Printer Friendly

Measurement of the energy-generating capacity of human muscle mitochondria: diagnostic procedure and application to human pathology.

The human mitochondrion contains at least 100 different proteins directly involved in the mitochondrial energy-generating system (MEGS) [3] (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 FAD[H.sub.2], 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 (4-11), 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 [sup.14]C[O.sub.2] production rates from oxidation of [1-[sup.14]C]pyruvate and carboxyl-[sup.14]C-labeled TCA cycle intermediates. We developed a unique set of incubations with 3 carboxyl-[sup.14]C-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

MATERIALS

[1-[sup.14]C]Sodium pyruvate (0.4-1.1 GBq/mmol) and L-[1,4(2,3)-[sup.14]C]malate ([U-[sup.14]C]malate; 1.5-2.3 GBq/mmol) were obtained from Amersham Life Sciences. [1,4-[sup.14]C]Succinate (0.55-1.11 GBq/mmol) and carbonyl cyanide 3-chlorophenyl hydrazone (CCCP) were from ICN. Sodium pyruvate, acetyl-n,L-carnitine hydrochloride, L-malate, L-carnitine hydrochloride, creatine, sodium-m-arsenite, atractyloside, and [p.sup.1],[p.sup.5]-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 [10.sup.4] 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-[micro]L aliquots in liquid nitrogen and kept at -80[degrees]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[degrees]C in 20-mL glass incubation vials closed with injection caps and rubber septa. The vials for measuring [sup.14]C-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 Mg[Cl.sub.2], 0.2 mmol/L [p.sup.1],[p.sup.5]-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-[sup.14]C]pyruvate, (with or without) 8.3 kBq of [U-[sup.14]C]malate, (with or without) 8.3 kBq of [1,4-[sup.14]C]succinate, 5 mmol/L L-carnitine, 2 mmol/L acetyl-D,L-carnitine, 2 mmol/L sodium arsenite, 5 mmol/L malonate, 2 [micro]mol/L CCCP, and 10 [micro]mol/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-[sup.14]C]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-[sup.14]C]malate were measured in the presence of malonate [inhibitor of succinate dehydrogenase (SDH)] to prevent the oxidation of [2,3-[sup.14]C]malate to proceed beyond 1 TCA cycle. The end product, [2,3-[sup.14]C]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 [micro]L 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 [sup.14]C[O.sub.2] 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[degrees]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 KHC[O.sub.3]. The mixtures were kept on ice for 15 min and frozen at -20[degrees]C.

ATP AND PHOSPHOCREATINE MEASUREMENTS

Samples were thawed, put on ice for 5 min, and centrifuged (2 min at 14 000g and 2[degrees]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

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-[sup.14]C]pyruvate, of an acetyl-CoA donor on the oxidation rate of [U-[sup.14]C]malate, and of malonate on the oxidation rate of [U-[sup.14]C]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

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.sup.+], and acetyl-CoA/CoA ratios (2). Therefore, a defect in the TCA cycle or RC will lead to a decreased oxidation rate for [1-[sup.14]C]pyruvate + malate as the result of an increase in the acetyl-CoA/CoA or NADH/[NAD.sup.+] ratio, respectively. A disturbance in complex V or ANT leads to a decreased oxidation rate for [1-[sup.14]C]pyruvate + malate as the result of an increase in the NADH/[NAD.sup.+] 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 FAD[H.sub.z] (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 acetyl-CoA donor. Without pyruvate, the oxidation rate of [U-[sup.14]C]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 [sup.14]C[O.sub.2] 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 [sup.14]C[O.sub.2] formed. Malonate, added to incubations 6 and 7, prevents FAD[H.sub.2] 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 [sup.14]C[O.sub.2] from oxidation of 1 mole of [U-[sup.14]C]malate. Incubation 8 yielded only 1 mole of [sup.14]C[O.sub.2] 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 acetylcamitine, 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-[sup.14]C]malate oxidation rates are independent of SDH and fumarase (Fig. 1), we included incubation 9, in which [sup.14]C[O.sub.2] production from oxidation of succinate is measured. A decreased oxidation rate of [1,4-[sup.14]C]succinate combined with moderately decreased or normal oxidation rates of [U-[sup.14]C]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-l- 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.sup.+] 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-ND4L [4] 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.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

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[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.

Received October 20, 2005; accepted February 22, 2006.

Previously published online at DOI: 10.1373/clinchem.2005.062414

References

(1.) Taylor SW, Fahy E, Ghosh SS. Global organellar proteomics. Trends Biotechnol 2003;21:82-8.

(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.

(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 I defects can be resolved by monoclonal antibody analysis into distinct subunit assembly patterns. J Biol Chem 2001;276:8892-7.

(5.) Benit P, Slama A, Cartault F, Giurgia I, Chretien D, Lebon S, et al. Mutant NDUFS3 subunit of mitochondrial complex I causes Leigh syndrome. J Med Genet 2004;41:14-7.

(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 I deficiency. J Clin Invest 2004;114:837-45.

(7.) Parfait B, Chretien D, Rotig 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;106:236-43.

(8.) Rustin P, Chretien D, Bourgeron T, Gerard B, Rotig A, Saudubray JM, et al. Biochemical and molecular investigations in respiratory chain deficiencies. Clin Chim Acta 1994;228:35-51.

(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;15:209-19.

(10.) Grafakou 0, 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: 714-8.

(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;53:273-7.

(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 I assembly in a Leigh syndrome patient with a novel missense mutation in the ND6 gene. Ann Neurol 2003;54:665-9.

(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;174:71-8.

(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.

(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: 766-70.

(17.) FischerJC, 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.

(18.) Srere PA. EC 4.1.3.7, citrate oxaloacetate-lyase (CoA-acetylating). Methods Enzymol 1969;X111:3-11.

(19.) Lowry OH, Rosebrough NJ, Farr AL, Randell RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-75.

(20.) Lamprecht W, Stein P, Heinz F, Weisser H. Phosphocreatine. Determination with creatine kinase, hexokinase and glucose-6-phosphate dehydrogenase. In: Bergmeyer HU, ed. Methods of enzymatic analysis, 2nd ed. New York: Academic Press, 1974: 1777-81.

(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;20:409-13.

(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 III) deficiency. Hum Genet 1999;104:460-6.

(23.) Vazquez-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;21:829-36.

(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;59:264-70.

(25.) Wolf NI, Smeitink JAM. Mitochondrial disorders. A proposal for consensus diagnostic criteria in infants and children. Neurology 2002;59:1402-5.

(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: 186-92.

(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;36:214-7.

(28.) Budde SM, van den Heuvel LP, Janssen AJ, Smeets RJ, Buskens CA, De Meirleir L, et al. Combined enzymatic complex I and III deficiency associated with mutations in the nuclear encoded NDUFS4 gene. Biochem Biophys Res Commun 2000;275: 63-8.

(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 (PSS7) nuclear-encoded subunit of complex I. Ann Neurol 1999;45:787-90.

(30.) Loeffen J, Smeitink J, Triepels R, Smeets R, Schuelke M, Sengers R, et al. The first nuclear encoded complex I mutation in a patient with Leigh syndrome. Am J Hum Genet 1998;63:1598-608.

(31.) Schuelke M, Smeitink J, Mariman E, Loeffen J, Plecko B, Trijbels F, et al. Mutant NDUFV1 subunit of mitochondrial complex I causes leukodystrophy and myoclonic epilepsy. Nat Genet 1999; 21:260-1.

(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; 163:467-71.

(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: 412-7.

ANTOON J.M. JANSSEN, [1] FRANS J.M. TRIJBELS, [1] ROB C.A. SENGERS, [1] LIESBETH T.M. WINTJES, [1] WIM RUITENBEEK, [1] JAN A.M. SMEITINK, [1] EVA MORAVA, [1] BAZIEL G.M. VAN ENGELEN [2] LAMBERT P. VAN DEN HEUVEL, [1] and RICHARD J.T. RODENBURG [1] *

[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.

[3] 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).

[4] 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.

* Address 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@cukz.umcn.nl.
Table 1. Oxidation rates of [1-.sup.14C]pyruvate, [U-.sup.14C]malate,
and [1,4-.sup.14C] succinate; ATP production rate from the oxidation
of pyruvate malate; incubation ratios; and intraassay variations in
control muscle biopsies. (a)

 Rate of oxidation

Incubation Substrate Mean (SD) Mean [+ or -] 2 SD

 1 [1-.sup.14C] 5.72 (1.15) 3.43-8.01
 Pyruvate + malate

 2 [1-.sup.14C] 6.09 (1.08) 3.92-8.26
 Pyruvate +
 carnitine

 3 [1-.sup.14C] 0.99 (0.35) 0.28-1.69
 Pyruvate +
 malate without
 ADP

 4 [1-.sup.14C] 5.69 (1.08) 3.53-7.86
 Pyruvate +
 malate-ADP +
 CCCP

 5 [1-.sup.14C] 0.50 (0.16) 0.18-0.81
 Pyruvate +
 malate-ADP +
 atractyloside

 6 [U-.sup.14C] 6.33 (1.30) 3.73-8.94
 Malate +
 pyruvate +
 malonate

 7 [U-.sup.14C] 3.93 (0.72) 2.50-5.36
 Malate +
 acetylcarnitine
 + malonate

 8 [U-.sup.14C] 2.21 (0.39) 1.43-3.00
 Malate +
 acetylcarnitine
 + arsenite

 9 [1,4-.sup.14C] 3.14 (0.55) 2.05-4.24
 Succinate +
 acetylcarnitine

 10 ATP + CrP from 56.2 (10.2) 35.9-76.5
 oxidation of
 pyruvate +
 malate

 Ratios

 ATP/Pyruvate 9.8 (1.6) 6.6-13.1
 (incubation
 10/incubation 1)

 Incubation 6.0 (1.5) 3.0-8.9
 1/Incubation 3
 (ADP stimulation)

 Incubation 1.1 (0.1) 0.8-1.3
 2/Incubation 1

 Incubation 1.0 (0.1) 0.7-1.3
 4/Incubation 1

 Incubation 2.0 (0.5) 1.1-3.0
 3/Incubation 5

 Incubation 1.1 (0.1) 0.8-1.4
 6/Incubation 1

 Incubation 0.6 (0.1) 0.4-0.8
 7/Incubation 6

 Incubation 1.8 (0.2) 1.5-2.1
 7/Incubation 8

 Rate of oxidation

Incubation Substrate Range n CV, %

 1 [1-.sup.14C] 3.45-7.99 43 7.4
 Pyruvate + malate

 2 [1-.sup.14C] 4.21-8.34 43 4.8
 Pyruvate +
 carnitine

 3 [1-.sup.14C] 0.45-2.31 43 4.9
 Pyruvate +
 malate without
 ADP

 4 [1-.sup.14C] 3.76-8.37 41 3.7
 Pyruvate +
 malate-ADP +
 CCCP

 5 [1-.sup.14C] 0.23-1.10 42 5.7
 Pyruvate +
 malate-ADP +
 atractyloside

 6 [U-.sup.14C] 3.28-8.80 42 6.3
 Malate +
 pyruvate +
 malonate

 7 [U-.sup.14C] 1.97-5.24 37 6.5
 Malate +
 acetylcarnitine
 + malonate

 8 [U-.sup.14C] 1.10-3.02 37 6.2
 Malate +
 acetylcarnitine
 + arsenite

 9 [1,4-.sup.14C] 2.03-4.18 34 6.8
 Succinate +
 acetylcarnitine

 10 ATP + CrP from 36.0-81.7 41 12
 oxidation of
 pyruvate +
 malate

 Ratios

 ATP/Pyruvate 6.6-12.1 41
 (incubation
 10/incubation 1)

 Incubation 3.5-8.5 43
 1/Incubation 3
 (ADP stimulation)

 Incubation 0.9-1.5 43
 2/Incubation 1

 Incubation 0.7-1.3 41
 4/Incubation 1

 Incubation 1.3-3.2 42
 3/Incubation 5

 Incubation 0.9-1.3 42
 6/Incubation 1

 Incubation 0.4-1.0 37
 7/Incubation 6

 Incubation 1.5-2.1 37
 7/Incubation 8

(a) Substrate oxidation rates and ATP production rates were measured
in a total of 43 control muscle samples, as described in the
Materials and Methods section, in the presence of ADP unless
indicated otherwise. Oxidation rates for [sup.14]C-labeled substrates
were calculated as nmol [sup.14]C[O.sub.2]/(h * mU CS). The ATP
production rate was calculated as nmol (ATP + CrP)/(h * mU CS).
The mean (SD) age of the controls was 28.9 (15.5) years (range,
2.2-57.6 years). The intraassay variations (CV), as described in
the Results section, are given in column 7.

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.a

Incubation Mean (SD), % n

[[U-.sup.14]C 104 (4) 9
Malate +
pyruvate without
malonate

ATP + CrP from 87 (5) 7
oxidation of
pyruvate + malate
malonate

[[U-.sup.14]C 6 (1) 4
Malate + malonate
without pyruvate

[[1.sup.-14]C 20 (3) 4
Pyruvate without
malate

(a) The [[U-.sup.14]C malate oxidation rates in the absence of either
malonate or pyruvate were related to those in the presence of
either pyruvate or malonate, which were set to 100%. The ATP
production rate in the presence of malonate was related to that in
the absence of malonate, which was set to 100%. The oxidation rate
of [[1.-sup.14]C pyruvate in the absence of malate was related to
that in the presence of malate, which was set to 100%. All
incubations were performed in the presence of ADP and as described
in the Materials and Methods section.

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

Patient (a) Observed enzyme Genetics (c)
(sex) deficiency (b)

1 (F) Complex I NDUFS2
 (Muscle, 20%; fibroblast, 26%) 1336G A (D446N)

2 (M) Complex I NDUFS4
 (Muscle, 14%; fibroblast, 60%) 316C T (premature
 stop)

3 (M) Complex I NDUFS7
 (Muscle, 23%; fibroblast, 59%) 364G A (V122M)

4 (F) Complex I NDUFS7
 (Muscle, 43%; fibroblast, 38%) 364G A (V122M)

5 (M) Complex I NDUFS7
 (Muscle, 34%; fibroblast, 24%) 364G A (V122M)

6 (M) Complex I NDUFS8 (C.H.)
 (Muscle, 29%; fibroblast, 63%) 236C T (P79L)
 305G A (R102H)

7 (M) Complex I NDUFV1 (C.H.) (d)
 (Muscle, 39%; fibroblast, 77%) 175C T (premature
 1268C T (T423M) stop)

8 (M) Complex I MT-ND2
 (Muscle, 51%; fibroblast, 37%) T4681C (L71P);
 100%) (muscle, 100%;
 fibroblast,

9 (F) Complex I MT-ND4L
 (Muscle, 66%; fibroblast, A10750G; (muscle,
 normal) 40%; blood, 40%)

10 (F) Complex I MT-ND5
 (Muscle, 43%; fibroblast, 85%) G13513A; (muscle,
 blood, 26%) 65%; fibroblast,
 25%;

11 (F) Complex I MELAS
 (Muscle, 13%; fibroblast, MELAS; (muscle, 40%;
 normal) fibroblast, 30%)

12 (F) Complex I + III mtDNA (A3243G)
 Complex I MELAS; (muscle, 81%)
 (Muscle, 18%)

 Complex III
 (Muscle, 84%)

13 (F) Complex II Genetic defect unknown
 (Muscle, 42%; fibroblast, 38%)

14 (M) Complex II Genetic defect unknown
 (Muscle, 8%; fibroblast, 10%)

15 (F) Complex III MT-CYB
 (Muscle, 7%; fibroblast, G15243A (G166E);
 normal) (muscle, 100%;
 fibroblast, 0%)

16 (F) Complex IV SURF1
 (Muscle, 35%) 312 insAT
 TCTGCCAGCC del

17 (M) PDHc PDHA1
 (Muscle, 83%) 784G > C (V262L)
 PDHc-E1
 (Muscle, 2%)

18 (F) PDHc PDHA1
 (Muscle, 68%; fibroblast, 82%) del926 AAGTAAG
 PDHc-E1
 (Muscle, 64%)

19 (F) PDHc PDHA1
 (Muscle, 89%; fibroblast, 85%) ins926 AAGTAAG
 PDHc-E1
 (Muscle, 29%)

20 (F) PDHc PDHA1
 (Muscle, 24%; fibroblast, 36%) 430G > A (G144S)

21 (F) PDHc PDHA1
 (Muscle, 51%; fibroblast, dupl859-862 (TACC)
 normal)

 PDHc-E1
 (Muscle, 31%)

22 (F) PDHc PDHA1
 (Muscle, 80%; fibroblast, 1133G > A (R378H)
 normal)
 PDHc-E1
 (Muscle, 46%)
23 (F) PDHc PDHA1
 (Muscle, 116%; fibroblast, 924G T > (Q308H)
 98%)

 PDHc-E1
 (Muscle, 81%)

24 (M) Complex V MT-ATP6
 (Muscle, 77%) T8993G; (muscle,
 >95%)

25 (F) Complex V MT-ATP6
 (Muscle, 50%) T8993G; (muscle,
 90%)

26 (F) Complex V MT-ATP6
 (Muscle, 49%) T8993C; (muscle
 >95%)

27 (M) ANT Genetic defect unknown

28 (F) ANT Genetic defect unknown

29 (M) ANT Genetic defect unknown

Patient (a) Age at biopsy (age at Clinical course
(sex) death) (d)

1 (F) 5 months (8 months) Leigh syndrome

2 (M) 4.5 months (4.5 months) Leigh syndrome

3 (M) 3.75 years (5 years) Leigh syndrome

4 (F) 40 years Spastic hemipareses;
 poor eyesight;
 blepharospasmus;
 exercise intolerance

5 (M) 2.25 years Leigh syndrome;
 progressive ataxia

6 (M) 2.3 months (2.5 months) Leigh syndrome

7 (M) 9 months (17 months) Brain atrophy; psychomotor
 retardation; myoclonic
 epilepsy

8 (M) 1.5 years (12 years) Leigh syndrome

9 (F) 46 years Exercise
 intolerance;
 migraine; disturbed
 cognitive function;
 progressive
 spasticity

10 (F) 22 years Leigh syndrome

11 (F) 20 years Muscle cramps;
 exercise intolerance;
 insufficient kidney
 function

12 (F) 21 years Psychomotor retardation;
 convulsions; sensory
 deafness

13 (F) 11 months Leigh syndrome

14 (M) 9 months Leigh syndrome

15 (F) 10.9 years Psychomotor retardation;
 exercise intolerance;
 muscle weakness;
 hypertrichosis

16 (F) 2.5 years Leigh syndrome

17 (M) 11.75 years Leigh syndrome

18 (F) 1.6 years Leigh syndrome

19 (F) 1.2 years Leigh syndrome

20 (F) 6 months Psychomotor retardation;
 hypotonia

21 (F) 1.1 years Psychomotor retardation;
 convulsions; lissencephaly

22 (F) 14.2 years Leigh syndrome

23 (F) 1.3 months Psychomotor retardation;
 convulsions; hypotonia;
 spasticity; microcephaly

24 (M) 4 years Leigh syndrome

25 (F) 10 months Leigh syndrome

26 (F) 9.7 years Leigh syndrome

27 (M) 1 year Sengers-like syndrome

28 (F) 1.7 months Sengers-like syndrome

29 (M) 5.2 years Exercise intolerance;
 motor retardation;
 slightly dystrophic

Patient (a) Reference
(sex)

1 (F)

2 (M) (28

3 (M) (29)

4 (F)

5 (M)

6 (M) (30)

7 (M) (31)

8 (M)

9 (F)

10 (F)

11 (F)

12 (F)

13 (F)

14 (M)

15 (F)

16 (F)

17 (M)

18 (F)

19 (F)

20 (F)

21 (F)

22 (F)

23 (F)

24 (M)

25 (F)

26 (F)

27 (M) (32)

28 (F) (32)

29 (M) (33)

(a) Patients 1-5 and 17 carried a homozygous mutation. Patients 6, 7,
and 16 carried a compound heterozygous mutation.

(b) Measured enzymatic activities in muscle tissue and cultured
fibroblasts are expressed as a percentage of the lowest control values.

(c) Affected genes and the mutation. In the case of a mtDNA mutation,
the percentage heteroplasmy in muscle tissue, fibroblasts, and blood,
is given. Human genes: NDUFS2, NADH dehydrogenase (ubiquinone) Fe-S
protein 2, 49kDa (NADH-coenzyme Q reductase); NDUFS4, NADH
dehydrogenase (ubiquinone) Fe-S protein 4, 18kDa (NADH-coenzyme Q
reductase); NDUFS7, NADH dehydrogenase (ubiquinone) Fe-S protein 7,
20kDa (NADH-coenzyme Q reductase); NDUFS8, NADH dehydrogenase
(ubiquinone) Fe-S protein 8, 23kDa (NADH-coenzyme Q reductase);
NDUFV1, NADH dehydrogenase (ubiquinone) flavoprotein 1, 51kDa; MT-ND2,
mitochondrially encoded NADH dehydrogenase 2; MT-ND4L, mitochondrially
encoded NADH dehydrogenase 4L; MT-ND5, mitochondrially encoded NADH
dehydrogenase 5; MELAS, mitochondrial encephalomyopathy, lactic
acidosis, and stroke-like episodes; MT-CYB, mitochondrially encoded
cytochrome b; SURF1, surfeit 1; PDHA1, pyruvate dehydrogenase
(lipoamide) alpha 1; MT-ATP6, mitochondrially encoded ATP synthase 6.

(d) Age of each patient at the time of the muscle biopsy. If the
patient died as a result of the mitochondrial disease, the age at the
time of death is given in parentheses.

(e) C. H., compound heterozygous; MELAS, mitochondrial myopathy,
encephalopathy, lactic acidosis, and stroke-like episodes; PDHc-E1,
enzymatic activity of the E1 subunit of PDHc.

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. (a)

 Oxidation rate in
 incubation:

Patient Deficiencyb (b) 1 2 3

 RC
deficiencies

 1 Complex I (NDUFS2) 16 32 26
 2 Complex I (NDUFS4) 7 13 26
 3 Complex I (NDUFS7) 19 29 40
 4 Complex I (NDUFS7) 62 94 91
 5 Complex I (NDUFS7) 19 37 31
 6 Complex I (NDUFS8) 12 23 52
 7 Complex I (NDUFV1) 16 24 49
 8 Complex I (MT-ND2) 35 72 95
 9 Complex I (MT-ND4L) 69 90 67
 10 Complex I (MT-ND5) 27 58 62
 11 Complex I (MELAS) 5 8 22
 12 Complex I (MELAS) 15 23 58
 13 Complex II 68 78 63
 14 Complex II 28 37 46
 15 Complex III (MT-CYB) 15 25 44
 16 Complex IV (SURF1) 55 69 55

PDHc deficiencies

 17 PDHc (PDHA1) 23 21 70
 18 PDHc (PDHA1) 15 15 23
 19 PDHc (PDHA1) 22 22 30
 20 PDHc (PDHA1) 12 15 18
 21 PDHc (PDHA1) 14 14 38
 22 PDHc (PDHA1) 39 39 63
 23 PDHc (PDHA1) 27 26 56

Complex V deficiencies

 24 Complex V (MT-ATP6) 14 ND 13
 25 Complex V (MT-ATP6) 18 31 34
 26 Complex V (MT-ATP6) 57 88 100

ANT deficiencies

 27 10 12 12
 28 3 3 3
 29 11 22 23

Controls

Mean (SD) 100 (20) 100 (18) 100 (35)
Range 60-140 69-137 45-233

 Oxidation rate in
 incubation:

Patient Deficiencyb (b) 4 5 6

 RC
deficiencies

 1 Complex I (NDUFS2) 14 28 11
 2 Complex I (NDUFS4) 4 24 7
 3 Complex I (NDUFS7) 20 72 15
 4 Complex I (NDUFS7) 101 92 55
 5 Complex I (NDUFS7) 26 16 20
 6 Complex I (NDUFS8) 9 52 9
 7 Complex I (NDUFV1) 20 NDd 14
 8 Complex I (MT-ND2) 51 110 30
 9 Complex I (MT-ND4L) 80 66 77
 10 Complex I (MT-ND5) 34 62 22
 11 Complex I (MELAS) 7 36 7
 12 Complex I (MELAS) 17 132 12
 13 Complex II 72 44 73
 14 Complex II 28 22 24
 15 Complex III (MT-CYB) 26 40 14
 16 Complex IV (SURF1) 68 56 50

PDHc deficiencies

 17 PDHc (PDHA1) 15 38 21
 18 PDHc (PDHA1) 14 14 ND
 19 PDHc (PDHA1) 18 18 20
 20 PDHc (PDHA1) 13 8 16
 21 PDHc (PDHA1) 13 50 16
 22 PDHc (PDHA1) 39 70 42
 23 PDHc (PDHA1) 41 34 27

Complex V deficiencies

 24 Complex V (MT-ATP6) 17 ND 26
 25 Complex V (MT-ATP6) 28 50 ND
 26 Complex V (MT-ATP6) 90 108 49

ANT deficiencies

 27 17 14 12
 28 5 6 ND
 29 62 44 8

Controls

Mean (SD) 100 (19) 100 (32) 100 (21
Range 66-147 46-220 52-139

 Oxidation rate in
 incubation:

Patient Deficiencyb (b) 7 8 9

 RC
deficiencies

 1 Complex I (NDUFS2) 30 31 24
 2 Complex I (NDUFS4) 13 22 12
 3 Complex I (NDUFS7) 27 27 21
 4 Complex I (NDUFS7) 86 95 81
 5 Complex I (NDUFS7) 39 52 30
 6 Complex I (NDUFS8) 17 38 12
 7 Complex I (NDUFV1) ND ND 11
 8 Complex I (MT-ND2) 74 ND ND
 9 Complex I (MT-ND4L) 102 93 86
 10 Complex I (MT-ND5) 49 66 43
 11 Complex I (MELAS) 14 41 27
 12 Complex I (MELAS) 28 46 26
 13 Complex II 91 117 68
 14 Complex II 38 41 9
 15 Complex III (MT-CYB) 31 48 14
 16 Complex IV (SURF1) 69 68 61

PDHc deficiencies

 17 PDHc (PDHA1) 83 93 89
 18 PDHc (PDHA1) ND ND ND
 19 PDHc (PDHA1) 43 59 27
 20 PDHc (PDHA1) 35 44 33
 21 PDHc (PDHA1) 54 71 45
 22 PDHc (PDHA1) 103 104 93
 23 PDHc (PDHA1) 51 51 40

Complex V deficiencies

 24 Complex V (MT-ATP6) 34 35 31
 25 Complex V (MT-ATP6) ND ND ND
 26 Complex V (MT-ATP6) 85 71 75

ANT deficiencies

 27 18 21 15
 28 ND ND ND
 29 19 37 7

Controls

Mean (SD) 100 (18) 100 (18) 100 (18)
Range 50-133 50-137 65-133

Patient Deficiencyb (b) production (c)

 RC
deficiencies

 1 Complex I (NDUFS2) 21
 2 Complex I (NDUFS4) 9
 3 Complex I (NDUFS7) 21
 4 Complex I (NDUFS7) 37
 5 Complex I (NDUFS7) 29
 6 Complex I (NDUFS8) 11
 7 Complex I (NDUFV1) 15
 8 Complex I (MT-ND2) 34
 9 Complex I (MT-ND4L) 77
 10 Complex I (MT-ND5) 28
 11 Complex I (MELAS) 14
 12 Complex I (MELAS) 17
 13 Complex II 81
 14 Complex II 19
 15 Complex III (MT-CYB) ND
 16 Complex IV (SURF1) 52

PDHc deficiencies

 17 PDHc (PDHA1) 23
 18 PDHc (PDHA1) 15
 19 PDHc (PDHA1) 17
 20 PDHc (PDHA1) 10
 21 PDHc (PDHA1) 25
 22 PDHc (PDHA1) 42
 23 PDHc (PDHA1) 21

Complex V deficiencies

 24 Complex V (MT-ATP6) 20
 25 Complex V (MT-ATP6) 13
 26 Complex V (MT-ATP6) 52

ANT deficiencies

 27 8
 28 3
 29 6

Controls

Mean (SD) 100 (18)
Range 64-145

 Incubation ratio

 ATP/Pyr ADP
 ratio stimulation (e)
 RC
deficiencies

 1 Complex I (NDUFS2) 135 57
 2 Complex I (NDUFS4) 133 25
 3 Complex I (NDUFS7) 107 47
 4 Complex I (NDUFS7) 60 65
 5 Complex I (NDUFS7) 160 57
 6 Complex I (NDUFS8) 97 22
 7 Complex I (NDUFV1) 92 32
 8 Complex I (MT-ND2) 97 37
 9 Complex I (MT-ND4L) 111 100
 10 Complex I (MT-ND5) 104 43
 11 Complex I (MELAS) 276 22
 12 Complex I (MELAS) 113 25
 13 Complex II 119 105
 14 Complex II 70 58
 15 Complex III (MT-CYB) ND 32
 16 Complex IV (SURF1) 94 98

PDHc deficiencies

 17 PDHc (PDHA1) 104 32
 18 PDHc (PDHA1) 101 60
 19 PDHc (PDHA1) 77 70
 20 PDHc (PDHA1) 89 63
 21 PDHc (PDHA1) 180 35
 22 PDHc (PDHA1) 108 60
 23 PDHc (PDHA1) 80 47

Complex V deficiencies

 24 Complex V (MT-ATP6) 142 107
 25 Complex V (MT-ATP6) 71 52
 26 Complex V (MT-ATP6) 90 55

ANT deficiencies

 27 81 85
 28 104 92
 29 55 45

Controls

 Mean (SD), % 100 (16) 100 (25)
 Range, % 67-123 58-142

 Incubation ratio

 2/1 4/1 3/5
 RC
deficiencies

 1 Complex I (NDUFS2) 199 88 93
 2 Complex I (NDUFS4) 182 54 108
 3 Complex I (NDUFS7) 143 102 56
 4 Complex I (NDUFS7) 148 162 98
 5 Complex I (NDUFS7) 196 142 194
 6 Complex I (NDUFS8) 190 78 98
 7 Complex I (NDUFV1) 148 125 ND
 8 Complex I (MT-ND2) 198 145 85
 9 Complex I (MT-ND4L) 126 115 100
 10 Complex I (MT-ND5) 206 124 98
 11 Complex I (MELAS) 160 128 61
 12 Complex I (MELAS) 147 108 43
 13 Complex II 112 105 141
 14 Complex II 129 101 209
 15 Complex III (MT-CYB) 162 179 110
 16 Complex IV (SURF1) 121 123 97

PDHc deficiencies

 17 PDHc (PDHA1) 91 68 182
 18 PDHc (PDHA1) 101 96 164
 19 PDHc (PDHA1) 98 82 167
 20 PDHc (PDHA1) 122 112 225
 21 PDHc (PDHA1) 98 89 76
 22 PDHc (PDHA1) 97 100 89
 23 PDHc (PDHA1) 93 153 162

Complex V deficiencies

 24 Complex V (MT-ATP6) ND 122 ND
 25 Complex V (MT-ATP6) 164 151 68
 26 Complex V (MT-ATP6) 148 156 92

ANT deficiencies

 27 109 159 86
 28 81 137 50
 29 195 582 52

Controls

 Mean (SD), % 100 (9) 10100 (10) 100 (25)
 Range, % 82-136 6 70-130 65-160

 Incubation ratio

 6/1 7/6 7/8
 RC
deficiencies

 1 Complex I (NDUFS2) 71 283 96
 2 Complex I (NDUFS4) 98 202 59
 3 Complex I (NDUFS7) 80 179 97
 4 Complex I (NDUFS7) 90 161 89
 5 Complex I (NDUFS7) 107 207 74
 6 Complex I (NDUFS8) 80 189 44
 7 Complex I (NDUFV1) 87 ND ND
 8 Complex I (MT-ND2) 86 256 ND
 9 Complex I (MT-ND4L) 112 136 108
 10 Complex I (MT-ND5) 82 229 74
 11 Complex I (MELAS) 147 199 35
 12 Complex I (MELAS) 82 230 59
 13 Complex II 108 129 77
 14 Complex II 89 160 91
 15 Complex III (MT-CYB) 93 234 63
 16 Complex IV (SURF1) 91 143 99

PDHc deficiencies

 17 PDHc (PDHA1) 95 405 88
 18 PDHc (PDHA1) ND ND ND
 19 PDHc (PDHA1) 92 227 73
 20 PDHc (PDHA1) 136 224 78
 21 PDHc (PDHA1) 114 348 76
 22 PDHc (PDHA1) 109 252 97
 23 PDHc (PDHA1) 100 197 98

Complex V deficiencies

 24 Complex V (MT-ATP6) 183 136 96
 25 Complex V (MT-ATP6) ND ND ND
 26 Complex V (MT-ATP6) 86 179 118

ANT deficiencies

 27 116 160 85
 28 ND ND ND
 29 77 244 52

Controls

 Mean (SD), % 100 (9) 100 (17) 100 (11)
 Range, % 82-118 67-167 83-117

(a) Incubations were performed as described in the Materials and
Methods section. Incubations are numbered as in Table 1.
The activities and ratios, including the respective control values,
are expressed as percentage of the mean control values given in
Table 1. The patient numbering is as in Table 3.

(b) Terms in parentheses indicate gene defects. Human genes: NDUFS2,
NADH dehydrogenase (ubiquinone) Fe-S protein 2, 49 kDa (NADH-coenzyme
Q reductase); NDUFS4, NADH dehydrogenase (ubiquinone) Fe-S
protein 4, 18kDa (NADH-coenzyme Q reductase); NDUFS7, NADH
dehydrogenase (ubiquinone) Fe-S protein 7, 20kDa (NADH-coenzyme Q
reductase); NDUFS8, NADH dehydrogenase (ubiquinone) Fe-S protein 8,
23kDa (NADH-coenzyme Q reductase); NDUFV1, NADH dehydrogenase
(ubiquinone) flavoprotein 1, 51kDa; MT-ND2, mitochondrially encoded
NADH dehydrogenase 2; MT-ND4L, mitochondrially encoded NADH
dehydrogenase 4L; MT-ND5, mitochondrially encoded NADH dehydrogenase
5; MELAS, mitochondrial encephalomyopathy, lactic acidosis, and
stroke-like episodes; MT-CYB, mitochondrially encoded cytochrome
b; SURF1, surfeit 1; PDHA1, pyruvate dehydrogenase (lipoamide) alpha
1; MT-ATP6, mitochondrially encoded ATP synthase 6.

(c) ATP production rate from oxidation of pyruvate + malate.

(d) ND, not determined; MELAS, mitochondrial myopathy, encephalopathy,
lactic acidosis, and stroke-like episodes.

(e) Stimulation of the oxidation rate of pyruvate + malate by
ADP (incubation 1/incubation 3 ratio).
COPYRIGHT 2006 American Association for Clinical Chemistry, Inc.
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2006 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Title Annotation:Endocrinology and Metabolism
Author:Janssen, Antoon J.M.; Trijbels, Frans J.M.; Sengers, Rob C.A.; Wintjes, Liesbeth T.M.; Ruitenbeek, W
Publication:Clinical Chemistry
Date:May 1, 2006
Words:8299
Previous Article:Relationship of adiponectin with markers of systemic inflammation, atherogenic dyslipidemia, and heart failure in patients with coronary heart...
Next Article:Sensitive quantitative analysis of C-peptide in human plasma by 2-dimensional liquid chromatography-mass spectrometry isotope-dilution assay.
Topics:

Terms of use | Privacy policy | Copyright © 2018 Farlex, Inc. | Feedback | For webmasters