Research Notes: Ethylmalonic Aciduria

Neuropediatrics. 2007 Apr.
Ethylmalonic encephalopathy: clinical and biochemical observations.
Zafeiriou DI, Augoustides-Savvopoulou P, Haas D, Smet J, Triantafyllou P, Vargiami E, Tamiolaki M, Gombakis N, van Coster R, Sewell AC, Vianey-Saban C, Gregersen N.
1Department of Pediatrics, Aristotle University of Thessaloniki, Thessaloniki, Greece.

Ethylmalonic encephalopathy (EE) is a rare, recently defined inborn error of metabolism which affects the brain, gastrointestinal system and peripheral blood vessels and is characterized by a unique constellation of clinical and biochemical features. A 7-month-old male, who presented with psychomotor retardation, chronic diarrhea and relapsing petechiae is described with the objective of highlighting the biochemical and neuroradiological features of this disorder as well as the effect of high-dose riboflavin therapy. Urinary organic acid analysis revealed markedly increased excretion of ethylmalonic acid, isobutyrylglycine, 2-methylbutyrylglycine and isovalerylglycine. Acylcarnitine analysis in dried blood spots showed increased butyrylcarnitine. Short-chain acyl-CoA dehydrogenase (SCAD) activity in muscle was normal as were mitochondrial OXPHOS enzyme activities in cultured skin fibroblasts. In skeletal muscle the catalytic activity of complex II was decreased. Brain MRI revealed bilateral and symmetrical atrophy in the fronto-temporal areas, massive enlargement of the subarachnoid spaces and hyperdensities on T (2) sequences of the basal ganglia. Mutation analysis of the ETHE1 gene demonstrated homozygosity for the Arg163Gly mutation, confirming the diagnosis of EE at a molecular level. On repeat MRI, a significant deterioration was seen, correlating well with the clinical deterioration of the patient.

Mol Genet Metab. 2006 Dec.
A case of ethylmalonic encephalopathy with atypical clinical and biochemical presentation.
Di Rocco M, Caruso U, Briem E, Rossi A, Allegri AE, Buzzi D, Tiranti V.
II Pediatric Unit, Gaslini Institute, Largo Gaslini 5, Genoa, Italy.

A child is reported presenting with a clinical picture suggestive of genetic connective tissue disorders (vascular fragility, articular hyperlaxity, delayed motor development, and normal cognitive development), an absence of pathological ethylmalonic acid excretion during inter-critical phases and a homozygous R163W mutation in the ETHE1 gene. This case suggests that ethylmalonic aciduria is not a constant biochemical marker of ethylmalonic encephalopathy and that its normal excretion outside of metabolic decompensation episodes does not exclude this metabolic disease.

Arch Neurol. 2004 Apr.
The role of methionine in ethylmalonic encephalopathy with petechiae.
McGowan KA, Nyhan WL, Barshop BA, Naviaux RK, Yu A, Haas RH, Townsend JJ.
Institute of Molecular Genetics and the Department of Pediatrics, University of California San Diego, La Jolla, USA.
[ Free full text ]

BACKGROUND: Among patients with ethylmalonic aciduria, a subgroup with encephalopathy, petechial skin lesions, and often death in infancy is distinct from those with short-chain acyl-coenzyme A dehydrogenase deficiency or multiple acyl-coenzyme A dehydrogenase deficiency. The nature of the molecular defect in this subgroup is unknown, and the source of the ethylmalonic acid has been unclear. OBJECTIVE: To determine whether the administration of candidate amino acids increased the excretion of ethylmalonic acid. DESIGN: Examination of patterns of organic acids excreted in the urine before and following loading doses of isoleucine and methionine. SETTING: General clinical research center. PATIENT: An infant with ethylmalonic aciduria, global developmental delay, acrocyanosis, and intermittent showers of petechiae. MAIN OUTCOME MEASURE: Excretion of ethylmalonic acid in the urine. RESULTS: Loading with methionine increased the excretion of ethylmalonic acid, whereas loading with isoleucine did not. Restriction of the dietary intake of methionine decreased ethylmalonic acid excretion. CONCLUSION: In ethylmalonic acid encephalopathy with petechiae, methionine is a precursor of ethylmalonic acid.

From the full text article:

Ethylmalonic encephalopathy with petechiae is a progressive degenerative disease in which acrocyanosis and petechial skin lesions are prominent. Acute episodic metabolic crises occur in which there is acidosis and mild hypoglycemia. Neuroimaging results have revealed extensive frontotemporal atrophy and areas of high T2-weighted intensity in the basal ganglia. The defining metabolic abnormality is the excretion of large amounts of ethylmalonic acid in the urine. Excretion of adipic acid is not increased in contrast to multiple acyl-coenzyme A dehydrogenase deficiency, which has been referred to as ethylmalonic-adipic aciduria. Ethylmalonic aciduria is also encountered in short-chain acyl-coenzyme A dehydrogenase deficiency in which an initial feature is hypoketotic hypoglycemia, which is characteristic of disorders of fatty acid oxidation.

The vascular manifestations of this disorder are unusual and characteristic features. Acrocyanosis appears to be its mildest manifestation. The development of showers of petechiae in response to intercurrent illness has led to investigation and treatment for presumptive meningococcemia. Ecchymoses and hemorrhagic streaks have also been seen, as has an infarct of the basal ganglia. Further evidence of bleeding abnormality are microscopic hematuria and hemoperitoneum. Biopsy of the skin lesions has shown only hemorrhage. Dilated tortuous retinal vessels are seen.

Cerebral abnormality is manifest early in hypotonia and delayed development and later in spasticity. Generalized seizures are associated with abnormal focal discharges on the electroencephalogram. Neurologic deterioration accelerates following intercurrent infectious illness, and most patients have died in the early years of life.

It is the purpose of this report to describe a patient in whom methionine loading led to a marked increase in the excretion of ethylmalonic acid and restriction of the intake of methionine led to decreased excretion.

Case 1

This patient was 8 months of age when she was admitted to the University of California San Diego General Clinical Research Center in San Diego. She had appeared to develop normally for 3 months after which she lost the ability to control her head, reach for objects, and sit with support. By 8 months of age, she was limp, disinterested in her surroundings, and spent most of her time sleeping. Infantile spasms began 1 month prior to hospital admission. She also had absence seizures in which the eyes rolled. Striking easy bruising began at 4 months of age. Hematological investigation at a university hospital revealed a normal platelet count, prothrombin time, partial thromboplastin time, factor VIII activity, ristocetin cofactor activity, and von Willebrand factor antigen. Lactate concentrations in serum taken on 2 occasions were 45.9 mg/dL (5.1 mmol/L) and 36.9 mg/dL (4.1 mmol/L). Her pyruvate level was 0.001 mg/dL (0.17 mmol/L). Her creatine kinase level was 2100 U/L. The electroencephalographic pattern was that of hypsarrythmia. There were several generalized epileptiform abnormalities. Magnetic resonance images revealed patchy T2-weighted hyperintensities in the putamen and cortical atrophy. Muscle biopsy findings showed increased lipid droplets but no ragged red fibers; electromicroscopy results showed there were mildly increased numbers of pleomorphic subsarcolemnal mitochondria. No deletions or point mutations were found in the mitochondrial DNA of the muscle. Enzyme analysis of frozen muscle at the Molecular Cardiology Institute, Highland Park, NJ, revealed reduced activity of complex III, IV, and V. Activity of citrate synthase was not increased.

The parents were cousins in whom the mother's paternal grandmother was the sister of the father's maternal grandfather (Figure 2). The patient had sisters 6 and 9 years of age who were well. A 13-year-old brother was well except for myopia. A brother (case 2) died at 8 months of age of what appeared to be the same disease as in case 1.

On examination, her weight was 7.1 g (10th percentile); length, 165 cm (5th percentile); and head circumference, 42.5 cm (25th percentile). She was very floppy and unresponsive and had virtually constant infantile spasms. She had epicanthal folds, upslanting palpebral fissures, and an upturned nose with a depressed nasal bridge. Her parents noted that her facial features were similar to those of her brother (case 2) and dissimilar to her other siblings. Deep tendon reflexes were markedly increased, and there was sustained ankle clonus at the slightest touch. There was a soft systolic precordial murmur. Studies of nerve conduction velocity revealed evidence of sensory neuropathy in the legs. There were multiple cutaneous petechiae.

Her hemoglobin level was 8.5 g/L, and her hematocrit level was 27.9%. Her serum lactate concentration was 53.4 mg/dL (5.7 mmol/L). Her bicarbonate level was 15 mEq/L. Her values for sodium, potassium, and chloride were 143 mEq/L, 4.0 mEq/L, and 112 mEq/L, respectively. Her uric acid level was 0.005 mg/dL (0.27 mmol/L). The patient had a normal prothrombin time (10.9 seconds; control subject, 10.3 seconds), partial thromboplastin time (24.2 seconds; control subject, 27 seconds), bleeding time (6 minutes, 30 seconds), and von Willebrand factor antigen. Platelet counts were normal (564 x 103/µL). The level of plasminogen activator inhibitor–1 was markedly elevated at 35 ng/mL (reference range, 3-5 ng/mL).

Concentrations of amino acids in the blood and cerebrospinal fluid were normal. Examination results of the mitochondrial DNA of the serum were negative for point mutations and deletions. The plasma concentration of free carnitine was low at 14 µmol/L (reference range, 21-53 µmol/L). Her urinary free carnitine level was 11 mmoL/mol of creatine, whereas her esterified carnitine level was 57 mmoL/mol of creatine, yielding an elevated esterified carnitine–urinary free carnitine ratio of 5.2 (reference range, 0-3). Analysis of the organic acids of the urine revealed ethylmalonic aciduria. The level was 276 mmoL/mol of creatinine (reference range, 0-11). The values for adipic and glutaric acids were 79 mmoL/mol of creatine and 11 mmoL/mol of creatine, respectively (reference ranges, 0-12 mmoL/mol of creatine and 0-5mmoL/mol of creatine, respectively). An acylcarnitine profile revealed increased C4 and C5 acylcarnitine esters, as well as increased C2.

Case 2

The infant in this case developed irritability at 2 months of age and hypotonia and myoclonic jerks a month later. He also had episodic petechiae, metabolic acidosis, and lactic acidemia. Magnetic resonance images revealed symmetric T2-weighted hyperintensities in the basal ganglia. Progressive neurodegeneration was followed by death at 8 months of age. At autopsy, the brain weighed 630 g. One half of the brain was frozen, and the other half was fixed in formaldehyde. On gross examination, the coronal sections of the fixed half of the brain appeared normal except for cortical thinning in the insular cortex. Microscopically, there was marked capillary proliferation in the substantia nigra, periaqueductal area, putamen, caudate, and medial thalamus (Figure 3). The endothelial cells were increased in number and size. There was a relative sparing of neurons and pallor of the background parenchyma that was quite prominent in the substantia nigra (Figure 4). There was no loss of the background parenchyma or cystic change. Only minimal astrocytosis was found. The insular cortex demonstrated pallor and capillary proliferation without good preservation of neurons. There was vacuolization of compacted white matter tracts. This was prominent in the crossing of the superior cerebellar peduncle and medial lemniscus. There were a few vacuoles in the optic chaism. The location of the lesions, vascular proliferation, and relative sparing of neurons were similar to what is found with subacute necrotizing encephalopathy (Leigh disease).

Loading with methionine and isoleucine was carried out in the General Clinical Research Center after the patient fasted for at least 4 hours. The dose of each was 100 mg/kg by mouth. Urine was obtained prior to and at 4 and 8 hours after the administration of the amino acid. Ethylmalonic acid concentration was determined by organic acid analysis performed as described by Hoffmann et al.8

The excretion levels of ethylmalonic acid following loading with isoleucine and methionine is presented in Table 1. Isoleucine loading was performed on 2 occasions because of consideration of the pathway by which isoleucine might be a precursor and because of the report by Malgorzata et al in which isoleucine appeared to be a precursor. However, on neither occasion did the excretion of ethylmalonic acid increase appreciably following isoleucine loading. On the second occasion, the plasma concentration of isoleucine rose from 0.62 mg/dL (47 µmol/L) to 6.28 mg/dL (479 µmol/L) 4 hours after the load. Alloisoleucine was not detected following isoleucine loading. On this occasion, the baseline urine was a 24-hour collection. Following isoleucine loading, the excretion levels of methylsuccinic acid were 28 mmol/mol of creatine, 29 mmol/mol of creatine, and 24 mmol/mol of creatine on the first load and increased from 19 mmol/mol of creatine to 32 mmol/mol of creatine on the second. 2-Methylbutyrylglycine levels were 20 mmol/mol of creatine and 25 mmol/mol of creatinine after isoleucine loading and 4 mmol/mol of creatinine after methionine loading. Isobutyrylglycine levels were 12 mmol/mol of creatine and 25 mmol/mol of creatinine before and after methionine loading, respectively. The serum concentration of lactic acid decreased from 21.4 mg/dL (2.4 mmol/L) to 17.6 mg/dL (2.0 mmol/L) after isoleucine loading and from 25.7 mg/dL (2.9 mmol/L) to 35.3 mg/dL (3.9 mmol/L) after methionine loading.

Following the methionine load, the excretion of ethylmalonic acid rose 1.7 times to a concentration of 648 mmol/mol of creatinine. Furthermore, the excretion of ethylmalonic acid was measured 8 times across 50 days, and the values ranged from 224 mmol/mol of creatinine to 384 mmol/mol of creatinine, indicating the value after methionine loading to be quite unusual. In addition, the concentration of methylsuccinic acid, which did not change following isoleucine loading and ranged generally from 21 mmol/mol of creatinine to 38 mmol/mol of creatinine, rose to 65 mmol/mol of creatinine following methionine loading. The concentration of methionine in serum had risen from 0.3 mg/dL (20 µmol/L) to 7.7 mg/dL (520 µmol/L) 4 hours after the methionine load. The urinary methionine level was 60 mmol/mol of creatinine 4 hours after the methionine load. A load of 0.5 g/kg of medium-chain triglyceride was followed, as expected, by a rise in ethylmalonic acid excretion from 280 mmol/mol of creatinine to 377 mmol/mol of creatinine. Both parents were given methionine loads. There was no detectable ethylmalonic acid in their urine before or after methionine loading.

Following the results of methionine loading, the patient was treated with a diet restricted in methionine. She was given a mixture of Similac (Ross; Columbus, Ohio), Propimex (Ross), and Moducal (Mead Johnson; Evansville, Ind), providing 111 cal/kg at a weight of 7.8 kg, 0.8 g/kg of whole protein, and 20.5 mg/kg of methionine. Solid foods were selected from a list to provide no more than 10 mg of methionine per day. For a healthy infant, a diet composed of Similac would provide 69 mg/kg per day of methionine. The excretion of ethylmalonic acid decreased with this regimen to 186 mmol/mol of creatinine. Her methylsuccinic acid level was 15 mmol/mol of creatinine. Baseline preloading urinary ethylmalonic acid levels had ranged in the month preceding the diet from 224 mmol/mol of creatinine to 367 mmol/mol of creatinine. The parents reported increased alertness and beginning vocalization. Concentrations of serum lactic acid and bicarbonate became normal. The patient developed an acute febrile illness diagnosed as sinusitis and died at 11 months of age.

A relationship between this syndrome of ethylmalonic aciduria and the metabolism of sulfur amino acids has been proposed by Duran et al, who found increased excretion levels of inorganic thiosulfite and an absence of detectable sulfite. They also reported 2 sulfur-containing acidic amino acids, S-sulfocysteine and S-sulfothiocysteine, each of which can be formed nonenzymatically from thiosulfate and cysteine. The increase in excretion of ethylmalonic acid in our patient following methionine loading is consistent with these observations. The mechanism and the site of the defect are not clear.

Methionine is converted normally to homoserine and cysteine. Homoserine is converted to 2-oxobutyric acid, which could be a source of ethylmalonic acid. Cysteine is converted to 2-mercaptopyruvic acid, which is metabolized to pyruvic acid, thiosulfate, and ultimately sulfate. Ethylmalonic acid can be formed via carboxylation of butyric acid, and this appears to be the source of ethylmalonic acid found in short-chain acyl-coenzyme A dehydrogenase deficiency and in multiple acyl-coenzyme A dehydrogenase deficiency. In our patient, loading with a medium-chain triglyceride did not greatly increase the excretion of ethylmalonic acid. Ethylmalonic acid could be a product of isoleucine metabolism after racemization of 2-oxo-3-methylvaleric acid, the precursor of alloisoleucine, from the S to the R form, which is then convertible to 2-methylbutyryl-coenzyme A, 2-ethyl-3-hydroxypropionyl-coenzyme A, and ethylmalonic semialdehyde and then to ethylmalonic acid. In our patient, loading with isoleucine did not change the excretion of ethylmalonic acid.

Elevated excretion of ethylmalonic acid and methylsuccinic acid after a load of isoleucine was reported by Malgorzata et al. In these studies, the patient was given 150 mg/kg of isoleucine following which they observed accumulation of 2-methylbutyrylglycine and levels of tiglylglycine lower than in 3 control subjects, but the level of 2-methylbutyrylglycine exceeded 2 SDs above the control mean only slightly and only on 1 time point and the tiglylglycine was quite close to the 2 SDs line for the control subjects. Furthermore, the increase in ethylmalonic acid levels in the urine is difficult to evaluate because the response to isoleucine loading was given in micromoles per liter, whereas the diagnostic level was given in the more conventional millimoles per mole of creatinine. In our patient, 2-methylbutyrylglycine was found in small amounts in the urine only after isoleucine loading. Malgorzata et al postulated a block at the levels of 2-methylbutyryl-coenzyme A dehydrogenase; however, the activity of this enzyme was studied and found to be normal. Furthermore, Ozand et al found the oxidation of 14C-isoleucine to 14CO2 to be normal in fibroblasts derived from typical patients. In these studies, the oxidation of 14C-butyrate was normal, consistent with our findings on triglyceride loading. Normal oxidation of fatty acids was also observed by Burlina et al. It is not clear why results were different following isoleucine administration in our patient and the patient of Malgorzata et al, but neither of the siblings reported by their group had petechiae, so they could have been studying a different disease.

A relationship between ethylmalonic aciduria and defective mitochondrial electron transport has been raised by a number of investigators, but the phenotype of these patients appears quite distinct from the ethylmalonic encephalopathy with petechiae syndrome. Hoffmann et al reported a fatal progressive pancytopenia with ethylmalonic aciduria in which there were paracrystalline inclusion bodies on electronmicroscopy findings and reduced activity of cytochrome c oxidase and reductase in muscle. Progressive neurologic disease and partial deficiency of cytochrome oxidase were reported by Lehnert and Ruitenbeek; neither patient had petechiae. A third patient they described who had hematuria had conflicting results on cytochrome c oxidase activity.

A number of typical patients with this syndrome have been found to have normal cytochrome c oxidase activity in fibroblasts. On the other hand, Garavaglia et al reported studies on 2 patients with the typical ethylmalonic encephalopathy with petechiae syndrome in whom cytochrome oxidase activity in fibroblasts was normal but in whose muscles it ranged from 7% to 35% of control. The activity of short-chain acyl-coenzyme A dehydrogenase in fibroblasts was normal. Normal alleles for the short-chain acyl-coenzyme A dehydrogenase gene were reported by Nowaczyk et al. Another patient with cytochrome oxidase deficiency had a pattern of urinary organic acids dominated by ethylmalonic acid but entirely consistent in pattern with multiple acyl-coenzyme A dehydrogenase deficiency. However, in contrast to cells from patients with multiple acyl-coenzyme A dehydrogenase deficiency, fibroblasts from this patient displayed normal oxidation of myristic acid. The clinical manifestations of patients with this syndrome are clearly very different from those usually associated with cytochrome c oxidase deficiency, and certainly most patients with cytochrome c oxidase deficiency do not have ethylmalonic aciduria.

The brother of our proband, case 2, doubtless had the same disease as his sister. Lesions in the brain were suggestive of subacute necrotizing encephalopathy; however, the typical dropout of the background parenchyma seen in Leigh disease, which creates a partially cystic lesion, was not present, and there was vacuolization of compacted white matter tracts, which is not typical of Leigh disease. Vacuolization or spongiosus in white matter tracts has been documented in aminoacidemias such as nonketotic hyperglycinemia. To our knowledge, case 2 represents the first patient with ethylmalonic encephalopathy in whom an autopsy has been performed.

Results from previous studies to elucidate the bleeding in this disease have been normal. Biopsy results of the petechiae revealed only hemorrhage. Findings from studies of prothrombin time, partial thromboplastin time fibrinogen levels, bleeding time, and platelet counts have been normal. Studies of platelet aggregation were normal. Tortuous retinal vessels have suggested a vascular pathogenesis. The elevated levels of plasminogen activator inhibitor found in our patient may be relevant. This protein, which inhibits tissue plasminogen activator and urokinase, might intuitively be expected to promote thrombosis, and levels have been correlated with myocardial infarction and deep venous thrombosis. Tissue plasminogen activator is a fibrinolytic agent that is used in the treatment of thrombotic disease and bleeding is a complication of its use, but levels may be positively or negatively associated with infarction.

Metabolism. 1998 Jul.
Ethylmalonic and methylsuccinic aciduria in ethylmalonic encephalopathy arise from abnormal isoleucine metabolism.
Nowaczyk MJ, Lehotay DC, Platt BA, Fisher L, Tan R, Phillips H, Clarke JT.
Department of Pediatrics, Hospital for Sick Children and University of Toronto, Ontario, Canada.

Ethylmalonic encephalopathy (EE), an organic aciduria of unknown etiology characterized by developmental delay, hypotonia, and vascular instability associated with lactic acidemia and urinary excretion of ethylmalonic acid (EMA) and methylsuccinic acid (MSA), has been described in 11 patients. To test the possibility that the underlying biochemical defect involves isoleucine catabolism, we determined the response to oral L-isoleucine (IIe) load (150 mg/kg) in a 5-year-old girl with EE and in three healthy, age- and sex-matched controls. Following IIe load in the patient, there was accumulation of 2-methylbutyrylglycine (2-MBG) and a delayed and lower peak urinary excretion of tiglylglycine (TGL), suggesting a partial defect in 2-methyl-branched chain acylcoenzyme A dehydrogenase (2M-BCAD). In vitro measurements 2M-BCAD activity in cultured skin fibroblasts from patients with EE have been reported to be normal. Our results show that isoleucine is a source for the elevated EMA and MSA in patients with EE, and suggest a functional, possibly secondary, deficiency of activity of 2M-BCAD in vivo.

Pediatr Neurol. 1997 Sep.
Syndrome of encephalopathy, petechiae, and ethylmalonic aciduria.
Garcia-Silva MT, Ribes A, Campos Y, Garavaglia B, Arenas J.
Department of Pediatrics; Hospital 12 Octubre; Madrid, Spain.

We report a boy 20 months of age with encephalopathy, petechiae, and ethylmalonic aciduria (EPEMA). Other clinical features were severe hypotonia, orthostatic acrocyanosis, and chronic diarrhea. Magnetic resonance imaging (MRI) of the brain demonstrated bilateral lesions in the lenticular and caudate nuclei, periaqueductal region, subcortical areas, white matter, and brainstem. Short and medium chain Acyl-CoA dehydrogenase and cytochrome c oxidase (COX) activities in fibroblasts were normal. Muscle histochemistry disclosed diffuse COX deficiency, and respiratory chain activities in muscle disclosed severe COX deficiency. Twelve other patients with similar clinical features have been reported. Muscle COX activity, studied only in four, demonstrated a clear-cut defect.

Brain Dev. 1994 Nov.
Ethylmalonic aciduria: an organic acidemia with CNS involvement and vasculopathy.
Ozand PT, Rashed M, Millington DS, Sakati N, Hazzaa S, Rahbeeni Z, al Odaib A, Youssef N, Mazrou A, Gascon GG, et al.
Department of Pediatrics, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia.

Five infants from 3 families, one Egyptian, two Yemeni, are described with a progressive encephalopathy, four of whom have been studied in detail. All patients showed vascular lesions of the skin, characterized by waxing and waning petechiae and ecchymoses. Acrocyanosis was present in three patients. All patients showed retinal lesions characterized by tortuous veins. Protracted diarrhea was not a consistent finding, although they had metabolic crisis in association with diarrhea. They did not show failure to thrive. The neurologic symptoms were indicative of a progressive pyramidal tract disease. Three patients died following sudden emergence of severe basal ganglia, putaminal and head of caudate lesions. In one patient the CT changes in brain were suggestive of infarction. The patients who died manifested pulmonary congestion, or wet lung, and respiratory difficulties during the terminal stage of the disease. In all patients before and during the terminal event, mild-to-moderate hematuria, and in two RBC in CSF, was observed. In one patient there was mild hemoperitoneum at the terminal event. The urine organic acids indicated increased excretion of ethylmalonic, methylsuccinic, glutaric, and adipic acids. The patients invariably showed lactic acidosis, but no ketosis, during and in between the acidotic attacks of the disease. The acylcarnitine profile in blood of two patients showed a pronounced increase in C4 and C5 carnitine esters. In three patients, biopsies from petechiae indicated absence of an immune event, showing only fresh hemorrhage. An immunologic study in one patient was normal for the suppressor:cytotoxic lymphocyte ratio and concentration of interleukin-2 receptor during and in between hemorrhagic attacks. The cytochrome c oxidase activity in fibroblasts was normal. The rate of oxidation of glucose, leucine, isoleucine, valine, propionate and butyrate by fibroblasts was normal. The disease is not responsive to treatment with riboflavin, ascorbic acid, vitamin E, glycine, or carnitine. One patient remained stable on prolonged large doses of methylprednisolone. The biochemical defect leading to ethylmalonic aciduria in this disease remains unknown.

J Pediatr. 1994 Jan.
A new syndrome with ethylmalonic aciduria and normal fatty acid oxidation in fibroblasts.
Burlina AB, Dionisi-Vici C, Bennett MJ, Gibson KM, Servidei S, Bertini E, Hale DE, Schmidt-Sommerfeld E, Sabetta G, Zacchello F, et al.
Department of Pediatrics, University of Padua, Italy.

We describe four Italian male infants with a novel clinical phenotype characterized by orthostatic acrocyanosis, relapsing petechiae, chronic diarrhea, progressive pyramidal signs, mental retardation, and brain magnetic resonance imaging abnormalities. The first symptoms appeared after the termination of breast-feeding and introduction of formula feeding. Marked persistent 2-ethylmalonic aciduria was associated with abnormal excretion of C4-C5(n-butyryl-, isobutyryl-, isovaleryl-, and 2-methylbutyryl-)acylglycines and acylcarnitines and with intermittent lactic acidosis. Short- and branched-chain plasma acylcarnitine levels were also elevated. 2-Ethylmalonic aciduria is generally regarded as being indicative of a defect in fatty acid oxidation. Extensive studies of cultured fibroblasts failed to reveal such a defect. The observation of intermittent urinary excretion of 2-ethylhydracrylic acid pointed to involvement of the isoleucine R pathway in ethylmalonate biosynthesis. This hypothesis was tentatively corroborated by the biochemical responses to an oral isoleucine challenge in two patients. However, fibroblast studies showed normal oxidation rates of (14C)isoleucine (ul), indicating that this is not a defect of isoleucine oxidation expressed in skin fibroblasts. In one of two patients tested, cytochrome c oxidase activity was partially reduced (45%) in cultured fibroblasts. This unique clinical and biochemical phenotype identifies a new metabolic encephalopathy of yet undetermined cause.

J Pediatr. 1993 Apr.
Effect of treatment with glycine and L-carnitine in medium-chain acyl-coenzyme A dehydrogenase deficiency.
Rinaldo P, Schmidt-Sommerfeld E, Posca AP, Heales SJ, Woolf DA, Leonard JV.
Department of Genetics, Yale University School of Medicine, New Haven, CT.

To assess the relative contribution of glycine and carnitine conjugation pathways to total acyl-group excretion, we investigated the excretion of C6 to C10 dicarboxylic acids, C6 to C8 acylglycines, and C6 to C8 acylcarnitines in five symptom-free patients with medium-chain acyl-coenzyme A dehydrogenase deficiency during sequential 1-week periods as follows: (1) no treatment, (2) oral supplementation with glycine, 250 mg/kg per day, (3) oral supplementation with L-carnitine, 100 mg/kg per day, and (4) oral supplementation with both combined. In untreated patients, acylglycines and acylcarnitines represented 60% and less than 1% of the total metabolite excretion, respectively; the average acylglycine/acylcarnitine ratio was 70:1. Oral supplementation with glycine did not alter the excretion of acylglycines or acylcarnitines. L-Carnitine supplementation increased the acylcarnitine excretion sixfold and caused a 60% reduction in acylglycine excretion (p < 0.001); however, even with carnitine supplementation, acylglycine excretion was still 10 times greater than that of acylcarnitines. The results suggest that glycine conjugation was the major pathway for the disposal of C6 to C8 acyl moieties and that oral L-carnitine supplements may inhibit glycine conjugation. The findings cast doubt on the value of long-term treatment of medium-chain acyl-coenzyme A dehydrogenase deficiency with L-carnitine.

Pediatr Res. 1991 Sep.
Ethylmalonic/adipic aciduria: effects of oral medium-chain triglycerides, carnitine, and glycine on urinary excretion of organic acids, acylcarnitines, and acylglycines.
Rinaldo P, Welch RD, Previs SF, Schmidt-Sommerfeld E, Gargus JJ, O'Shea JJ, Zinn AB.
Department of Genetics, Yale University, New Haven, Connecticut.

A 9-y-old girl with ethylmalonic/adipic aciduria was hospitalized to determine the possible therapeutic efficacy of oral carnitine and glycine supplementation. To provoke a mild metabolic stress, her diet was supplemented with 440 mg/kg/d of medium-chain triglycerides. She was treated successively with carnitine (100 mg/kg/d) for 5 d, neither carnitine nor glycine for 2 d, and then glycine (250 mg/kg/d) for 6 d. Consecutive 12-h urine collections were obtained throughout the entire period. The urinary excretion of eight organic acids, four acylglycines, and four acylcarnitines, which accumulate as a result of a metabolic block of five mitochondrial acyl-CoA dehydrogenases, were quantitatively determined by capillary gas chromatography, stable isotope dilution gas chromatography/mass spectrometry, and radioisotopic exchange HPLC, respectively. The excretion of each group of metabolites was calculated as the mean percentage of total output (mumol/24 h) during the four phases of the protocol (organic acids/acylglycines/acylcarnitines = 100.0%): 1) regular diet (3 d); 88.1/10.8/1.1; 2) medium-chain triglyceride supplementation (4); 82.5/15.6/1.9; 3) medium-chain triglycerides plus carnitine (5); 79.2/8.2/12.6; and 4) medium-chain triglycerides plus glycine (6); 81.0/18.7/0.3. Comparison between total and individual excretion of acylglycines and acylcarnitines indicates that oral glycine supplementation enhanced the conjugation and excretion of fatty acyl-CoA intermediates as efficiently as carnitine. We propose that oral glycine supplementation should be considered in the treatment of other inborn errors of metabolism associated with abnormal urinary excretion of acylglycines.

J Inherit Metab Dis. 1991.
Possible deleterious effect of L-carnitine supplementation in a patient with mild multiple acyl-CoA dehydrogenation deficiency (ethylmalonic-adipic aciduria).
Green A, Preece MA, de Sousa C, Pollitt RJ.
Department of Clinical Chemistry, Children's Hospital, Ladywood Middleway, Birmingham, UK.

A patient with riboflavin-responsive mild multiple acyl-CoA dehydrogenation deficiency of the ethylmalonic-adipic aciduria type experienced a recurrence of spontaneous hypoglycaemic episodes whilst being given supplementary L-carnitine. This phenomenon is explicable in terms of the known biochemical features of this condition and suggests caution in the carnitine supplementation of patients with defective oxidation of medium- or short-chain fatty acyl-CoA esters. This patient excreted excessive phenylpropionylglycine after an oral phenylpropionic acid load. Thus the phenylpropionic acid loading test is not completely specific for primary medium-chain acyl-CoA dehydrogenase deficiency as has been supposed.