| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Endocrinology and Metabolism |
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 IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. 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
.
|
|
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
.
|
|
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
) 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.
|
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 2
). We measured malate and succinate oxidation rates in the presence of pyruvate or acetylcarnitine (Fig. 2
).
|
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 1
) 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 2
), 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 1
). 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. 1
). 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. 1
), 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 1
), 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 3
), 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 3
). 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 2
). 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
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 |
|---|
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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
subunit gene (PDHA1) in patients with a pyruvate dehydrogenase complex deficiency. Hum Mutat 2000;15:209-219.[CrossRef][Web of Science][Medline]
[Order article via Infotrieve]The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  A. I. Jonckheere, M. Huigsloot, A. J.M. Janssen, A. J.H. Kappen, J. A.M. Smeitink, and R. J.T. Rodenburg High-Throughput Assay to Measure Oxygen Consumption in Digitonin-Permeabilized Cells of Patients with Mitochondrial Disorders Clin. Chem., March 1, 2010; 56(3): 424 - 431. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. I Jonckheere, M. Hogeveen, L. Nijtmans, M. van den Brand, A. Janssen, H. Diepstra, F. van den Brandt, B. van den Heuvel, F. Hol, T. Hofste, et al. A novel mitochondrial ATP8 gene mutation in a patient with apical hypertrophic cardiomyopathy and neuropathy BMJ Case Reports, January 22, 2009; 2009(jan21_1): bcr0720080504 - bcr0720080504. [Abstract] [Full Text]
|
|
|
|
 |
|
 S. B. Wortmann, R. J. T. Rodenburg, A. Jonckheere, M. C. de Vries, M. Huizing, K. Heldt, L. P. van den Heuvel, U. Wendel, L. A. Kluijtmans, U. F. Engelke, et al. Biochemical and genetic analysis of 3-methylglutaconic aciduria type IV: a diagnostic strategy Brain, January 1, 2009; 132(1): 136 - 146. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A I Jonckheere, M Hogeveen, L G J Nijtmans, M A M van den Brand, A J M Janssen, J H S Diepstra, F C A van den Brandt, L P van den Heuvel, F A Hol, T G J Hofste, et al. A novel mitochondrial ATP8 gene mutation in a patient with apical hypertrophic cardiomyopathy and neuropathy J. Med. Genet., March 1, 2008; 45(3): 129 - 133. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. J.M. Janssen, F. J.M. Trijbels, R. C.A. Sengers, J. A.M. Smeitink, L. P. van den Heuvel, L. T.M. Wintjes, B. J.M. Stoltenborg-Hogenkamp, and R. J.T. Rodenburg Spectrophotometric Assay for Complex I of the Respiratory Chain in Tissue Samples and Cultured Fibroblasts Clin. Chem., April 1, 2007; 53(4): 729 - 734. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Carrozzo, C. Dionisi-Vici, U. Steuerwald, S. Lucioli, F. Deodato, S. Di Giandomenico, E. Bertini, B. Franke, L. A. J. Kluijtmans, M. C. Meschini, et al. SUCLA2 mutations are associated with mild methylmalonic aciduria, Leigh-like encephalomyopathy, dystonia and deafness Brain, March 1, 2007; 130(3): 862 - 874. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Morava, L. van den Heuvel, F. Hol, M. C. de Vries, M. Hogeveen, R. J. Rodenburg, and J.A.M. Smeitink Mitochondrial disease criteria: Diagnostic applications in children Neurology, November 28, 2006; 67(10): 1823 - 1826. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
