Oxidative phosphorylation analysis: assessing the integrated functional activity of human skeletal muscle mitochondria—case studies

Oxidative phosphorylation analysis, performed on freshly-isolated mitochondria, assesses the integrated function of the electron transport chain (ETC) coupled to ATP synthesis, membrane transport, dehydrogenase activities, and the structural integrity of the mitochondria. In this review, a case study approach is employed to highlight detection of defects in the adenine nucleotide translocator, the pyruvate dehydrogenase complex, fumarase, coenzyme Q function, fatty acid metabolism, and mitochondrial membrane integrity. Our approach uses the substrates glutamate, pyruvate, 2-ketoglutarate (coupled with malonate), malate, and fatty acid substrates (palmitoylcarnitine, octanoylcarnitine, palmitoyl-CoA (with carnitine), octanoyl-CoA (with carnitine), octanoate and acetylcarnitine) in addition to succinate, durohydroquinone and TMPD/ascorbate to uncover metabolic defects that would not be apparent from ETC assays performed on detergent-solubilized mitochondria.     

Oxidative phosphorylation analysis: assessing the integrated functional activity of human skeletal muscle mitochondria--case studies

Oxidative phosphorylation analysis, performed on freshly-isolated mitochondria, assesses the integrated function of the electron transport chain (ETC) coupled to ATP synthesis, membrane transport, dehydrogenase activities, and the structural integrity of the mitochondria. In this review, a case study approach is employed to highlight detection of defects in the adenine nucleotide translocator, the pyruvate dehydrogenase complex, fumarase, coenzyme Q function, fatty acid metabolism, and mitochondrial membrane integrity. Our approach uses the substrates glutamate, pyruvate, 2-ketoglutarate (coupled with malonate), malate, and fatty acid substrates (palmitoylcarnitine, octanoylcarnitine, palmitoyl-CoA (with carnitine), octanoyl-CoA (with carnitine), octanoate and acetylcarnitine) in addition to succinate, durohydroquinone and TMPD/ascorbate to uncover metabolic defects that would not be apparent from ETC assays performed on detergent-solubilized mitochondria.     

Quantitation of the efflux of acylcarnitines from rat heart, brain, and liver mitochondria

The efflux of individual short-chain and medium-chain acylcarnitines from rat liver, heart, and brain mitochondria metabolizing several substrates has been measured. The acylcarnitine efflux profiles depend on the substrate, the source of mitochondria, and the incubation conditions. The largest amount of any acylcarnitine effluxing per mg of protein was acetylcarnitine produced by heart mitochondria from pyruvate. This efflux of acetylcarnitine from heart mitochondria is almost 5 times greater with 1 mM than 0.2 mM carnitine. Apparently the acetyl-CoA generated from pyruvate by pyruvate dehydrogenase is very accessible to carnitine acetyltransferase. Very little acetylcarnitine effluxes from heart mitochondria when octanoate is the substrate except in the presence of malonate. Acetylcarnitine production from some substrates peaks and then declines, indicating uptake and utilization. The unequivocal demonstration that considerable amounts of propionylcarnitine or isobutyrylcarnitine efflux from heart mitochondria metabolizing alpha-ketoisovalerate and alpha-keto-beta-methylvalerate provides evidence for a role (via removal of non-metabolizable propionyl-CoA or slowly metabolizable acyl-CoAs) for carnitine in tissues which have limited capacity to metabolize propionyl-CoA. These results also show propionyl-CoA must be formed during the metabolism of alpha-ketoisovalerate and that extra-mitochondrial free carnitine rapidly interacts with matrix short-chain aliphatic acyl-CoA generated from alpha-keto acids of branched-chain amino acids and pyruvate in the presence and absence of malate.     

Quantitation of the effect of L-carnitine on the levels of acid-soluble short-chain acyl-CoA and CoASH in rat heart and liver mitochondria

The steady state levels of mitochondrial acyl-CoAs produced during the oxidation of pyruvate, alpha-ketoisovalerate, alpha-ketoisocaproate, and octanoate during state 3 and state 4 respiration by rat heart and liver mitochondria were determined. Addition of carnitine lowered the amounts of individual short-chain acyl-CoAs and increased CoASH in a manner that was both tissue- and substrate-dependent. The largest effects were on acetyl-CoA derived from pyruvate in heart mitochondria using either state 3 or state 4 oxidative conditions. Carnitine greatly reduced the amounts of propionyl-CoA derived from alpha-ketoisovalerate, while smaller effects were obtained on the branched-chain acyl-CoA levels, consistent with the latter acyl moieties being poorer substrates for carnitine acetyltransferase and also poorer substrates for the carnitine/acylcarnitine translocase. The levels of acetyl-CoA in heart and liver mitochondria oxidizing octanoate during state 3 respiration were lower than those obtained with pyruvate. The rate of acetylcarnitine efflux from heart mitochondria during state 3 (with pyruvate or octanoate as substrate, in the presence or absence of malate with 0.2 mM carnitine) shows a linear response to the acetyl-CoA/CoASH ratio generated in the absence of carnitine. This relationship is different for liver mitochondria. These data demonstrate that carnitine can modulate the aliphatic short-chain acyl-CoA/CoA ratio in heart and liver mitochondria and indicate that the degree of modulation varies with the aliphatic acyl moiety.     

Specific inhibition of mitochondrial fatty acid oxidation by 2-bromopalmitate and its co-enzyme A and carnitine esters

1. The CoA and carnitine esters of 2-bromopalmitate are extremely powerful and specific inhibitors of mitochondrial fatty acid oxidation. 2. 2-Bromopalmitoyl-CoA, added as such or formed from 2-bromopalmitate, inhibits the carnitine-dependent oxidation of palmitate or palmitoyl-CoA, but not the oxidation of palmitoylcarnitine, by intact liver mitochondria. 3. 2-Bromopalmitoylcarnitine inhibits the oxidation of palmitoylcarnitine as well as that of palmitate or palmitoyl-CoA. It has no effect on succinate oxidation, but inhibits that of pyruvate, 2-oxoglutarate or hexanoate; however, the oxidation of these substrates (but not of palmitate, palmitoyl-CoA or palmitoyl-carnitine) is restored by carnitine. 4. In damaged mitochondria, added 2-bromopalmitoyl-CoA does inhibit palmitoylcarnitine oxidation; pyruvate oxidation is unaffected by the inhibitor alone, but is impaired if palmitoylcarnitine is subsequently added. 5. The findings have been interpreted as follows. 2-Bromopalmitoyl-CoA inactivates (in a carnitine-dependent manner) a pool of carnitine palmitoyltransferase which is accessible to external acyl-CoA. This results in inhibition of palmitate or palmitoyl-CoA oxidation. A second pool of carnitine palmitoyltransferase, inaccessible to added acyl-CoA in intact mitochondria, can generate bromopalmitoyl-CoA within the matrix from external 2-bromopalmitoylcarnitine; this reaction is reversible. Such internal 2-bromopalmitoyl-CoA inactivates long-chain beta-oxidation (as does added 2-bromopalmitoyl-CoA if the mitochondria are damaged) and its formation also sequesters intramitochondrial CoA. Since this CoA is shared by pyruvate and 2-oxoglutarate dehydrogenases, the oxidation of their substrates is depressed by 2-bromopalmitoylcarnitine, unless free carnitine is available to act as a ;sink' for long-chain acyl groups. 6. These effects are compared with those reported for other inhibitors of fatty acid oxidation.     

Effects of chronic caloric restriction on mitochondrial respiration in the ischemic reperfused rat heart

Dietary restriction increases life span and delays the development of age-related diseases in rodents. We have recently demonstrated that chronic dietary restriction is beneficial on recovery of heart function following ischemia. We studied whether the metabolic basis of this benefit is associated with alterations in mitochondrial respiration. Male Wistar rats were assigned to an ad libitum-fed (AL) group and a food restricted (FR) group, in which food intake was reduced to 55% of the amount consumed by the AL group. Following an 8-month period of restricted caloric intake, isolated working hearts perfused with glucose and high levels of fatty acids were subjected to global ischemia followed by reperfusion. At the end of reperfusion, total heart mitochondria was respiration was assessed in the presence of pyruvate, tricarboxylic acid intermediates, and palmitoylcarnitine. Recovery of heart function following ischemia was greater in FR hearts compared to AL hearts. Paralleling these changes in heart function was in increase in state 3 respiration with pyruvate. The respiratory control ratios in the presence of pyruvate and tricarboxylic acid intermediates were higher in FR hearts compared to AL hearts, indicating well-coupled mitochondria. Overall energy production, expressed as the ADP:O ratio and the oxidative phosphorylation rate, was also improved in FR hearts. Our results indicate that the beneficial effect of FR on recovery of heart function following ischemia is associated with changes in mitochondrial respiration.     

Lipid oxidation by heart mitochondria from young adult and senescent rats

1. State-3 (i.e. ADP-stimulated) rates of O(2) uptake with palmitoylcarnitine, palmitoyl-CoA plus carnitine, pyruvate plus malonate plus carnitine and octanoate as respiratory substrate were all diminished in heart mitochondria isolated from senescent (24-month-old) rats compared with mitochondria from young adults (6 months old). By contrast, State-3 rates of O(2) uptake with pyruvate plus malate or glutamate plus malate were the same for mitochondria from each age group. 2. Measurements of enzyme activities in disrupted mitochondria showed a decline with senescence in the activity of acyl-CoA synthetase (EC 6.2.1.2 and 6.2.1.3), carnitine acetyltransferase (EC 2.3.1.7) and 3-hydroxy-acyl-CoA dehydrogenase (EC 1.1.1.35), but no change in the activity of carnitine palmitoyltransferase (EC 2.3.1.21) or acyl-CoA dehydrogenase (EC 1.3.99.3). 3. Measurement of dl-[(3)H]carnitine (in)/acetyl-l-carnitine (out) exchange in intact mitochondria showed decreased rates when the animals used were senescent. However, this followed from a decreased intramitochondrial pool of exchangeable carnitine, such that calculated first-order rate constants for exchange were identical in mitochondria from the two age groups. 4. The decline in acyl-CoA synthetase activity is thought to be the reason for the diminished rate of O(2) uptake with octanoate in senescence. The decline in carnitine acetyltransferase activity is considered to be the cause of the diminished rate of O(2) uptake with acetylcarnitine or with pyruvate plus malonate plus carnitine as substrate. The mechanism of the diminished rate of O(2) uptake with palmitoylcarnitine in senescence is discussed.     

Active-site probes of carnitine acyltransferases. Inhibition of carnitine acetyltransferase by hemiacetylcarnitinium, a reaction intermediate analogue

Hemiacetylcarnitinium (2S,6R:2R,65)-6-carboxymethyl-2-hydroxy-2,4,4- trimethylmorpholinium) chloride is a relatively potent competitive inhibitor (Ki = 0.89 mM) of pigeon breast carnitine acetyltransferase (CAT) and of the crude rat liver CAT (Ki = 4.72 mM) but is neither an inhibitor nor an effective substrate for purified rat liver carnitine palmitoyltransferase (CPT). It does not inhibit state 3 oxygen consumption in isolated hepatic mitochondria using palmitoyl-CoA or palmitoylcarnitine as substrates. This compound is a reaction intermediate analogue of the proposed tetrahedral intermediate for acetyl transfer between acetylcarnitine and CoASH. Because the hemiketal carbon is chiral, a suggestion is made that one of the enantiomers has the same relative configuration as the proposed tetrahedral intermediate.     

Substrate-dependent differential regulation of mitochondrial bioenergetics in the heart and kidney cortex and outer medulla

The kinetics and efficiency of mitochondrial oxidative phosphorylation (OxPhos) can depend on the choice of respiratory substrates. Furthermore, potential differences in this substrate dependency among different tissues are not well-understood. Here, we determined the effects of different substrates on the kinetics and efficiency of OxPhos in isolated mitochondria from the heart and kidney cortex and outer medulla (OM) of Sprague-Dawley rats. The substrates were pyruvate+malate, glutamate+malate, palmitoyl-carnitine+malate, alpha-ketoglutarate+malate, and succinate±rotenone at saturating concentrations. The kinetics of OxPhos were interrogated by measuring mitochondrial bioenergetics under different ADP perturbations. Results show that the kinetics and efficiency of OxPhos are highly dependent on the substrates used, and this dependency is distinctly different between heart and kidney. Heart mitochondria showed higher respiratory rates and OxPhos efficiencies for all substrates in comparison to kidney mitochondria. Cortex mitochondria respiratory rates were higher than OM mitochondria, but OM mitochondria OxPhos efficiencies were higher than cortex mitochondria. State 3 respiration was low in heart mitochondria with succinate but increased significantly in the presence of rotenone, unlike kidney mitochondria. Similar differences were observed in mitochondrial membrane potential. Differences in H₂O₂ emission in the presence of succinate±rotenone were observed in heart mitochondria and to a lesser extent in OM mitochondria, but not in cortex mitochondria. Bioenergetics and H₂O₂ emission data with succinate±rotenone indicate that oxaloacetate accumulation and reverse electron transfer may play a more prominent regulatory role in heart mitochondria than kidney mitochondria. These studies provide novel quantitative data demonstrating that the choice of respiratory substrates affects mitochondrial responses in a tissue-specific manner.     

The action of tellurite, a reagent for thiol groups, on mitochondria oxidative processes

Tellurite, a reagent for thiol groups, added to rat kidney, or liver mitochondria at the concentration of 1 mM, selectively inhibits the oxidation of NAD dependent substrates (pyruvate, α-ketoglutarate, glutamate, etc.) without affecting the oxidation of succinate, α-glycerophosphate and ascorbate. Moreover NADH oxidation by rat heart or by aged liver mitochondria is not affected by tellurite.     

Studies of energy-linked reactions. Inhibition of oxidative phosphorylation by DL-8-methyldihydrolipoate.

1. DL-8-Methyldihydrolipoate was shown to be a potent inhibitor of mitochondrial oxidative phosphorylation and ATP-driven energy-linked reactions. 2. ADP-stimulated respiration utilizing pyruvate + malate and succinate in both ox heart and rat liver mitochondria is inhibited; oxidative phosphorylation using pyruvate + malate, succinate and ascorbate + NNN'N'-tetramethyl-p-phenylenediamine as substrates is also inhibited; uncoupler-stimulated respiration is unaffected regardless of the substrate used. 3. Mitochondrial oligomycin-sensitive adenosine triphosphatase is inhibited in both the membrane-bound form and the purified detergent-dispersed preparation. 4. ATP-driven transhydrogenase and the ATP-driven energy-linked reduction of NAD+ by succinate in ox heart submitochondrial particles are inhibited, whereas the respiratory-chain-driven transhydrogenase is unaffected. 5. DL-8-Methyl-lipoate has no immediate effect on the above reactions, demonstrating the requirement for the reduced form for inhibition. 6. The inhibitory properties of DL-8-methyldihydrolipoate are analogous to those of oligomycin and provide further evidence of a role for lipoic acid in oxidative phosphorylation.     

Cellular energy metabolism during fetal development. II. Fatty acid oxidation by the developing heart

In view of the importance of fatty acids as substrates for the mature heart, fatty acid oxidation by fetal and calf heart mitochondria has been investigated. Free fatty acids of 10 carbon units or less which exhibit carnitine-independent transport into mitochondria were effective substrates for oxidative phosphorylation in both fetal and calf heart mitochondria. Efficient oxidative phosphorylation with these substrates was dependent upon the presence of bovine serum albumin in the assay medium to reverse the uncoupling effects of the fatty acids. In the presence of bovine serum albumin, ADP/0 ratios were in the range of 3 when short-chain fatty acids and carnitine esters of short- and long-chain fatty acids were substrates. Compared with calf heart mitochondria, fetal heart mitochondria showed decreased carnitine-dependent oxidation of palmityl-CoA. However, the oxidation of palmitylcarnitine was identical in both. These data suggest that the formation of palmitylcarnitine is rate limiting for palmityl-CoA oxidation by the fetal heart mitochondria and that long-chain fatty acids are not readily oxidized by the fetal heart.     

Purification and properties of tauropine dehydrogenase from the shell adductor muscle of the ormer, Haliotis lamellosa

Tauropine dehydrogenase (tauropine:NAD oxidoreductase) was purified from the shell adductor muscle of the ormer, Haliotis lamellosa. The enzyme was found to utilize stoichiometrically NADH as co-enzyme and pyruvate and taurine as substrates producing tauropine [rhodoic acid; N-(D-1-carboxyethyl)-taurine]. The enzyme was purified to a specific activity of 463 units/mg protein using a combination of ammonium sulphate fractionation, ion-exchange and affinity chromatography. The relative molecular mass was 38,000 +/- 1000 when assessed by gel filtration on Ultrogel AcA 54 and 42,000 +/- 150 by electrophoresis on 5-10% polyacrylamide gels in the presence of 1% sodium dodecyl sulphate; the data suggest a monomeric structure. Tauropine and pyruvate were found to be the preferred substrates. Among the amino acids tested for activity with the enzyme, only alanine is used as an alternative substrate, but with a rate less than 6% of the enzyme activity with taurine. Of the oxo acids tested, 2-oxobutyrate and 2-oxovalerate were also found to be substrates. Apparent Km values for the substrates NADH, pyruvate and taurine are 0.022 +/- 0.003 mM, 0.64 +/- 0.07 mM and 64.7 +/- 5.4 mM, respectively, at pH 7.0 and for the products, NAD+ and tauropine, are 0.29 +/- 0.01 mM and 9.04 +/- 1.27 mM, respectively, at pH 8.3. Apparent Km values for both pyruvate and taurine decrease with increasing co-substrate (taurine or pyruvate) concentration. NAD+ and tauropine were found to be product inhibitors of the forward reaction. NAD+ was a competitive inhibitor of NADH, whereas tauropine gave a mixed type of inhibition with respect to pyruvate and taurine. Succinate was found to inhibit non-competitively with respect to taurine and pyruvate with an apparent Ki value in the physiological range of this anaerobic end product. The inhibition by L-lactate, not an end product in the ormer, was competitive with respect to pyruvate. The physiological role or tauropine dehydrogenase during anaerobiosis is discussed.     

High resolution respirometry of permeabilized skeletal muscle fibers in the diagnosis of neuromuscular disorders

High resolution respirometry in combination with the skinned fiber technique offers the possibility to study mitochondrial function routinely in small amounts of human muscle. During a period of 2 years, we investigated mitochondrial function in skeletal muscle tissue of 13 patients (average age = 5.8 years). In all of them, an open muscle biopsy was performed for diagnosis of their neuromuscular disorder. Mitochondrial oxidation rates were measured with a highly sensitive respirometer. Multiple substrate-inhibitor titration was applied for investigation of mitochondrial function. About 50 mg fibers were sufficient to obtain maximal respiratory rates for seven different substrates (pyruvate/malate, glutamate/malate, octanoylcarnitine/malate, palmitoylcarnitine /malate, succinate, durochinol and ascorbate/TMPD). Decreased respiration rates with reference to the wet weight of the permeabilized fiber could immediately be detected during the course of measurements.In 4 patients with mitochondrial encephalomyopathy (MEM) the respiration pattern indicated a specific mitochondrial enzyme defect, which was confirmed in every patient by measurements of the individual enzymes (one patient with PDHC deficiency, one with complex I deficiency and two patients with combined complex I and IV deficiency). In the 6 patients with spinal muscular atrophy (SMA) oxidation rates were found to be decreased to 23 ± 5% of controls. The normalized respiration pattern was comparable to that of the controls indicating a decreased content of mitochondria in SMA muscle with normal functional properties. Also in the 3 patients with Duchenne muscular dystrophy (DMD) decreased oxidation rates (42 ± 5%) were detected. In addition a low RCI (1.2) indicated a loose coupling of oxidative phosphorylation in the mitochondria of these patients.It is concluded that investigation of mitochondrial function in saponin skinned muscle fibers using high resolution respirometry in combination with multiple substrate titration offers a valuable tool for evaluation of mitochondrial alterations in muscle biopsies of children suffering from neuromuscular disorders. (Mol Cell Biochem 174: 71–78, 1997)     

The disposition of citric acid cycle intermediates by isolated rat heart mitochondria

The mechanism of depletion of tricarboxylic acid cycle intermediates by isolated rat heart mitochondria was studied using hydroxymalonate (an inhibitor of malic enzymes) and mercaptopicolinate (an inhibitor of phosphoenolpyruvate carboxykinase) as tools. Hydroxymalonate inhibited the respiration rate of isolated mitochondria in state 3 by 40% when 2 mM malate was the only external substrate, but no inhibition was found with 2 mM malate plus 0.5 mM pyruvate as substrates. In the prescence od bicarbonate, arsenite and ATP, propionate was converted to pyruvate and malate at the rates of 14.0 ± 2.9 and 2.8 ± 1.8 nmol/mg protein in 5 min, respectively. Under these conditions, 0.1 mM mercaptopicolinate did not affect this conversion, but 2 mM hydroxymalonate inhibited pyruvate formation completely and resulted in an accumulation of malate up to 13.2 ± 2.9 nmol/mg protein. No accumulation of phosphoenolpyruvate was found under any condition tested. It is concluded that malic enzymes but not phosphoenolpyruvate carboxykinase, are involved in conversion of propionate to pyruvate in isolated rat heart mitochondria.     

Inhibitory effect of gentamicin on gluconeogenesis from pyruvate, propionate, and lactate in isolated rabbit kidney-cortex tubules

The effect of gentamicin on glucose production in isolated rabbit renal tubules was studied with lactate, propionate, malate, 2-oxoglutarate, and succinate as substrates. This antibiotic at 5 mM concentration inhibited gluconeogenesis from lactate by about 60% and that from either pyruvate or propionate by about 30%. In contrast, it did not alter the rate of glucose formation from other substrates studied. The rate of gluconeogenesis was higher at 1 mM propionate than at increasing concentrations of this substrate and was stimulated in the presence of 1 mM carnitine. However, the addition of carnitine did not affect the degree of inhibition of glucose formation by gentamicin. Since the mitochondrial free coenzyme A level was significantly lower in the presence of 10 than 1 mM propionate and increased on the addition of carnitine to the reaction medium, the inhibitory effect of propionate concentrations above 1 mM on gluconeogenesis in rabbit renal tubules may be due to a depletion of the free mitochondrial coenzyme A level, resulting in an inhibition of the mitochondrial coenzyme A-dependent reactions. In intact rabbit kidney cortex mitochondria incubated in State 4 as well as in Triton X-100-treated mitochondria, 5 mM gentamicin inhibited by about 30-40% the incorporation of 14CO2 into both pyruvate and propionate. The results indicate that the inhibitory effect of gentamicin on glucose formation in isolated kidney tubules incubated with lactate, pyruvate, or propionate is likely due to a decrease of the rate of carboxylation reactions.     

Studies on mitochondria from dystrophic skeletal muscle of mice

Mitochondrial respiration and oxidative phosphorylation were compared in normal and dystrophic mouse skeletal muscles. To obtain the maximum respiration control ratio (RCR) and adenosine diphosphate/oxygen (ADP/O) ratio from isolated muscle mitochondria, it is found that there is an advantage in having a low concentration of proteinase and EGTA present in the medium during preparation of mitochondria by centrifugation fractionation. Using pyruvate, acetylcarnitine, and palmitylcarnitine as substrates for oxidation, a highly significant reduction (40-60%) is shown in oxygen uptake by dystrophic muscle mitochondria as compared to normal muscle mitochondria. Studies of the integrity of the oxidative phosphorylation apparatus in these samples showed that there is a reduction of the RCR and ADP/O ratio in dystrophic muscle mitochondria as compared to normal muscle mitochondria.     

Changes in urinary taurine and hypotaurine excretion after two-thirds hepatectomy in the rat

Summary This study followed the time course of urinary taurine and hypotaurine excretion after two-thirds hepatectomy in rats. The excretion of both taurine and hypotaurine was elevated during 18th following the hepatectomy, with maximal excretion during the first 6h. Twelve and 24h after partial hepatectomy, the hepatic hypotaurine concentration was increased but liver taurine did not differ significantly from controls. No changes were observed in hypotaurine and taurine concentrations of heart, kidney, lung, muscle tissue and spleen. We postulate that partial hepatectomy induces a rapid increase of hepatic (hypo)taurine synthesis from precursor amino acids. The increased (hypo)taurine concentrations spill over into urine.     

Uncoupled oxidation in rat heart mitochondria

Isolated heart mitochondria possessing a high phosphorylation efficiency with pyruvate and malate as substrates oxidize NADH and ascorbate unassociated with ADP phosphorylation. This uncoupled pathway is expressed partially when succinate or NAD-linked substrates are oxidized. The uncoupled oxidation is likely to be the result of the presence of a mitochondrial population with the high-permeable inner membrane in intact tissues. The nature and origin of a uncoupled respiratory system and its role in the thermoproduction of endotherms are discussed.     

Rapid Communication Muscle Mitochondrial ATP Production in Progressive Supranuclear Palsy

Abstract: Six patients with progressive supranuclear palsy (PSP) and 12 age-matched disease-free subjects participated in this study designed to compare rates of ATP production by intact mitochondria from biopsied skeletal muscle. When pyruvate and malate were used as metabolic substrates, rates of ATP production were 0.184 ± 0.025 μmol/min/U of citrate synthase (CS) activity (a mitochondrial marker) in control subjects and 0.131 ± 0.051 μmol/min/U of CS in PSP patients. In the presence of succinate, rates of ATP formation were 0.137 + 0.02 μmol/min/U of CS in controls and 0.109 ± 0.04 /4mUmol/min/U of CS in patients. With N,N,N',N' -tetramethyl- p -phenylenediamine (TMPD) and ascorbate, rates were 0.034 ± 0.008 μm Umol/min/U of CS in controls and 0.022 ± 0.01 μmol/min/U of CS in PSP subjects. Differences between the control and PSP populations reached statistical significance with pyruvate/malate and TMPD/ascorbate. No differences in either muscle histopathology or histochemistry were found between patient and control subjects. Results of this study suggest that oxidative phosphorylation defects occur in muscle mitochondria from patients with PSP.