Elevated cerebrospinal fluid lactate/pyruvate ratio in Machado-Joseph disease

To identify the metabolic alterations related to mitochondrial functions in Machado-Joseph disease (MJD), we analyzed the cerebrospinal fluid (CSF) levels of lactate, pyruvate, and citric acid cycle intermediates by high performance liquid chromatography (HPLC) in 7 Japanese patients with that disease and then measured some mitochondrial enzymes. Their mean age was 46 years. Diseased controls were matched by age to the patients studied. The CSF level of lactate was significantly elevated, pyruvate was significantly decreased, and the lactate/pyruvate (L/P) ratio was significantly elevated in the patients with MJD. There were no significant differences of citric acid cycle intermediates of the CSF between the patients and the controls. We measured the native and dichloroacetate (DCA)-activated pyruvate dehydrogenase complex (PDHC) activities, and mitochondrial electron transport activities in 3 patients with MJD, and found these activities to be normal. Therefore, the increased CSF lactate, increased lactate/pyruvate ratio, and decreased pyruvate may reflect the decreased regional cerebral blood flow rather than metabolic derangement of the mitochondria.     

Gender-specific occurrence of West syndrome in patients with pyruvate dehydrogenase complex deficiency

Pyruvate dehydrogenase complex (PDHC) deficiency, a major cause of congenital lactic acidemia in children, usually is complicated by seizures, and, in some patients, West syndrome has occurred. We diagnosed 60 patients with PDHC deficiency, including equal numbers of affected males and females. We studied the clinical features in 10 patients with West syndrome caused by PDHC deficiency, and examined the relation to the mutation of the E(1)alpha subunit, representing the great majority of PDHC deficiencies. Among 30 boys and 30 girls with PDHC deficiency,1 boy and 9 girls had West syndrome, even though overall West syndrome shows a slight male preponderance. Therefore, West syndrome associated with PDHC deficiency occurred in 9 of 30 female patients (33%), but in only 1 of 30 male patients (3%).The frequency of West syndrome in patients with PDHC deficiency was significantly higher in females than in males(p<0.05). Lactate concentrations in blood and CSF should be measured in female patients with West syndrome as a screening test for PDHC deficiency, because of gender-specific occurrence of West syndrome caused by PDHC deficiency.     

Diagnosis and molecular analysis of three male patients with thiamine-responsive pyruvate dehydrogenase complex deficiency

Pyruvate dehydrogenase complex (PDHC) deficiency is a major cause of congenital lactic acidemia in children. PDHC catalyzes the thiamine-dependent decarboxylation of pyruvate. Thiamine treatment was effective for some patients with PDHC deficiency. We reexamined 30 patients with congenital lactic acidemia of unknown origin who had normal PDHC activity in their cultured fibroblasts using a routine assay with a high (0.4 mM) thiamine pyrophosphate (TPP) concentration. We measured the activity of PDHC in the presence of a low (1x10(-4) mM) TPP concentration, and analyzed for mutations in the E1alpha subunit gene. Three males had low PDHC activity in the presence of 1x10(-4) mM TPP. The DNA sequence of these three patients' X-linked E1alpha subunit revealed a substitution of alanine for valine at position 71 (V71A) in exon 3, phenylalanine for cysteine at position 101 (C101F) in exon 4, and glycine for arginine at position 263 (R263G) in exon 8, respectively. Thiamine treatment was effective in these three patients. Therefore, they had a thiamine-responsive PDHC deficiency due to a point mutation in the E1alpha subunit gene. PDHC activity should be measured at a low TPP concentration to detect thiamine-responsive PDHC deficiency so that thiamine treatment can be initiated as soon as possible.     

Dichloroacetate exerts therapeutic effects in transgenic mouse models of Huntington's disease.

Dichloroacetate (DCA) stimulates pyruvate dehydrogenase complex (PDHC) activity and lowers cerebral lactate concentrations. In the R6/2 and N171-82Q transgenic mouse models of Huntington's disease (HD), DCA significantly increased survival, improved motor function, delayed loss of body weight, attenuated the development of striatal neuron atrophy, and prevented diabetes. The percentage of PDHC in the active form was significantly reduced in R6/2 mice at 12 weeks of age, and DCA ameliorated the deficit. These results provide further evidence for a role of energy dysfunction in HD pathogenesis and suggest that DCA may exert therapeutic benefits in HD.     

Pyruvate dehydrogenase activity in the rat cerebral cortex following cerebral ischemia

The effect of cerebral ischemia on the activity of pyruvate dehydrogenase (PDH) enzyme complex (PDHC) was investigated in homogenates of frozen rat cerebral cortex following 15 min of bilateral common carotid occlusion ischemia and following 15 min, 60 min, and 6 h of recirculation after 15 min of ischemia. In frozen cortical tissue from the same animals, the levels of labile phosphate compounds, glucose, glycogen, lactate, and pyruvate was determined. In cortex from control animals, the rate of [1(-14)C]pyruvate decarboxylation was 9.6 +/- 0.5 nmol CO2/(min-mg protein) or 40% of the total PDHC activity. This fraction increased to 89% at the end of 15 min of ischemia. At 15 min of recirculation following 15 min of ischemia, the PDHC activity decreased to 50% of control levels and was depressed for up to 6 h post ischemia. This decrease in activity was not due to a decrease in total PDHC activity. Apart from a reduction in ATP levels, the acute changes in the levels of energy metabolites were essentially normalized at 6 h of recovery. Dichloroacetate (DCA), an inhibitor of PDH kinase, given to rats at 250 mg/kg i.p. four times over 2 h, significantly decreased blood glucose levels from 7.4 +/- 0.6 to 5.1 +/- 0.3 mmol/L and fully activated PDHC. In animals in which the plasma glucose level was maintained at control levels of 8.3 +/- 0.5 mumol/g by intravenous infusion of glucose, the active portion of PDHC increased to 95 +/- 4%. In contrast, the depressed PDHC activity at 15 min following ischemia was not affected by the DCA treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of dichloroacetate on brain lactate levels following incomplete ischemia in the hyperglycemic rat

Dichloroacetate (DCA) is known to prevent the phosphorylation of the pyruvate dehydrogenase complex (PDHC) by blocking the action of PDH kinase. This action allows the active PDHC to exert its effect on the metabolism of glucose, lactate and alanine to acetyl CoA. DCA has been shown to reduce serum lactate levels in humans and animals in such conditions as diabetes, phenformin-induced hepatic failure, exercise, and endotoxin-induced shock. Lactic acidosis in the brain has often been postulated as a cause of neuronal damage following ischemia and hypoxia. Therefore, we examined the effect of intravenously administered DCA (100 mg/kg) in rats that were rendered hyperglycemic by intravenous glucose (2 g/kg), and then made to undergo 15 minutes of incomplete cerebral ischemia by bilateral carotid ligation and systemic hypotension (mean arterial pressure of 50 mm Hg). DCA significantly reduced serum lactate levels pre-ischemia, but had no effect on serum lactate levels after ischemia induction. Brain levels of lactate, ATP and PCr after 15 minutes of incomplete ischemia were unaffected by DCA. We conclude that in this in-vivo model the control of PDHC activity in the brain may be different than that in the periphery, and that DCA was not effective in reducing brain tissue lactate levels.     

Pyruvate dehydrogenase-E1α deficiency presenting as recurrent acute proximal muscle weakness of upper and lower extremities in an 8-year-old boy

The mitochondrial pyruvate dehydrogenase enzyme complex (PDHC) plays an important role in aerobic energy metabolism and acid–base equilibrium. PDHC contains of 5 enzymes, 3 catalytic (E1, E2, E3) and 2 regulatory, as well as 3 cofactors and an additional protein (E3-binding protein) encoded by nuclear genes. The clinical presentation of PDHC deficiency ranges from fatal neonatal lactic acidosis to chronic neurologic dysfunction without lactic acidosis. Paroxysmal neurologic problems such as intermittent ataxia, episodic weakness, exercise-induced dystonia and recurrent demyelination may also be seen although they are rare. Here, we present an 8-year-old boy complaining of acute proximal muscle weakness of upper and lower extremities with normal mental status. He had a history of Guillain–Barré-like syndrome at the age of 2 years. Electrophysiologic studies showed sensorial polyneuropathy findings in the first attack and sensorimotor axonal polyneuropathy findings in the last attack. The genetic analysis revealed a previously reported hemizygote novel mutation of the PDHA1 gene (p.A353T/c.1057G > A), which encodes the E1α subunit of PDHC. Thiamine was ordered (15 mg/kg/day), dietary carbohydrates were restricted and clinical findings improved in a few weeks. This rare phenotype of PDHC deficiency is discussed.     

The Rett syndrome and CSF lactic acid patterns.

We investigated both blood and cerebrospinal fluid (CSF) lactate and pyruvate levels in seven girls with the Rett syndrome (RS) and evaluated the relationship between CSF lactate and pyruvate levels and the clinical manifestations, particularly seizures, anticonvulsant medication, and breathing dysfunction including breath holding, apnea and hyperventilation. Elevated lactate and pyruvate levels in CSF with normal serum lactate were found in two RS patients. Elevated CSF lactate correlated significantly with the clinical occurrence of hyperventilation (P0 = 0.048, Fisher exact probability). We measured native and dichloroacetate (DCA)-activated pyruvate dehydrogenase (PDH) complex activities in two patients (#1 and 2) using cultured lymphoblastoid cell lines which were transformed by EB virus and the results were normal. We also analyzed CSF citric acid intermediates from 7 RS patients including citric acid, cis-aconitate, alpha-ketoglutarate, succinate, fumarate, malate and oxaloacetate. These concentrations were not significantly different from those control patients (N = 21). An elevated lactate level may be a clue to clarify the etiology of RS.     

Abnormalities of mitochondrial enzymes in hereditary ataxias

The activity of 7 mitochondrial enzymes, fumarase, NAD-malate dehydrogenase (MDH), citrate synthase (CS), valine dehydrogenase (VDH), succinate dehydrogenase (SDH), glutamate dehydrogenase (GDH), pyruvate dehydrogenase complex (PDHC) has been measured in platelet preparations from patients affected by Friedreich's ataxia (FA), dominant and non-dominant olivopontocerebellar atrophy (DOPCA, NDOPCA) and normal individuals. Significant decreases of GDH (P less than 0.01), PDHC (P less than 0.01), VDH (P less than 0.05) and SDH (P less than 0.05) activities were observed in FA patients. Significant decreases of GDH (P less than 0.01), PDHC (P less than 0.01), VDH (P less than 0.05), SDH (P less than 0.05) and CS (P less than 0.05) activities were Observed in ND-OPCA patients, whereas in DOPCA patients only GDH activity was significantly (P less than 0.05) decreased. In 8 of 10 patients with FA and in all patients with NDOPCA the activity of one or more of 4 enzymes, i.e. GDH, VDH, SDH, PDHC, was lower than the lowest of control values. Four of 6 patients with DOPCA had GDH activity lower than the lowest of control values. These results indicate that abnormalities of mitochondrial metabolism is a constant element in hereditary ataxia and suggest that the alteration primary leading to the different types of ataxias should be related to mitochondrial oxidative metabolism, at least at a regulatory level.     

Deficiency of pyruvate dehydrogenase complex (PDHC) in Leigh's disease fibroblasts: an abnormality in lipoamide dehydrogenase affecting PDHC activation

Several groups have reported abnormalities of the pyruvate dehydrogenase complex (PDHC) in cultured cells or other tissues from patients with Leigh's disease (subacute necrotizing encephalomyelopathy). We therefore undertook studies to elucidate the molecular basis of the defect of PDHC in cultured skin fibroblasts from two patients with Leigh's disease. The deficit of total PDHC activity in homogenates of Leigh's disease fibroblasts could be restored by adding exogenous lipoamide dehydrogenase (LAD, E3), the third component of PDHC. The LAD in these Leigh's disease cells had a markedly reduced ability (less than 20% of normal LAD) to reconstitute with other PDHC components to form active enzyme complex. A polyclonal antibody to pig heart LAD inhibited LAD activity in control cells more efficiently than in Leigh's disease cells. Other mitochondrial enzyme activities and growth of these two Leigh's disease cells appeared normal. These results suggest that the deficiency of PDHC in these two patients with Leigh's disease was due to a structural abnormality of the LAD component of PDHC.     

Beneficial effect of pyruvate therapy on Leigh syndrome due to a novel mutation in PDH E1α gene

Leigh syndrome (LS) is a progressive untreatable degenerating mitochondrial disorder caused by either mitochondrial or nuclear DNA mutations. A patient was a second child of unconsanguineous parents. On the third day of birth, he was transferred to neonatal intensive care units because of severe lactic acidosis. Since he was showing continuous lactic acidosis, the oral supplementation of dichloroacetate (DCA) was introduced on 31st day of birth at initial dose of 50 mg/kg, followed by maintenance dose of 25 mg/kg/every 12 h. The patient was diagnosed with LS due to a point mutation of an A-C at nucleotide 599 in exon 6 in the pyruvate dehydrogenase E1α gene, resulting in the substitution of aspartate for threonine at position 200 (N200T). Although the concentrations of lactate and pyruvate in blood were slightly decreased, his clinical conditions were deteriorating progressively. In order to overcome the mitochondrial or cytosolic energy crisis indicated by lactic acidosis as well as clinical symptoms, we terminated the DCA and administered 0.5 g/kg/day TID of sodium pyruvate orally. We analyzed the therapeutic effects of DCA or sodium pyruvate in the patient, and found that pyruvate therapy significantly decreased lactate, pyruvate and alanine levels, showed no adverse effects such as severe neuropathy seen in DCA, and had better clinical response on development and epilepsy. Though the efficacy of pyruvate on LS will be evaluated by randomized double-blind placebo-controlled study design in future, pyruvate therapy is a possible candidate for therapeutic choice for currently incurable mitochondrial disorders such as LS.     

Intravenous ketogenic diet therapy for neonatal-onset pyruvate dehydrogenase complex deficiency

BackgroundPyruvate dehydrogenase complex (PDHC) deficiency is an inborn error of metabolism that causes lactic acidosis and neurodevelopmental changes. Five causative genes have been identified: PDHA1, PDHB, DLAT, DLD, and PDHX. Four neurological phenotypes have been reported: neonatal encephalopathy with lactic acidosis, non-progressive infantile encephalopathy, Leigh syndrome, and relapsing ataxia. Of these, neonatal encephalopathy has the worst mortality and morbidity and there is no effective treatment.Subjects and methodsWe studied two girls who were clinically diagnosed with PDHC deficiency as neonates; they were subsequently found to have PDHA1 mutations. The clinical diagnosis was based on white matter loss and a lateral ventricular septum on fetal MRI, spasticity of the lower extremities, and lactic acidosis worsening after birth. Intravenous ketogenic diets were started within 24 h after birth. The ketogenic ratio was increased until the blood lactate level was controlled, while monitoring for side effects.ResultsIn both cases, the lactic acidosis improved immediately with no apparent side effects. Both children had better developmental outcomes than previously reported cases; neither exhibited epilepsy.ConclusionsIntravenous ketogenic diet therapy is a treatment option for neonatal-onset PDHC deficiency. Further studies are needed to optimize this therapy.     

Inhibition of lactate-induced swelling by dichloroacetate in human astrocytoma cells.

High levels of tissue lactate exacerbate tissue damage that results from cerebral ischemia and reperfusion injury that follows. Post-ischemic treatment with dichloroacetate (DCA) facilitates a decrease in lactate in the central nervous system (CNS) of animals during reperfusion following experimental ischemia, thus it may help to ameliorate ischemic cell damage. It has been suggested that the lactate lowering effect is mediated through a stimulatory effect of DCA on pyruvate dehydrogenase (PDHC) activity. We have studied such a hypothesis in a human astrocytoma derived cell line, UC-11MG. Under conditions resembling those of the ischemic tissue (i.e. high lactate and low pH) these cells accumulate lactate, driven by the inwardly directed proton gradient, and swell as a consequence of the osmotic effect of intracellular lactate. We have demonstrated that DCA increases PDHC activity and also reduces lactate-induced swelling. However, we also found that these two effects could be uncoupled and that the ability of DCA to prevent swelling is still present in the absence of any stimulation of PDHC. We also demonstrated that DCA competitively inhibits the uptake of lactate (Ki = 1.9 mM) and increases the efflux of lactate in a trans-acting manner that suggests the presence of a lactate-DCA exchange. We present a mechanism by which reduction in the rate of lactate uptake could account for the observed inhibition of swelling. This effect of DCA on lactate transport indicates another possible mechanism of action for DCA in facilitating the decrease in lactate observed in vivo during reperfusion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of sodium dichloroacetate on human pyruvate metabolism

Sodium dichloroacetate (DCA) was administered orally at doses of 12.5 to 50 mg/kg body weight twice or three times per day to a patient with mitochondrial encephalomyopathy associated with congenital lactic acidemia. During therapy, the rates of decarboxylation of (1-14C) pyruvate and (3-14C) pyruvate, which represent the activity of the pyruvate dehydrogenase (PDH) complex and the function of the TCA cycle, respectively, were markedly increased in the platelets and increases in the lactate levels in the blood and urine during exercise were markedly reduced. These results suggest that oral administration of DCA causes significant increases in the activities of the PDH complex and TCA cycle not only in the platelets but also in various tissues of humans, which is important as a pathway for production of energy, resulting in decreases in the lactate and pyruvate levels in the blood and cerebrospinal fluid.     

Dichloroacetate treatment for mitochondrial cytopathy: long-term effects in MELAS

The long-term effects of the sodium salt of dichloroacetic acid (DCA) were evaluated in four patients with mitochondrial encephalomyelopathy with lactic acidosis and stroke-like episodes (MELAS) carrying A3243G mutation. Oral administration of DCA in MELAS patients was followed for an average of 5 years 4 months. Serum levels of lactate and pyruvate were maintained at around 10 and 0.6 mg/dl, respectively. Serum levels of DCA were 40-136 microg/ml. Symptoms responding to treatment included persistent headache, abdominal pain, muscle weakness, and stroke-like episodes. In contrast, no improvements in mental status, deafness, short stature, or neuroelectrophysiological findings were observed. Adverse effects included mild liver dysfunction in all patients, hypocalcemia in three and peripheral neuropathy in one. None of these adverse events was severe enough to require discontinuation of treatment. To determine suitable indications for DCA therapy, analysis of many more patients who have undergone DCA administration is required.     

Thiamine-responsive congenital lactic acidosis: clinical and biochemical studies

We studied six infants with thiamine-responsive congenital lactic acidosis and normal pyruvate dehydrogenase complex activity in vitro, through clinical and biochemical analysis. In addition to elevated lactate and pyruvate levels, the data revealed increased urinary excretion of alpha-ketoglutarate, alpha-ketoadipate, and branched chain ketoacids, indicating functional impairment of thiamine-requiring enzymes, such as pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, alpha-ketoadipate dehydrogenase, and branched chain amino acid dehydrogenase. The metabolism of thiamine has not been investigated in patients with thiamine-responsive congenital lactic acidosis. We evaluated two specific transport systems, THTR-1 (SLC19A2) and THTR-2 (SLC19A3), and a pyrophosphorylating enzyme of thiamine, thiamine pyrophosphokinase (hTPK 1), in addition to pyruvate dehydrogenase complex and alpha-ketoglutarate dehydrogenase complex activity; no abnormality was found. Although the clinical features of thiamine-responsive congenital lactic acidosis are heterogeneous and clinical responses to thiamine administration vary, we emphasize the importance of early diagnosis and initiation of thiamine therapy before the occurrence of permanent brain damage. Careful monitoring of lactate and pyruvate would be useful in determining thiamine dosage.     

An immunochemical study of the pyruvate dehydrogenase deficit in Alzheimer's disease brain

The activity of the pyruvate dehydrogenase complex (PDHC; EC 1.2.4.1, EC 2.3.1.12, and EC 1.6.4.3) was reduced to about 30% of control values in histologically unaffected occipital cortex of the brains of patients with Alzheimer's disease, as well as in histologically affected frontal cortex. In contrast, activity of another mitochondrial enzyme, glutamate dehydrogenase, was normal. Neither age nor time until postmortem study correlated significantly with PDHC activity in either Alzheimer or control samples, and PDHC was not inactivated significantly on incubation with homogenates of either Alzheimer or control brain. Antibodies against the highly purified bovine PDHC inhibited Alzheimer and control PDHC equally per unit of enzyme activity. Immunoblots also indicated that the PDHC antigens were not different in normal and Alzheimer brains. This antibody, however, inhibited Alzheimer PDHC more effectively than it did control PDHC, based on milligrams of protein, suggesting a reduced amount of normal PDHC protein. Other data suggest that the PDHC deficiency is related to mitochondrial damage and to impaired calcium homeostasis in Alzheimer nerve cells, which may then mediate a variety of other cellular impairments.     

Diagnosis of partial deficiency of the pyruvate dehydrogenase complex in biopsied muscle

We have measured the total activity of pyruvate dehydrogenase (PDH) complex, by in vitro activation with a broad specificity protein phosphatase, and the basal activity, supposed to be present in vivo, in biopsied muscles from three patients with PDH complex deficiency and 11 patients with lactic acidemia. Results showed that the total PDH complex activity must be determined in biopsied muscles for the diagnosis, because the basal activities of two of three patients with PDH complex deficiency overlapped those of two patients with lactic acidemia whose total activities were within normal range.     

Inhibition of pyruvate dehydrogenase complex (PDHC) by antipsychotic drugs

The effects of 11 antipsychotic drugs on the pyruvate dehydrogenase complex (PDHC) prepared from bovine heart and rat brain were investigated. All inhibited PDHC to varying extents. With clinically equivalent doses, chlorpromazine and thioridazine inhibited the most and fluphenazine and thiothixene the least. The relationship of degree of inhibition of PDHC by neuroleptics to clinical improvement of 32 outpatients treated with acetazolamide and thiamine (A + T) ancillary therapy for chronic mental illness suggests that patients treated with psychoactive drugs that inhibit PDHC the least are most likely to have a favorable response with A + T treatment.     

Acute flaccid paralysis as initial symptom in 4 patients with novel E1alpha mutations of the pyruvate dehydrogenase complex

We report on 4 boys from 3 families presenting initially in infancy with an acute onset of flaccid tetraparesis and areflexia, resembling Guillain-Barré syndrome (GBS). However, the cerebrospinal fluid (CSF) protein was normal, while serum and CSF lactate were elevated. All patients had recurrent similar episodes, usually associated with infections. Brain MRI showed T (2) hyperintensities in the basal ganglia in two boys, in one of them at the first clinical presentation; the other one had a normal brain MRI during the first episode. A third boy had a normal MRI twice but an increased lactate peak in the basal ganglia in (1)H-MR spectroscopy. Motor nerve conduction velocities (NCV) were normal in all patients. Biochemical analyses of muscle tissue, performed in two patients, revealed a deficiency of the pyruvate dehydrogenase (PDH). Molecular genetic analysis of the X-chromosomal E1alpha subunit of PDH showed three new mutations in phylogenetically conserved areas of the protein: Glu358Lys in patient 1; Arg88Lys in patient 2 and 3 (brothers); and Leu216Ser in patient 4. In conclusion, children with "atypical GBS" should be evaluated for a mitochondrial disorder, including pyruvate dehydrogenase deficiency, even after a first episode.