Hum Genet (1984) 68:51-53

© Springer-Verlag 1984

Pyruvate dehydrogenase activity is not deficient in the brain of three autopsied cases with Leigh disease (subacute necrotizing encephalomyelopathy, SNE)

B. Kustermann-Kuhn 1, K. Harzer 1, R. Schr6der 2, W. Permanetter 3 , and J. Peiffer 1

lInstitut f/ir Hirnforschung, Universitfit TiJbingen, Belthlestr. 15, D-7400 Tfibingen 2Department of Pathology, University of Cologne 3Department of Pathology, University of Munich, Federal Republic of Germany

Summary. In autopsied brain tissue from three cases with Leigh disease (subacute necrotizing encephalomyelitis, SNE) and controls, the activity of pyruvate dehydrogenase complex (PDHC) was determined under different conditions. It was found to be at the control level or increased, but not deficient. The activities of succinate dehydrogenase, fumarase, succi- nate cytochrome c reductase, cytochrome c oxidase, and glu- tamate dehydrogenase were measured as additional mito- chondrial markers and showed no essential differences be- tween SNE and control tissue. The metabolic defect in SNE remains unknown. According to the literature, the defect may be localized to the mitochondrial systems. However, the reported results indicate that it cannot be ascribed to PDHC function. Extensive biochemical studies are necessary for understanding of the pathogenesis in the fatal genetic meta- bolic disease.

Introduction

Subacute necrotizing encephalomyelopathy (SNE) is a reces- sively inherited fatal neurometabolic disease defined neuro- pathologically (Jellinger and Seitelberger 1970) by character- istic symmetrical lesions in the tegmentum of the midbrain and pons, but also in the basal ganglia, medulla, and some- times in the spinal cord. Varying cell losses, demyelination, and capillary proliferation are essential histologic findings. Most patients die before the age of three years, but very much longer clinical courses have been observed.

The primary metabolic defect in SNE is unknown. Various results have been reported which suggest a defect in pyruvate oxidation or carboxylation (for reviews, see Evans 1981; Gil- bert et al. 1983), or in thiamine triphosphate synthesis (for review, see Murphy and Craig 1975). Defective functions of the Krebs cycle or respiratory chain have also been discussed.

Most cases of SNE are diagnosed at autopsy only and post- mortem biochemical studies are therefore complicated by the artifacts occurring in the autopsied tissues before and during deep-freeze storage. However, the activities of different mito- chondrial enzymes can be measured in autopsied tissue, if the artifactual activity losses are controlled by the use of appro- priate reference tissues matched with the tissues under study for the periods between the death of patients and freeze stor-

Offprint requests to: B. Kustermann-Kuhn, Institut ffir Hirnfor- schung, Universit~it Ttibingen, Belthlestr. 15, D-7400 Ttibingen

age, and during freeze storage. We were able to cotlect three pairs of SNE (confirmed at autopsy) and control material which sufficiently fulfilled those criteria. In one of the pairs, the ages of patients were not matched with each other. We investigated this pair as well, since no substantial age-de- pendence of the activity of the enzymes studied is known.

We determined the activity of the pyruvate dehydrogenase complex (PDHC) including its activity (PDHCra) attained after inhibition by ATP and spontaneous reactivation (DeVivo et al. 1979). We could not confirm the reported abnormalities of PDHC in SNE (Evans 1981). However, occa- sional cases of the disease (e.g., Toshima et al. 1982) may have defective PDHC. Other cases may have other mitochon- drial enzyme abnormalities (e.g., Willems et al. 1977). The following enzyme activities were measured as additional mito- chondrial markers: succinate dehydrogenase (SDH), fuma- rase (FU), succinate cytochrome c reductase (SCCR), cyto- chrome c oxidase (CCO), and glutamate dehydrogenase (GDH).

Materials and methods

Brain autopsy tissues

The autopsy tissues used and the patients are characterized in Table 1.

Homogenates

The enzyme activities were measured with homogenates (except for GDH, see below) prepared with a cutting homo- genizer ("Ultra-Turrax"; lmin) to yield a 10% (wet weight basis) concentration in the buffers of the respective assay methods immediately before the assays were started.

Enzyme assays

Pyruvate dehydrogenase complex (PDHC) activity was meas- ured with the assay described by Cremer and Teal (1974) using 50mg brain tissue per assay ( lml) which was overlaid with l m l paraffin oil immediately after initiating the reaction by addition of the radioactive pyruvate (1.4~tCi = 0.06gmol 1-14C-pyruvate, The Radiochemical Centre Amersham U.K., no. CFA.85, and 19.94gmol unlabeled pyruvate). Nitrogen was constantly bubbled (lml/s) through the assay mixture shaken in a 37°C water-bath. The 14CO2 released was transfer-

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Table 1. Patients and autopsy material (brain frontal lobes)

Age at death Hours before (years/ tissue freezing months) (but cooling

confirmed)

Months of tissue storage at - 35 ° C

Neuropathologic diagnosis

Pair 1

SNE Case 1 2/10 24 16 control no. 1 2/6 18 22

Pair 2

SNE Case 2 7/1 about 20 11 control no. 2 9/0 29 7

Typical SNE Demyelinating disease clearly different from SNE

Typical SNE Brain tumor (neuroblastoma or embryonal sarcoma; frontal lobes not involved)

Pair 3

SNE Case 3 1/7 about 20 28 control no. 3 16/0 23 24

Additional control

control no. 4 19/0 35 7 Neuropathologically normal (peritonitis)

Table 2. Mitochondrial enzyme activities in brain autopsy tissue (nmol substrate utilized per h and g wet tissue; mean of three determinations, methodologic variation coefficient 8-19%); for abbreviations, see Introduction, for patients, Table 1

PDHC PDHCra SDH FU GDH SCCR CCO

SNE without substantial capillary proliferation (resting stage) Neuroaxonal dystrophy; no signs of SNE

Pair 1

SNE Case 1 17.6 17.0 571 642 12270 122 407 control no. 1 16.0 14.8 578 607 8300 124 604

Pair 2

SNE Case 2 43.0 40.5 784 864 14870 254 1045 control no. 2 27.1 22.0 883 622 9265 238 749

Pair 3

SNE Case 3 34.3 35.0 1110 910 11400 313 669 control no. 3 23.9 20.9 1270 670 8320 358 782

Additional control

control no. 4 29.5 27.5 1125 593 9025 381 920

red with the nitrogen flow to a series of nine plastic vials each containing 1.5 ml hyamine hydroxide as a carbon dioxide trap. Each vial was bubbled for exactly 10rain by the tubing with the 14COJnitrogen flow. Contents of vials were counted for radioactivity with PPO/POPOP/toluene by liquid scintillation. Counting rates were sufficiently constant in the 4th to 6th vial (PDHC fully active). Therefore, PDHC activity was calculat- ed from the mean radioactivity in the 4th to 6th vial. Blank values with water instead of homogenate were subtracted. PDHCra activity (see Introduction) was calculated from the mean radioactivity in the 7th to 9th vial in experiments where 0.1mMol ATP (neutralized with KOH) was added to the assay after the first 30min of reaction time. It was confirmed that the added ATP immediately depressed the 14C02 produc- tion by at least 50% (radioactivity in the 4th comPared to the 3rd vial) before the production spontaneously increased again.

Succinate dehydrogenase (SDH) activity was measured with potassium hexacyanoferrate (III) according to the method of DeRobertis et al. (1962) following the decrease of absorption at 405nm for 45min in the presence of 10mg brain tissue per 1 ml assay. Turbidities were removed by centrifugation ( lmin 20,000 x g) before reading absorptions.

Fumarase (FU) activity was determined by the method de- scribed by Bergmeyer et al. (1970). The amount of fumarate produced from malate was monitored by the increase of absorption at 240nm for 30min in the presence of 5mg brain tissue (1.5 ml assay). Turbidities were removed as described in the SDH assay.

Glutamate dehydrogenase (GDH) activity was assayed accord- ing to the method of Bergmeyer et al. (1970) with freshly dis- solved NADH and the extract of lmg brain tissue in lml.

Succinate cytochrome c reductase (SCCR) activity was deter- mined essentially as described by Darley-Usmar et al. (1983) following the reduction of ferricytochrome c at 550nm during 5min; 2mg brain tissue were used per assay (lml).

Inhibition of SCCR activity was studied with 2raM antimycin A present in the assay to exclude non-SCCR-catalyzed cyto- chrome c~reduction,

Cytochrome c Oxidase (CCO) activity was measured according to the method of Darley-Usmar et al: (1983) following the oxi- dation of ferrocytochrome c at 550nm for 10min in the pre- sence of lmg brain tissue per lml assay and 0.5% Triton X-100. Ferrocytochrome c was prepared as described pre-

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viously (DiMauro et al. 1983). Inhibition of C C O activity was tested with 1.5raM K C N in the assay to show the absence of non-enzymatic fer rocytochrome c oxidation.

Results

The mitochondrial enzyme activities listed in Table 2 show no major differences be tween SNE and control brains. P D H C and PDHCra (= reactivated, see Methods) activities are slightly increased rather than deficient in the SNE cases. The activities of F U and G D H also show the tendency to be increased in the SNE cases. The other enzyme activities are not significantly decreased in SNE. The activities of S C C R and C C O could be completely inhibited in both SNE and con- trols by the addition of antimycin A and K C N respectively (see Methods) to the assays, whereby non-enzymatic reduc- tion or oxidation of cytochrome c was ruled out. All enzyme activities studied showed a sufficiently linear dependence on enzyme concentrat ion and reaction t ime within the limits used.

Discussion

The purpose of this study was to show that, in contrast to repor ted findings, the activity of P D H C (measured also after inhibition by A T P and spontaneous reactivation of the com- plex according to the method of DeVivo et al. 1979; PDHCra in Table 2) is not defective in SNE, at least not in the brain (Table 2) and the liver (results with this organ not shown) from three cases with the disease proven at autopsy. We can- not confirm that "decreased P D C activity, ei ther primary or secondary, is the common metabolic feature in patients with SNE . . . " (Evans 1981; P D C = pyruvate decarboxylase = enzyme 1 of the complex). The other enzyme activities (Table 2) were determined as additional markers of preserved mito- chondrial activity in the autopsy material . However , pyruvate carboxylase with its suggested role in SNE (Gilbert et al. 1983) was not measurable in the autopsy tissues.

The more or less marked elevation of P D H C , FU, and G D H activity in the SNE cases (Table 2) over the controls may be due to an unspecific mitochondrial activation in the severe neurometabol ic disease. The activity of CCO was de- scribed to be low in muscle tissue of one patient with SNE (Willems et al. 1977). In the brain of our SNE cases, CCO activity was essentially at the control level or slightly increas- ed. Also the activity of S C C R was found to be not substan- tially different f rom the control values in the SNE brains. This enzyme was repor ted to have low activity in cases with "mito- chondrial encephalomyopathies" (Sengers et al. 1983; Riggs et al. 1984). According to Egger et al. (1982), SNE can also present as mitochondrial encephalomyopathy which term may serve as a working hypothesis. Al though in our opinion all biochemical findings repor ted up to now for SNE cases have failed to show a pr imary mitochondrial defect in brain, muscle, or other tissues of patients, some indirect indication of such defect in SNE can hardly be denied. Toshima et al. (1982) reviewed the l i terature on disturbances of pyruvate metabol ism in SNE cases. Since P D H C , Krebs cycle, respira-

tory chain, and oxidative phosphorylat ion can all influence the pyruvate metabol ism (Egger et al. 1982), extensive studies of these metabolic pathways in SNE are necessary. In the pre- sent preliminary study we were able to give only a few exam- ples of enzyme studies in this unclear fatal genetic disease.

Acknowledgments. Drs. H. Budka and K. Kitz (Neurological Institute of the University of Vienna) generously provided us with the brain material from two SNE cases (R.M. = no. 2 in Table 1, and B.R., see Note added in proof). We are grateful to the Deutsche Forschungsge- meinschaft (Ha 845/7) for financial support.

References

Bergmeyer HU, Gawehn K, Grass1 M (1970) Enzyme. In: Bergmeyer HU (ed) Methoden der enzymatischen Analyse. Verlag Chemie, Weinheim/Bergstrage, pp 411-412, 607-613

Cremer JE, Teal HM (1974) The activity of pyruvate dehydrogenase in rat brain during postnatal development. FEBS Lett 39 : 17-20

Darley-Usmar VM, Kennaway NG, Buist NRM, Capaldi RA (1983) Deficiency in ubiquinone cytochrome c reductase in a patient with mitochondrial myopathy and lactic acidosis. Proc Natl Acad Sci USA 80 : 5103-5106

DeRobertis E, Pellegrino de Iraldi A, Rodriguez de Lores Arnaiz G, Salganicoff L (1962) Cholinergic and non-cholinergic nerve endings in rat brain. I. Isolation and subcellular distribution of acetylcholine and acetylcholinesterase. J Neurochem 9 : 23-35

DeVivo DC, Haymond MW, Obert KA, Nelson JS, Pagliara AS (1979) Defective activation of the pyruvate dehydrogenase com- plex in subacute necrotizing encephalomyelopathy (Leigh disease). Ann Neurol 6 : 483-494

DiMauro S, Nicholson JF, Hays AP, Eastwood AB, Papadimitriou A, Koenigsberger R, DeVivo DC (1983) Benign infantile mito- chondrial myopathy due to reversible cytochrome c oxidase defi- ciency. Ann Neurol 14:226-234

Egger J, Wynne-Williams CJE, Erdohazi M (1982) Mitochondrial cytopathy or Leigh's syndrome? Mitochondrial abnormalities in spongiform encephalopathies. Neuropediatrics 13 : 219-224

Evans O (1981) Pyruvate decarboxylase deficiency in subacute ne- crotizing encephalomyopathy. Arch Neurol 38 : 515-519

Gilbert EF, Arya S, Chun R (1983) Leigh's necrotizing encephalo- myelopathy with pyruvate carboxylase deficiency. Arch Pathol Lab Med 107 : 162-166

Jellinger K, Seitelberger F (1970) Subacute necrotizing encephalo- myelopathy (Leigh). In: Heilmeyer L, Muller AF, Prader A, Schoen R (eds) Ergebnisse der Inneren Medizin und Kinderheil- kunde, vol 29. Springer, Berlin, pp 155-219

Murphy JV, Craig L (1975) Leigh's disease: significance of the bio- chemical changes in brain. J Neurol Neurosurg Psychiatry 38:1100-1103

Riggs JE, Schochet SS, Fakadej AV, Papadimitriou A, DiMauro S, Crosby TW, Gutmann L, Moxley RT (1984) Mitochondrial ence- phalomyopathy with decreased succinate-cytochrome c reductase activity. Neurology (NY) 34 : 48-53

Sengers RCA, Fischer JC, Trijbels JMF, Ruitenbeek W, Stadhouders AM, ter Laak HJ, Jaspar HHJ (1983) A mitochondrial myopathy with a defective respiratory chain and carnitine deficiency. Eur J Pediatr 140 : 332-337

Toshima K, Kuroda Y, Hashimoto T, Ito M, Watanabe T, Miyao M, II K (1982) Enzymologic studies and therapy in Leigh's disease associated with pyruvate decarboxylase deficiency. Pediatr Res 16: 430-435

Willems JL, Monnens LHA, Trijbels JMF, Veerkamp JH, Meyer AEFH, VanDam K, VanHaelst U (1977) Leigh's encephalomyo- pathy in a patient with cytochrome c oxidase deficiency in muscle tissue. Pediatrics 60 : 850-857

Received June 6, 1984

Note added in proof

Recently, we could study two additional SNE cases (B.R., 11 months old, showing not only typical brain stem lesions, but also severe central demyelination and spongy degeneration; and A.W., 16 months old, with typical SNE). PDHC activities were clearly within control limits.