Pyruvate-dehydrogenase complex in ataxic patients: Enzyme deficiency in ataxic encephalopathy plus lactic acidosis and normal activity in Friedreich ataxia

Ital. J. Neurol. Sci. 4." 317-321, 1982

Pyruvate-dehydrogenase complex in ataxic patients: enzyme deficiency in ataxic encephalopathy plus lactic acidosis and normal activity in Friedreich ataxia Uziel G., Bottacchi E., Moschen G.*, Giovanardi-Rossi P.*, Cardace G., and Di Donato S.

Laboratorio Neuropatie Metaboliche, Istituto Neurologico "C. Besta'" Milano *Clinica Neuropsichiatrica Infantile Bologna.

Pyruvate dehydrogenase complex (PDHC) activity was measured in cultured fibroblasts from 12 patients with Friedreich's ataxia (FA), and in 1 patient with lactic acidosis and ataxia. The activities obtained after extraction of PDHC by different methods were compared. Triton-X-lO0 extraction yielded enzyme activities 5 to 10 times greater than those obtained with the older methods. With this sensitive technique, PDHC activity was markedly deficient in fibroblasts from the patient with lactic acidosis and ataxia but it was normal in the fibroblasts from FA patients. Mg++activation of the PDHC in FA fibroblasts was normal. Key-Words: Pyruvate-dehydrogenase, Friedreich ataxia, lactic acidosis.

Introduction The pyruvate dehydrogenase complex in mammalian tissues consists of three catalytic and two regulatory enzymes [6]. The catalytic enzymes are pyruvate dehydrogenase, PDH (EC 1.2.4.1.), lipoate transacetylase, LAT (EC 2.3.1.12) and lipoamide dehydrogenase, LAD (EC 1.6.4.3.). The two regulatory enzymes are PDH-phosphatase and PDH-Kinase [17]. Several patients with deficiencies of PDHC, either its catalytic [10, 14] or its regulatory components [13, 9], have been reported. According to Blass [3], some of these patients suffer from

severe encephalopathies manifestating in the first months of life with lactic acidosis: their tissues and cultured fibroblasts show marked deficiency of P D H C (less than 15% of the normal values). Other patients have less severe encephalopathies, generally manifesting as ataxia in the first years of fife: these patients have slight-to-moderate deficiencies of P D H C in their cells (20 to 35% of the normal values) [3]. P D H C activity has also been reported to be low in cultured fibroblasts from patients with Friedreich's ataxia [4, 11]. These data for the FA patients, however, were not confirmed by other laboratories [16, 7]. In our hands [2], P D H C 317

The Italian Journal of Neurological Sciences

activity in cultured fibroblasts from patients with FA was found to be normal when sonication [16] was used to solubilize the P D H C and low when homogenization in glycerol [4] was used. These conflicting data stimulated us to develop a third reliable and reproducible method to measure the full acitivity of the P D H C in cultured cells and to apply it to a study of strains of human fibroblasts from patients with suspected or proved P D H C deficiencies.

Case Reports and Methods

Ataxic encephalopathy plus lactic acidosis The propositus was a 15-year-old male born of healthy unrelated parents. The only brother of the patient had died at age 4 years and 6 months with a clinical history of progressive encephalopathy. The propositus had marked hypotonia at birth. At age 7 months he had generalized seizures. At age 2 years he was admitted to a county hospital because of delayed psychomotor development: lower limb spasticity and ataxia of gait were observed. The severity of these was reported to vary with time. At age 5 years physical examination revealed inexpressive face, dorsolumbar kypho-scoliosis, increased deep tendon reflexes, spasticity of the lower limbs with bilateral Babinski sign, ataxia of gait. Comprehension was very poor. Laboratory investigations were normal except for blood lactate and pyruvate, which were high to 3.1 mM and 0.23 mM (normal values for lactate < 1.8 mM, for pyruvate < 0.18 raM). Alanine was high in both plasma and urine. Repeated EEGs showed diffuse theta activity, prevalent in the left temporal lobe. CT scan revealed a moderate enlargement of the 3rd and 4th ventricles and a mild degree of cortical atrophy. In the following years the patient had several generalized seizures. At age ! 5 years his blood lactate was 4.0 mM and blood pyruvate 0.3 raM. He was given thiamine therapy at that time, 600 rag/day by mouth. The patients mother said that during therapy his general condition and lower limb spasticity improved. After 4 months, the therapy was discontinued because no further progress was observed. At age 15 years and 6 months the results of physical examination were similar to those of the examination made before thiarnJne therapy: blood lactate was 8.8 mM and blood pyruvate 0.18 mM. At age 16 years the patient had a generalized seizure with head trauma: he was admitted to a country hospital, where he developed epileptic status and died. Autopsy was refused by the parents. 318

Friedreich ataxia 12 patients with Friedreich's ataxia, selected according to strict clinical criteria [8] (patients 1-12 of this reference) were studied. Fibroblasts for culture were obtained by skin biopsy from the patient in section 1, the FA patients and 14 controls (6 normal subjects and 8 patients with genetic metabolic diseases unrelated to pyruvate metabolism). Ceils were grown as previously described. [2] To measure P D H C activity, fibroblasts, harvested by trypsinization, were washed twice in phosphate- buffered saline, (PBS) pH 7.4, centrifuged and suspended in PBS to a cell concentration of 1 to 2 mg cell protein/ml. PDHC was assayed by a radiochemical method [4]: 50 to 300/zg of cell extract, obtained by the different techniques described below, were incubated in a final volume of 320/~1 in the presence of adequate amounts of cofactors and labelled pyruvate, in K-phosphate buffer, pH 7.4, in scintillation vials stoppered with rubber caps from which strips of paper moistened with 50/~1 of hyamine hydroxide in methanol were hung, in an oscillating bath at 37~ The reaction was terminated by placing the vials in a vortex incubator precooled to 0~ and shaking for 20' to collect the labelled C0~ produced from the pyruvate. Blanks were obtained with boiled cells. The entrapped labelled CO 2 was quantitated in a Tricarb scintillation counter (Packard). Cell extracts were obtained by exposure of cell suspensions to various concentrations of Triton-X-100 for 5' at 37 ~ C, as in a method described by Atkin et al [1] for pyruvate carboxylase, or by sonication [16] or by glycerol homogenization [4]. The a-ketogluterate dehydrogenase complex ( a - K G D H C ) was measured in glycerol homogenates as described by Blass [4]. Pyruvate dehydrogenase, PDH, and a-ketoglutarate dehydrogenase, a - K G D H were measured in cell sonicates with potassium ferricyanide as electron acceptor [16]. Lipoate transacetylase (LAT) was measured by a radiochemical method modified from that of Butterworth et al. [5]. Lipoamide dehydrogenase was measured spectrophotometrically [16]. Protein was measured by the method of Lowry et al [12]. Results and Discussion

Method Preliminary experiments showed that the extraction of cell suspension with Triton-X-100 yielded specific P D H C activities greater than those obtained after glycerol homogenization or sonication. This method of extracting PDHC

Uziel: Pyruvate-dehydrogenase complex in ataxic patients

TABLEI. Activities* of a-KGDHC and P D H C in cultured fibroblasts from a patient with lactic acidosis ~lus ataxia. Enzyme***

Controls

Patient

0.67~0.17 (10)

0.60-0.79

0.23•

(12)

0.02-0.03

0.56__+0.13 (12)

0.06-0.12

PDHC

3.24-+-0.62 (10)

0.07-0.18

(3)

0.07

PDHC total**

3.43__+0.41 (3)

0.12

a-KGDHC PDHC PDHC

glycerol

sonicate

glycerol

triton PDHC active**

1.94•

Enzyme activities are expressed as nanomoles of labelled COa p r o d u c e d / m i n / m g cell protein ~ SD PDHC active = enzyme activity determined without Mg § PDHC total = enzyme activity determined with 10 mM Mg ++ * * * Oe~'ls of enzyme determination are reported in Methods. Number of controls in parentheses. *

**

TABLE II. Activities* ataxia.

of 2-oxoacid-dehydrogenase

Enzyme a-KGDH PDHE1

components in a patient with lactic acidosis plus

Controls E1

Patient

0.15•

(4)

0.17

0.085•

(9)

0.008-0.012

LAT

3.56~0.89 (11)

5.21

LAD

7.73•

(14)

5.74

LAD

1.8 •

(8)

1.5

10.20•

(8)

6.80

LAD

km lipoamide, mM

Vmax

Activities are exxpressed as nanomoles substrate m i n / m g cell protein ___SD Number of control in parentheses.

was therefore chosen for the further studies. At a saturating concentration of pyruvate (0.4 mM), PDHC activity was linear with time up to 20' of incubation. At the cell protein concentrations commonly used in PDHC assay (100-200 #g), higher enzyme activities were observed when the concentration of Triton-X-100 was 80/~g/incubation mixture. However, with this amount of Triton, the specific PDHC activity was not linear with the protein but declined when the protein content was increased over 200/Lg. Since these data suggested the dependence of PDHC activity on the protein/Triton ratio, we studied the optimal protein/Triton ratio and found that it should be maintained between 1 and 2. Optimal cofactor concentrations for NAD§ CoASH, thiamine pyrophosphate (TPP) and MgC1, in the Triton assay were found to be 2.5 mM, 6~.5 mM, 0.3 mM and 10 mM. PDHC in ataxic encephalopathy plus lactic acidosis Table I shows that the Triton method yielded

PDHC activities in controls 5 to 10 greater than those obtained with the older methods [4, 16]. Moreover, this table shows that with the three different methods of extracting the enzyme complex from fibroblasts, PDHC was markedly deficient in the patient's cells (from 3 to 20% of controls, with the different methods); while, the activity of a - K G D H C was normal. The Mg§ § dependent activation of the residual PDHC activity in the patient's cells was comparable to that of control cells. (Table I) Table 2 shows that PDHC deficiency was due to a defect of the first component of the complex, pyruvate-dehydrogenase: PDH activity was in fact reduced to about 12% of the control mean in the patient's cells; LAT and LAD specific enzyme activities were in the normal range and the kinetics of LAD with lipoamide were comparable in c6ntrol and in the patient's fibroblasts. P D H C in Friedreieh ataxia The figure shows that PDHC activities in controls cells (mean activity 3.13___0.65 SD nano319

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of Neurological

Sciences

TABLE IlI. M g ++ activation of P D H C in FA and control cells. Cell strain

PDHC activity*

E

active

total

active/ total %

0.68 0.69 1.63 0.92 0.96 1.19 1.11 1.13 0.68 0.96 1.42 1.11 1.07 1.83 1.25 1.74

2.70 2.69 4.81 2.63 2.60 3.16 2.56 2.63 3.14 3.09 3.80 2.82 2.71 4.23 2.44 3.32

21.1 25.6 33.9 35.0 36.9 37.6 43.4 44.7 21.6 31.1 37.3 39.4 39.5 43.3 51.2 52.4

e-

E

Control o

"6

9

E 0 et~ e-

% 0

9

o

1

2~

"g t~

.1a e~

FA

1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8

l-ig. 1. Activity of pyruvate-dehydrogenase complex in cultured fibroblasts from F.A. patients and Controls. Activities are expressed as nanomoles/ rain~rag cell protein. Mean and S D are given in Results and Comment. Closed symbols = controls Open symbols = FA patients

Eight experiments were d o n e in triplicate. In each experiment fibroblast from one control and one FA strain were grown to confluency and were examined in parallel for active and total PDHC activity. * Legend as in Table I.

moles/min/mg cell protein) and FA cells (2.95__0.63) were in the same range. Table III shows that Mg + + activation of PDHC was about equal in the 8 FA and 8 control cell strains studied: the ratio of active to total PDHC activity (see Table) varied between 21.1% to 44.7% in control's and between 21.6% to 52.4% in FA cells. Controversial results have been reported [4, 11, 16, 7] for PDHC activity in cells from patients with inherited ataxic diseases. The data in this paper show that with the sensitive Triton-X- 100 method we could not find any differences in active and total PDHC activity between fibroblasts from controls and those from FA patients (figure 3 and Table III). It was recently suggested by Blass [3] that various degrees of PDHC deficiency are associated with different clinical phenotypes. To make a statement of this kind one would have to know that the techniques that had been used measured all the PDHC activity. Our data show that the methods reported in the literature for extracting PDHC from human cells (sonications, homo-

genization in glycerol or buffer) in fact yield considerably lower PDHC activities from cultured fibroblasts than are measured after exposing the cells to Triton-X-100. (see Table I) This method yields PDHC activities similar to those recently obtained in cultured cells by Sheu et al (1981) [151. In the patient with ataxia and lactic acidosis, the enzyme defect was clearly shown with the Triton method, showing that the method can detect PDHC deficiencies due to defects in the catalytic components of the enzyme. (Table I, II) Since Mg ++ dependent activation of PDHC was found to be comparable in the ataxic patient and controls (Table II and III), we suggest that this method is also reliable for studies of PDHC deficiences due to defects in the regulatory components of the complex. One possible pitfall in this method is that there are considerable variations in enzyme activity when the protein content or the protein/Triton ratio in the incubation mixture are suboptimal. This may be a serious problem when the amount of tissue available is limited.

320

Uziel: Pyruvate-dehydrogenase complex in ataxic patients

Sommario II complesso enzimatico piruvico-deidrogenasi ( P D H C ) ~ stato misurato in fibroblasti coltivati da 12 pazienti con atassia di Friedreich ed 1 paziente con acidosi lattica e atassia. Le attivitd enzimatiche ottenute con diversi metodi di estrazione del P D H C sono stati confrontati. La estrazione con Triton-X-l OOpermetteva di ottenere attivitgt enzimatiche da 5 a 10 volte pifi elevate di quelle ottenute con i rnetodi precedenti. Con questo sensibile metodo l'attivita P D H C era molto ridotta nei fibroblasti del paziente con acidosi lattica e atassia: l'attivitg~ era invece normale nei fibroblasti dei pazienti con atassia di Friedreich. La attivazione del P D H C nei fibroblasti dei pazienti con Friedreich eta not'male. A d d r e s s r e p r i n t r e q u e s t s to: Dr. G r a z i e l l a U z i e l Laboratorio Neuropatie Metaboliche

Istituto Neurologico "C. Besta" Via C e l o r i a 11 - - 2 0 1 3 3 M i l a n o

ase complex in subacute necrotizing encephalomyelopathy (Leigh's disease). Ann N eurol 6: 483-

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