147
Leigh Syndrome Associated with a Deficiency of the Pyruvate Dehydrogenase Complex: Results of Treatment with a Ketogenic Diet By F. A. Wijburg l , P. G. Barth l , L. A. Bindoff, M. A. Birch-Machin2,]. F. van der Blij3, W. Ruitenbeek4, D. M. Turnbu1l2 and R. B. H. Schutgens l 1Department of Pediatrics, University Hospital Amsterdam (AMC), Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands; 2Division of Clinical Neuroscience, University of Newcastle upon Tyne, Newcastle upon Tyne, U.K.; 3Department of Pediatrics, Medical Centre Alkmaar, Alkmaar, The Netherlands, 4Department of Pediatrics, University of Nijmegen, Nijmegen, The Netherlands
A one-year-old boy suffering from intermittent lactic acidosis, muscular hypotonia, horizontal gaze paralysis and spasticity in both legs had low activity of the pyruvate dehydrogenase complex associated with low amounts of immunoreactive EIn and EIß. Leigh syndrome was diagnosed on the basis of the clinical and biochemical abnormalities and the typicallesions observed on MRI of the brain. Treatment with a ketogenic diet was associated with clinical and biochemical amelioration. A striking improvement of the cerebralIesions was observed by neuro-imaging.
Keywords Leigh syndrome - Lactic acidosis - Pyruvate dehydrogenase complex - Ketogenic diet
Zusammenfassung Bei einem 12 Monate alten Jungen mit intermittierender Laktatazidose, muskulärer Hypotonie und spastischer Parese der Beine fanden wir einen Mangel der E Inund E lß-Subunits des Pyruvatdehydrogenasekomplexes. Diese klinischen und biochemischen Befunde sowie der Nachweis typischer Hirnläsionen im MRI führten zu der Diagnose Leigh-Syndrom. Die Behandlung mit einer ketogenen Diät führte zur klinischen Besserung und Normalisierung des Laktatwertes im Blut. Eine erstaunliche Rückbildung der Hirnläsionen wurde im MRI beobachtet.
The pyruvate dehydrogenase complex (PDHc) is an intra-mitochondrial multienzyme complex consisting of multiple copies of four different subuits: E 1 (pyruvate dehydrogenase) which is a tetramer of two n and two ß subunits (E In and EIß), E2 (dihydrolipoyl transacetylase), E3 (dihydrolipoyl dehydrogenase), an additional protein X and two specific regulatory enzymes, pyruvate dehydrogenase phosphatase and kinase. PDHc catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA. Acetyl-CoA can enter the citric acid cycle by reacting with oxaloacetate to yield citrate and CoA. Because PDHc is the key enzyme between the glycolytic pathway and the citric acid cycle, a deficiency of one of the subunits of PDHc results in an impaired oxidation of pyruvate with increased blood lactate and pyruvate concentrations and decreased oxidative ATP synthesis. Deficiency of PDHc has been described in association with, sometimes fatal, lactic acidosis (3, 24, 29, 31) and different neurological syndromes as intermittent ataxias (2, 4) and Leigh syndrome (subacute necrotizing encephalomyelopathy) (13, 14, 17, 20). We report the results of the clinical and biochemical studies in a patient with intermittent lactic acidosis and neurological deterioration due to a deficiency of the EI subunit of PDHc. Leigh syndrome was diagnosed on the basis of the clinical and biochemical abnormalities and the cerebral lesions observed on magnetic resonance imaging (MRI) of the brain. Treatment with a ketogenic diet was followed by clinical and biochemical amelioration and a striking improvement of the abnormalities on MRI of the brain.
Case report
Received May 4, 1991; accepted August 12, 1991 Neuropediatrics 23 (1992) 147-152 © Hippokrates Verlag Stuttgart
The patient, a boy, was born at term after an uneventful pregnancy and delivery as the second child of healthy, non-consanguineous parents. There is no family history of neurological or metabolic disease. Psycho-motor development was normal until, at the age of 5 months, he became ill with a cough and an exanthemateous rash. He was admitted to hospital because of rapidly progressive generalized muscular hypotonia.
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Introduction Abstract
148 N europediatrics 23 (1992)
Fig. 1 a and b MRI (SE sequence, TR = 3000, TE = 128) in the transverse plane at the age of 1 year, before the start of the ketogenic diet. Myelination is at a level of 6 months. Bilateral, symmetrical areas of increased signal intensity in the globus pallidus, crus posterior of the capsula interna and cerebellum.
a
b
On admission the child was somnolent. There was a severe generalized muscular hypotonia with a complete head lag, and a marked tachypnea. Blood gas analysis showed mild metabolie acidosis. Blood glucose, electrolytes, liver enzymes and serum creatinine were normal. Erythrocyte sedimentation rate was increased (21 mrn/hr); leucocyte count was normal Lumbar puncture revealed no pleiocytosis and normal glucose and protein concentrations (lactate was not measured at that time). Further investigation revealed a high blood lactate concentration (6.1 mmol/l, normal< 2.0) with an increased pyruvate concentration (0.18 mmol/l, normal< 0.13). Antibiotics were administered for 4 days (blood and CSF cultures remained negative). Muscle tone and power gradually improved, however, blood lactate concentrations remained elevated (2.2 3.7 mmol/l). CSF lactate und pyruvate concentrations were also increased (3.32 and 0.42 mmol/l respectively). A 24 hr fast resulted in a fall of blood lactate from 3.7 to 2.5 mmol/l without hypoglycemia. The following investigations were normal: CT scan of the brain, EEG, nerve conduction velocity, brainstem auditory evoked potentials and visual evoked potentials. A magnetic resonance imaging (MRI) of the brain demonstrated delayed cerebral myelination without other abnormalities. The child recovered completely in aperiod of 5 weeks. Lactate and pyruvate in blood and CSF were normal. Subsequent psychomotor development was normal up to 12 months: he could sit without support at the age of 9 months, crawled at 10 months and showed normal, symmetrieal, intentional movements. Myotatic reflexes could not be elicited, all other reflexes were normal. At 12 months of age spasticity in both legs was noticed. Lactate and pyruvate concentrations were elevated in blood (5.4 and 0.25 mmol/l respectively) and CSF (4.0 and 0.35 mmol/l respectively). Total and free carnitine concentrations in serum were normal. Organic acid analysis of the urine showed severe lactic aciduria. Amino acid analysis of the urine revealed an increased concentration of alanine (5.2 mmol/g creatinine, normal 2.9 ± 1.1 [mean ± SD]). Brainstem auditory evoked
potentials demonstrated an increased 1-111 interval at the left side. Visual evoked potentials, EEG and fundoscopy were normaL
A second MRI of the brain at the age of 13 months demonstrated delayed myelination and bilateral symmetrie lesions with increased signal intensity in the globus pallidus and the posterior crus of the capsula interna as weH as in the cerebellum on T2-weighted images (Fig. 1 a and 1 b). The child's clinical condition deteriorated; he became somnolent, tachypnoeic and had an intermittent, conjugate, horizontal gaze paralysis to the right side. Because a deficiency of the pyruvate dehydrogenase complex was suspected on the basis of increased lactate and pyruvate concentrations in the blood and CSF (with normal lactate to pyruvate ratios), a trial with a ketogenic diet was started. At the age of 18 months, open biopsies of skeletal museie (m. quadriceps femoris) and liver were obtained for biochemical and immunochemical studies after obtaining informed consent of the parents. Morphological examination of the museIe biopsy material revealed no abnormalities.
Materials and methods
Spectrophotometric assay ofmitochondrial substrate oxidations Mitochondrial fractions were prepared from 1 g of skeletal museie as previously described (3). Liver mitochondrial fractions were prepared from 1 g of liver as described by Gatley and Sherratt (11). Substrate oxidation rates by mitochondrial fractions (0.2-0.4 mg/mI final assay protein concentration) were recorded spectrophotometrically (34). Rotenone was included when succinate was the substrate. PDHc activity wq.s measured spectrophotometrically in museie and liver mitochondrial extracts as previously described (3). Protein was measured according to the method of Lowry et al (23). Citrate synthase activity was measured spectrophotometrically, according to the method of Shepherd and Gar-
land (30).
Downloaded by: National University of Singapore. Copyrighted material.
F. A. Wijburg et al
Fig. 1 c and d MRI (SE sequence, TR = 2500, TE = 128) at a slightly different angle in the transverse plane at the age of 2 years. Myelination at a level of 1 year. No more areas of increased signal intensity. Normal aspect of globus pallidus and cerebellum. d
c
Immunochemical studies Muscle mitochondrial fractions were studied by immunoblot analysis. SDS-polyacrylamide gel electrophoresis was performed using 10 % gels as described by Laemmli (22). The separated proteins were transferred to nitrocellulose filter as described by Towbin et al (33). Nitrocellulose filters were washed and incubated with antiserum raised in rabbits against purified oxheart PDHc and subunit E 1 (8). Antigen-antibody complexes were visualized using the immunoperoxidase method with 4-chloro-1-naphthol as substrate.
Table 1 Results of substrate oxidation in isolated muscle and liver mitochondria. Substrate
Patient
Succinate Glutamine + malate Pyruvate + malate Oxogl uta rate
262
98 n.d. 55.7
n.d.: not detectable Results are nmoles ferricyanide reduced. min-1.(mg proteinyl. The control values are mean SO for 5 subjects (muscle) and 3 subjects (liver).
±
Ketogenic diet A ketogenic diet was introduced gradually over 10 days; the diet contained 66 % fat, 23 % carbohydrates and 11 % protein (percentages of total caloric intake).
Results
Substrate oxidations The oxidation rates of all substrates by muscle and liver mitochondrial fractions from the patient were low compared with controls, with a severely decreased oxidation rate of pyruvate (Table 1). Citrate synthase activity in muscle of the patient was low (0.48 units/mg protein; controls (n = 6): 1.1 ± 0.2 (mean ± SD). Correction for the low yield of muscle mitochondria, or less purification of the mitochondria, by expressing the oxidation rates in relation to citrate synthase activity, resulted in a better agreement of the oxidation rates of other substrates compared with controls (data not shown). The oxidation of pyruvate is very low compared to other substrates in both skeletal muscle and liver. Activity of PDHc was decreased in muscle and liver: 0.011 and 0.003 units enzyme activity/mg protein respectively (control ranges: muscle: 0.092-0.16 [n = 3]; liver: 0.006-0.010 [n = 3]).
Immunochemical studies Antibodies against ox-heart PDHc reacted with all the components of the PDHc in human muscle mitochondrial fractions. Immunoblot analysis of muscle mitochondrial fractions, using antibodies against the holoenzyme, showed markedly lowered concentrations of immunoreactive E 1a and EIß in the patient compared to controls (Fig. 2). Immunoblotting with antibodies against the E 1 subunits revealed a good reaction with E 1a and E 1ß subunits in controls, while in the patient low concentrations of immunoreactive E 1a with no detectable E 1ß can be observed (Fig. 2).
Ketogenic diet Introduction of a ketogenic diet resulted in a fall of blood lactate concentration to near normal values (Fig.3). The ß-hydroxybutyrate and acetoacetate concentrations increased to moderately elevated levels (0.3 - 0.85 mmol/ 1, normal< 0.2). The child's clinical condition improved considerably: the tachypnoea, apathy and horizontal gaze paralysis disappeared in 9 days. When the diet contained 66 % fat and 23 % carbohydrates a mild keto-acidosis was noted and the diet was changed to 58 % fat and 31 % carbohydrates. A short peri-
149
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Neuropediatrics 23 (1992)
Leigh Syndrome Associated with PDHc Deficiency
150 Neuropediatrics 23 (1992) "··",,,,·,'t····;..,..,.,,:,.·:·.
F. A. Wijburg et al
Leigh Syndrome Associated with a Deficiency of the Pyruvate Dehydrogenase Complex: Results of Treatment with a Ketogenic Diet By F. A. Wijburg l , P. G. Barth l , L. A. Bindoff, M. A. Birch-Machin2,]. F. van der Blij3, W. Ruitenbeek4, D. M. Turnbu1l2 and R. B. H. Schutgens l 1Department of Pediatrics, University Hospital Amsterdam (AMC), Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands; 2Division of Clinical Neuroscience, University of Newcastle upon Tyne, Newcastle upon Tyne, U.K.; 3Department of Pediatrics, Medical Centre Alkmaar, Alkmaar, The Netherlands, 4Department of Pediatrics, University of Nijmegen, Nijmegen, The Netherlands
A one-year-old boy suffering from intermittent lactic acidosis, muscular hypotonia, horizontal gaze paralysis and spasticity in both legs had low activity of the pyruvate dehydrogenase complex associated with low amounts of immunoreactive EIn and EIß. Leigh syndrome was diagnosed on the basis of the clinical and biochemical abnormalities and the typicallesions observed on MRI of the brain. Treatment with a ketogenic diet was associated with clinical and biochemical amelioration. A striking improvement of the cerebralIesions was observed by neuro-imaging.
Keywords Leigh syndrome - Lactic acidosis - Pyruvate dehydrogenase complex - Ketogenic diet
Zusammenfassung Bei einem 12 Monate alten Jungen mit intermittierender Laktatazidose, muskulärer Hypotonie und spastischer Parese der Beine fanden wir einen Mangel der E Inund E lß-Subunits des Pyruvatdehydrogenasekomplexes. Diese klinischen und biochemischen Befunde sowie der Nachweis typischer Hirnläsionen im MRI führten zu der Diagnose Leigh-Syndrom. Die Behandlung mit einer ketogenen Diät führte zur klinischen Besserung und Normalisierung des Laktatwertes im Blut. Eine erstaunliche Rückbildung der Hirnläsionen wurde im MRI beobachtet.
The pyruvate dehydrogenase complex (PDHc) is an intra-mitochondrial multienzyme complex consisting of multiple copies of four different subuits: E 1 (pyruvate dehydrogenase) which is a tetramer of two n and two ß subunits (E In and EIß), E2 (dihydrolipoyl transacetylase), E3 (dihydrolipoyl dehydrogenase), an additional protein X and two specific regulatory enzymes, pyruvate dehydrogenase phosphatase and kinase. PDHc catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA. Acetyl-CoA can enter the citric acid cycle by reacting with oxaloacetate to yield citrate and CoA. Because PDHc is the key enzyme between the glycolytic pathway and the citric acid cycle, a deficiency of one of the subunits of PDHc results in an impaired oxidation of pyruvate with increased blood lactate and pyruvate concentrations and decreased oxidative ATP synthesis. Deficiency of PDHc has been described in association with, sometimes fatal, lactic acidosis (3, 24, 29, 31) and different neurological syndromes as intermittent ataxias (2, 4) and Leigh syndrome (subacute necrotizing encephalomyelopathy) (13, 14, 17, 20). We report the results of the clinical and biochemical studies in a patient with intermittent lactic acidosis and neurological deterioration due to a deficiency of the EI subunit of PDHc. Leigh syndrome was diagnosed on the basis of the clinical and biochemical abnormalities and the cerebral lesions observed on magnetic resonance imaging (MRI) of the brain. Treatment with a ketogenic diet was followed by clinical and biochemical amelioration and a striking improvement of the abnormalities on MRI of the brain.
Case report
Received May 4, 1991; accepted August 12, 1991 Neuropediatrics 23 (1992) 147-152 © Hippokrates Verlag Stuttgart
The patient, a boy, was born at term after an uneventful pregnancy and delivery as the second child of healthy, non-consanguineous parents. There is no family history of neurological or metabolic disease. Psycho-motor development was normal until, at the age of 5 months, he became ill with a cough and an exanthemateous rash. He was admitted to hospital because of rapidly progressive generalized muscular hypotonia.
Downloaded by: National University of Singapore. Copyrighted material.
Introduction Abstract
148 N europediatrics 23 (1992)
Fig. 1 a and b MRI (SE sequence, TR = 3000, TE = 128) in the transverse plane at the age of 1 year, before the start of the ketogenic diet. Myelination is at a level of 6 months. Bilateral, symmetrical areas of increased signal intensity in the globus pallidus, crus posterior of the capsula interna and cerebellum.
a
b
On admission the child was somnolent. There was a severe generalized muscular hypotonia with a complete head lag, and a marked tachypnea. Blood gas analysis showed mild metabolie acidosis. Blood glucose, electrolytes, liver enzymes and serum creatinine were normal. Erythrocyte sedimentation rate was increased (21 mrn/hr); leucocyte count was normal Lumbar puncture revealed no pleiocytosis and normal glucose and protein concentrations (lactate was not measured at that time). Further investigation revealed a high blood lactate concentration (6.1 mmol/l, normal< 2.0) with an increased pyruvate concentration (0.18 mmol/l, normal< 0.13). Antibiotics were administered for 4 days (blood and CSF cultures remained negative). Muscle tone and power gradually improved, however, blood lactate concentrations remained elevated (2.2 3.7 mmol/l). CSF lactate und pyruvate concentrations were also increased (3.32 and 0.42 mmol/l respectively). A 24 hr fast resulted in a fall of blood lactate from 3.7 to 2.5 mmol/l without hypoglycemia. The following investigations were normal: CT scan of the brain, EEG, nerve conduction velocity, brainstem auditory evoked potentials and visual evoked potentials. A magnetic resonance imaging (MRI) of the brain demonstrated delayed cerebral myelination without other abnormalities. The child recovered completely in aperiod of 5 weeks. Lactate and pyruvate in blood and CSF were normal. Subsequent psychomotor development was normal up to 12 months: he could sit without support at the age of 9 months, crawled at 10 months and showed normal, symmetrieal, intentional movements. Myotatic reflexes could not be elicited, all other reflexes were normal. At 12 months of age spasticity in both legs was noticed. Lactate and pyruvate concentrations were elevated in blood (5.4 and 0.25 mmol/l respectively) and CSF (4.0 and 0.35 mmol/l respectively). Total and free carnitine concentrations in serum were normal. Organic acid analysis of the urine showed severe lactic aciduria. Amino acid analysis of the urine revealed an increased concentration of alanine (5.2 mmol/g creatinine, normal 2.9 ± 1.1 [mean ± SD]). Brainstem auditory evoked
potentials demonstrated an increased 1-111 interval at the left side. Visual evoked potentials, EEG and fundoscopy were normaL
A second MRI of the brain at the age of 13 months demonstrated delayed myelination and bilateral symmetrie lesions with increased signal intensity in the globus pallidus and the posterior crus of the capsula interna as weH as in the cerebellum on T2-weighted images (Fig. 1 a and 1 b). The child's clinical condition deteriorated; he became somnolent, tachypnoeic and had an intermittent, conjugate, horizontal gaze paralysis to the right side. Because a deficiency of the pyruvate dehydrogenase complex was suspected on the basis of increased lactate and pyruvate concentrations in the blood and CSF (with normal lactate to pyruvate ratios), a trial with a ketogenic diet was started. At the age of 18 months, open biopsies of skeletal museie (m. quadriceps femoris) and liver were obtained for biochemical and immunochemical studies after obtaining informed consent of the parents. Morphological examination of the museIe biopsy material revealed no abnormalities.
Materials and methods
Spectrophotometric assay ofmitochondrial substrate oxidations Mitochondrial fractions were prepared from 1 g of skeletal museie as previously described (3). Liver mitochondrial fractions were prepared from 1 g of liver as described by Gatley and Sherratt (11). Substrate oxidation rates by mitochondrial fractions (0.2-0.4 mg/mI final assay protein concentration) were recorded spectrophotometrically (34). Rotenone was included when succinate was the substrate. PDHc activity wq.s measured spectrophotometrically in museie and liver mitochondrial extracts as previously described (3). Protein was measured according to the method of Lowry et al (23). Citrate synthase activity was measured spectrophotometrically, according to the method of Shepherd and Gar-
land (30).
Downloaded by: National University of Singapore. Copyrighted material.
F. A. Wijburg et al
Fig. 1 c and d MRI (SE sequence, TR = 2500, TE = 128) at a slightly different angle in the transverse plane at the age of 2 years. Myelination at a level of 1 year. No more areas of increased signal intensity. Normal aspect of globus pallidus and cerebellum. d
c
Immunochemical studies Muscle mitochondrial fractions were studied by immunoblot analysis. SDS-polyacrylamide gel electrophoresis was performed using 10 % gels as described by Laemmli (22). The separated proteins were transferred to nitrocellulose filter as described by Towbin et al (33). Nitrocellulose filters were washed and incubated with antiserum raised in rabbits against purified oxheart PDHc and subunit E 1 (8). Antigen-antibody complexes were visualized using the immunoperoxidase method with 4-chloro-1-naphthol as substrate.
Table 1 Results of substrate oxidation in isolated muscle and liver mitochondria. Substrate
Patient
Succinate Glutamine + malate Pyruvate + malate Oxogl uta rate
262
98 n.d. 55.7
n.d.: not detectable Results are nmoles ferricyanide reduced. min-1.(mg proteinyl. The control values are mean SO for 5 subjects (muscle) and 3 subjects (liver).
±
Ketogenic diet A ketogenic diet was introduced gradually over 10 days; the diet contained 66 % fat, 23 % carbohydrates and 11 % protein (percentages of total caloric intake).
Results
Substrate oxidations The oxidation rates of all substrates by muscle and liver mitochondrial fractions from the patient were low compared with controls, with a severely decreased oxidation rate of pyruvate (Table 1). Citrate synthase activity in muscle of the patient was low (0.48 units/mg protein; controls (n = 6): 1.1 ± 0.2 (mean ± SD). Correction for the low yield of muscle mitochondria, or less purification of the mitochondria, by expressing the oxidation rates in relation to citrate synthase activity, resulted in a better agreement of the oxidation rates of other substrates compared with controls (data not shown). The oxidation of pyruvate is very low compared to other substrates in both skeletal muscle and liver. Activity of PDHc was decreased in muscle and liver: 0.011 and 0.003 units enzyme activity/mg protein respectively (control ranges: muscle: 0.092-0.16 [n = 3]; liver: 0.006-0.010 [n = 3]).
Immunochemical studies Antibodies against ox-heart PDHc reacted with all the components of the PDHc in human muscle mitochondrial fractions. Immunoblot analysis of muscle mitochondrial fractions, using antibodies against the holoenzyme, showed markedly lowered concentrations of immunoreactive E 1a and EIß in the patient compared to controls (Fig. 2). Immunoblotting with antibodies against the E 1 subunits revealed a good reaction with E 1a and E 1ß subunits in controls, while in the patient low concentrations of immunoreactive E 1a with no detectable E 1ß can be observed (Fig. 2).
Ketogenic diet Introduction of a ketogenic diet resulted in a fall of blood lactate concentration to near normal values (Fig.3). The ß-hydroxybutyrate and acetoacetate concentrations increased to moderately elevated levels (0.3 - 0.85 mmol/ 1, normal< 0.2). The child's clinical condition improved considerably: the tachypnoea, apathy and horizontal gaze paralysis disappeared in 9 days. When the diet contained 66 % fat and 23 % carbohydrates a mild keto-acidosis was noted and the diet was changed to 58 % fat and 31 % carbohydrates. A short peri-
149
Downloaded by: National University of Singapore. Copyrighted material.
Neuropediatrics 23 (1992)
Leigh Syndrome Associated with PDHc Deficiency
150 Neuropediatrics 23 (1992) "··",,,,·,'t····;..,..,.,,:,.·:·.
F. A. Wijburg et al
