proton-motive activity of complex I in these patients. In 9 of 11 complex-I–deficient patients, the ratio of incubation 7 to incubation 6 was increased, which is indicative of decreased PDHc activity. We measured PDHc activity in 7 of these patients and found that it was decreased in 5 patients [patients 1 (80% of the lowest control value), 2 (83%), 5 (74%), 6 (80%), and 12 (68%)]. In 6 of 10 patients, the ratio of incubation 7 to incubation 8 was decreased, indicative of decreased 2-ODHc activity. We found decreased 2-ODHc activity in 2 of these patients: patient 2 (49% of the lowest control value) and patient 11 (47% of the lowest control value). Complex I deficiency probably leads to a high NADH/NAD+ ratio, inhibiting PDHc and 2-ODHc (2). Complex-I–deficient patients 3, 4, and 5, who carry the same mutation, showed variability in clinical phenotype correlating with the biochemical phenotype (Table 3Up Up ), suggesting that additional factors influence the phenotype of these patients. Patient 9 showed normal substrate oxidation rates and ATP production, but diminished complex I activity (Table 3Up Up ). The mtDNA A10750G mutation in the MT-ND4L2 gene has been described as a polymorphism (21). Our data seem to support this, because the MEGS capacity is not affected, although the diminished complex I activity in muscle tissue still needs to be elucidated.

This study includes 2 genetically uncharacterized complex-II–deficient patients with dissimilar biochemical characteristics. Patient 13 had a complex II deficiency in muscle tissue and cultured fibroblasts. Succinate cytochrome c oxidoreductase (complex II + III) (SCC) activity was decreased in muscle tissue (60% of the lowest control value) and borderline in cultured fibroblasts (98% of the lowest control value). She had normal ATP production, borderline oxidation rate in incubation 1, and normal oxidation rates for all other substrates. This is compatible with the observation that blocking of complex II with malonate had little or no effect on the oxidation rate of malate + pyruvate and ATP production (Table 2Up ). We have no explanation for the normal oxidation rate obtained in incubation 9. Patient 14 had a complex II deficiency in muscle tissue and cultured fibroblasts. SCC activity in muscle tissue and cultured fibroblasts was decreased (13% and 46% of the lowest control value, respectively). The oxidation rates for incubations 1, 2, 6, 7, and 8, and ATP production ranged from 19% to 41% of the mean control value, whereas the oxidation rate of incubation 9 was strongly decreased (9% of the mean control value).

Patient 15, with a complex III deficiency attributable to a mutation in the MT-CYB gene, had decreased substrate oxidation rates and disturbed incubation 1/incubation 2, incubation 7/incubation 6, and incubation 7/incubation 8 ratios. The mutation has been described by Valnot et al. (22). The increased ratio of incubation 4 to incubation 1 in patient 15 indicates that CCCP inhibits the proton-motive activity of complex III in this patient, similar to complex I in patients 4, 5, and 8, who have complex I deficiency. Patient 16, with a complex IV deficiency attributable to a SURF1 mutation, displayed a relatively mild biochemical phenotype with oxidation rates and ATP production around 60% of the mean control values and normal incubation 2/incubation 1, incubation 7/incubation 6, and incubation 7/incubation 8 ratios.

Seven patients with a PDHc deficiency (PDHA1 gene mutation) were studied. The gene encoding the PDHc E1{alpha} subunit is located on the X chromosome. Although the affected females included in this study are heterozygous, they showed features of a PDHc deficiency attributable to X-linked inactivation (9). In all patients, the oxidation rates in incubations 1, 2, and 4 and the ATP production were decreased, with a normal incubation 2/incubation 1 ratio. Six patients had decreased oxidation rates in incubation 6 and an increased incubation 7/incubation 6 ratio. Therefore, decreased oxidation rates in all pyruvate-containing incubations, with a normal incubation 2/incubation 1 ratio and an increased incubation 7/incubation 6 ratio are indicative of a PDHc deficiency.

Three patients with complex V deficiency (Leigh/NARP T8993G/C mutation in the MT-ATP6 gene) showed a decreased oxidation rate in incubation 1, decreased ATP production, decreased oxidation rate in incubation 6 in patients 24 and 26, and an increased incubation 4/incubation 1 ratio in patients 25 and 26. The decreased oxidation rates are in agreement with observations in fibroblasts and platelets from patients carrying a T8993G/C mutation (23)(24). In 2 patients, the ratio of incubation 2 to incubation 1 was increased, indicating diminished RC activity. Three patients with ANT deficiency had decreased oxidation rates in incubation 1 and 2 and decreased ATP production, with an increased incubation 4/incubation 1 ratio. In 2 patients, the oxidation rates in incubations 6 and 7 were decreased. In patients 28 and 29, the ratio of incubation 3 to incubation 5 was decreased. This indicates that the combination of decreased oxidation rates and decreased ATP production, with an increased incubation 4/incubation 1 ratio and a decreased incubation 3/incubation 5 ratio, are indicative of an ANT deficiency.

A muscle biopsy is an invasive, uncomfortable procedure; it is therefore important to obtain maximal information from the biochemical examinations. A fresh muscle biopsy allows for measurement of both the MEGS capacity and individual enzyme activities. In each patient, 10 oxidation rates are measured and 8 ratios are calculated. From a statistical point of view, one of these values could be outside the 95% confidence interval. One deviating value, not fitting in the total biochemical picture, is not considered proof of mitochondrial dysfunction. Substrate oxidation rates and ATP production are always evaluated in the context of the total biochemical picture, whereas ratios are used as an additional diagnostic tool (25). We calculated 216 ratios in 29 patients. Six did not fit in our theory (ATP/pyruvate ratio in patients 5, 11, and 21; ratio of incubation 3 to incubation 5 in patients 3, 11, and 12). Although ratios are derived values and should not be regarded as diagnostic in their own right, this illustrates that the results agree with our theory about the substrate oxidations. We have observed decreased oxidation rates for one or more substrates and decreased ATP production rates in ~50% of the fresh muscle biopsies examined in our laboratory. In ~30% of these biopsies, the measured activities for all OXPHOS enzymes and PDHc have been normal. In a subset of these patients, the primary defect is probably caused by disturbances in uncharacterized proteins directly involved in the MEGS. The mitochondrial carrier proteins are among the likely candidates. Recently, the first patient with a pyruvate carrier deficiency was described (26). A small subset of patients in which we found secondary MEGS dysfunction are those with non-MEGS diagnoses, including spinal muscle atrophy, Duchenne muscular dystrophy, and Rett, Cockayne, and Joubert syndromes (27). This illustrates that decreased substrate oxidation rates and ATP production rate are not enough to diagnose primary MEGS dysfunction and that at definite diagnosis can be achieved only by combining biochemical, clinical, metabolic, and morphologic data (25). The method has been used in our center for many years and has demonstrated its merit in the diagnosis of patients suffering from a mitochondriopathy.


   Footnotes
 
1 Nonstandard abbreviations: MEGS, mitochondrial energy-generating system; PDHc, pyruvate dehydrogenase complex; TCA, tricarboxylic acid; OXPHOS, oxidative phosphorylation; RC, respiratory chain; ANT, adenine nucleotide translocator; mtDNA, mitochondrial DNA; CCCP, carbonyl cyanide 3-chlorophenyl hydrazone; CS, citrate synthase; SDH, succinate dehydrogenase; CrP, phosphocreatine; 2-ODHc, 2-oxoglutarate dehydrogenase complex; and SCC, succinate cytochrome c oxidoreductase (complex II + III).

2 Human genes: MT-ND4L, mitochondrially encoded NADH dehydrogenase 4L; MT-CYB, mitochondrially encoded cytochrome b; SURF1, surfeit 1; PDHA1, pyruvate dehydrogenase (lipoamide) alpha 1; MT-ATP6, mitochondrially encoded ATP synthase 6.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Taylor SW, Fahy E, Ghosh SS. Global organellar proteomics. Trends Biotechnol 2003;21:82-88.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  2. Janssen AJM, Smeitink JAM, van den Heuvel LP. Some practical aspects of providing a diagnostic service for respiratory chain defects. Ann Clin Biochem 2003;40:3-8.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  3. De Meirleir L. Defects of pyruvate metabolism and the Krebs cycle. J Child Neurol 2002;17:26-33.
  4. Triepels RH, Hanson BJ, van den Heuvel LPWJ, Sundell L, Marusich MF, Smeitink JAM, et al. Human complex l defects can be resolved by monoclonal antibody analysis into distinct subunit assembly patterns. J Biol Chem 2001;276:8892-8897.[Abstract/Free Full Text]
  5. Bénit P, Slama A, Cartault F, Giurgia I, Chretien D, Lebon S, et al. Mutant NDUFS3 subunit of mitochondrial complex l causes Leigh syndrome. J Med Genet 2004;41:14-17.[Abstract/Free Full Text]
  6. Kirby DM, Salemi R, Sugiana C, Othake A, Parry L, Bell KM, et al. NDUFS6 mutations are a novel cause of lethal neonatal mitochondrial complex l deficiency. J Clin Invest 2004;114:837-845.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  7. Parfait B, Chretien D, Rötig A, Marsac C, Munnich A, Rustin P. Compound heterozygous mutations in the flavoprotein gene of the respiratory chain complex II in a patient with Leigh syndrome. Hum Genet 2000;106:236-243.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  8. Rustin P, Chretien D, Bourgeron T, Gérard B, Rötig A, Saudubray JM, et al. Biochemical and molecular investigations in respiratory chain deficiencies. Clin Chim Acta 1994;228:35-51.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  9. Lissens W, De Meirleir L, Seneca S, Liebaers I, Brown GK, Brown RM, et al. Mutations in the X-linked pyruvate dehydrogenase (E1) {alpha} subunit gene (PDHA1) in patients with a pyruvate dehydrogenase complex deficiency. Hum Mutat 2000;15:209-219.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  10. Grafakou O, Oexle C, van den Heuvel L, Smeets R, Trijbels F, Goebel HH, et al. Leigh syndrome due to compound heterozygosity of dihydrolipoamide dehydrogenase gene mutations. Description of the first E3 splice site mutation. Eur J Pediatr 2003;162:714-718.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  11. Dey R, Mine M, Desguerre I, Slama A, Van Den Berghe L, Brivet M, et al. A new case of pyruvate dehydrogenase deficiency due to a novel mutation in the PDX1 gene. Ann Neurol 2003;53:273-277.[Medline] [Order article via Infotrieve]
  12. MITOMAP: a human mitochondrial genome database.http://www.mitomap.org (accessed October 1, 2005)..
  13. Ugalde C, Triepels RH, Coenen MJ, van de Heuvel LP, Smeets R, Uusima J, et al. Impaired complex l assembly in a Leigh syndrome patient with a novel missense mutation in the ND6 gene. Ann Neurol 2003;54:665-669.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  14. Sperl W, Skladal D, Gnaiger E, Wyss M, Mayr U, Hager J, et al. High resolution respirometry of permeabilized skeletal muscle fibres in the diagnosis of neuromuscular disorders. Mol Cell Biochem 1997;174:71-78.[CrossRef][Medline] [Order article via Infotrieve]
  15. Tarba C, Cracium C. A comparative study of the effects of procaine, lidocaine, tetracaine and dibucaine on the functions and ultrastructure of isolated rat liver mitochondria. Biochim Biophys Acta 1990;1019:19-28.[Medline] [Order article via Infotrieve]
  16. Tsutsumi Y, Oshita S, Kawano T, Kitahata H, Tomiyama Y, Kuroda Y, et al. Lidocaine and mexiletine inhibit mitochondrial oxidation in rat ventricular myocytes. Anesthesiology 2001;95:766-770.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  17. Fischer JC, Ruitenbeek W, Stadhouders AM, Trijbels JMF, Sengers RCA, Janssen AJM, et al. Investigation of mitochondrial metabolism in small human skeletal muscle biopsy specimens. Improvement of preparation procedure. Clin Chim Acta 1985;145:89-100.[CrossRef][Medline] [Order article via Infotrieve]
  18. Srere PA. EC 4.1.3.7, citrate oxaloacetate-lyase (CoA-acetylating). Methods Enzymol 1969;XIII:3-11.
  19. Lowry OH, Rosebrough NJ, Farr AL, Randell RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-275.[Free Full Text]
  20. Lamprecht W, Stein P, Heinz F, Weisser H. Phosphocreatine. Determination with creatine kinase, hexokinase and glucose-6-phosphate dehydrogenase. Bergmeyer HU eds. Methods of enzymatic analysis, 2nd ed 1974:1777-1781 Academic Press New York. .
  21. Crimi M, Sciacco M, Galbiati S, Bordoni A, Malferrari G, Del Bo R, et al. A collection of 33 novel human mtDNA homoplasmic variants. Hum Mutat 2002;20:409-413.[Medline] [Order article via Infotrieve]
  22. Valnot I, Kassis J, Chretien D, de Lonlay P, Parfait B, Munnich A, et al. A mitochondrial cytochrome b mutation but no mutations of nuclearly encoded subunits in ubiquinol cytochrome c reductase (complex lll) deficiency. Hum Genet 1999;104:460-466.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  23. Vázquez-Memije ME, Shanske S, Santorelli FM, Kranz-Eble P, DeVivo DC, DiMauro S. Comparative biochemical studies of ATPases in cells from patients with the T8993G or T8993C mitochondrial DNA mutations. J Inherit Metab Dis 1998;21:829-836.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  24. Carelli V, Baracca A, Barogi S, Pallotti F, Valentino ML, Montagna P, et al. Biochemical-clinical correlation in patients with different loads of the mitochondrial DNA T8993G mutation. Arch Neurol 2002;59:264-270.[Abstract/Free Full Text]
  25. Wolf NI, Smeitink JAM. Mitochondrial disorders. A proposal for consensus diagnostic criteria in infants and children. Neurology 2002;59:1402-1405.[Abstract/Free Full Text]
  26. Brivet M, Garcia-Cazorla A, Lyonnet S, Dumez Y, Nassogne MC, Slama A, et al. Impaired mitochondrial pyruvate importation in a patient and a fetus at risk. Mol Genet Metab 2003;78:186-192.[CrossRef][Medline] [Order article via Infotrieve]
  27. Morava E, Dinopoulos A, Kroes HY, Rodenburg RJ, van Bokhoven H, van den Heuvel LP, et al. Mitochondrial dysfunction in a patient with Joubert syndrome. Neuropediatrics 2005;36:214-217.[Medline] [Order article via Infotrieve]
  28. Budde SM, van den Heuvel LP, Janssen AJ, Smeets RJ, Buskens CA, De Meirleir L, et al. Combined enzymatic complex l and lll deficiency associated with mutations in the nuclear encoded NDUFS4 gene. Biochem Biophys Res Commun 2000;275:63-68.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  29. Triepels RH, van den Heuvel LPWJ, Loeffen JLCM, Buskens CAF, Smeets RJP, Rubio Gozalbo ME, et al. Leigh syndrome associated with a mutation in the NDUFS7 (PSST) nuclear-encoded subunit of complex l. Ann Neurol 1999;45:787-790.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  30. Loeffen J, Smeitink J, Triepels R, Smeets R, Schuelke M, Sengers R, et al. The first nuclear encoded complex l mutation in a patient with Leigh syndrome. Am J Hum Genet 1998;63:1598-1608.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  31. Schuelke M, Smeitink J, Mariman E, Loeffen J, Plecko B, Trijbels F, et al. Mutant NDUFV1 subunit of mitochondrial complex l causes leukodystrophy and myoclonic epilepsy. Nat Genet 1999;21:260-261.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  32. Morava E, Sengers R, ter Laak H, van den Heuvel L, Janssen A, Trijbels F, et al. Congenital hypertrophic cardiomyopathy, cataract, mitochondrial myopathy and defective oxidative phosphorylation in two siblings with Sengers-like syndrome. Eur J Pediatr 2004;163:467-471.[Medline] [Order article via Infotrieve]
  33. Bakker HD, Scholte HR, van den Bogert C, Ruitenbeek W, Jeneson JAL, Wanders RJA, et al. Deficiency of the adenine nucleotide translocator in muscle of a patient with myopathy and lactic acidosis: a new mitochondrial defect. Pediatr Res 1993;33:412-417.[ISI][Medline] [Order article via Infotrieve]



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