Clinical and biochemical phenotype in 11 patients with mevalonic aciduria.

OBJECTIVE: Mevalonic aciduria is a consequence of the deficiency of mevalonate kinase, the first enzyme after 3-hydroxy-3-methylglutaryl-coenzyme A reductase in the biosynthesis of cholesterol and nonsterol isoprenes. To establish the clinical and biochemical phenotype of mevalonic aciduria, the authors assembled their experience with 11 patients including attempts at therapeutic interventions. METHODS: Mevalonic acid in body fluids was determined by stable isotope dilution gas chromatography/mass spectroscopy with selected ion monitoring, ubiquinone-10 concentrations by reversed-phase high-pressure liquid chromatography. RESULTS: Varying degrees of severity of clinical illness were observed despite uniform, virtual absence of residual activity of the enzyme. The most severely affected patients have had profound developmental delay, dysmorphic features, cataracts, hepatosplenomegaly, lymphadenopathy, and anemia, as well as diarrhea and malabsorption, and have died in infancy. Less severely affected patients have had psychomotor retardation, hypotonia, myopathy, and ataxia. All patients have had recurrent crises in which there was fever, lymphadenopathy, increase in size of liver and spleen, arthralgia, edema, and a morbilliform rash. Neuroimaging studies revealed selective and progressive atrophy of the cerebellum. Mevalonic acid concentrations were found to be grossly elevated in body fluids of all patients. Concentrations of plasma cholesterol were normal or only slightly reduced. Concentrations of ubiquinone-10 in plasma were found to be decreased in most patients. Abnormalities such as hypoglycemia, metabolic acidosis, or lactic acidemia, the usual concomitants of disorders of organic acid metabolism, were conspicuously absent. CONCLUSIONS: These observations establish the broad range of clinical symptoms and biochemical findings in mevalonic aciduria. It is concluded that although patients with mevalonic aciduria have a recognizable phenotype of serious clinical manifestations, some patients are likely to remain undiagnosed and may be found in a variety of subspecialty clinics, including neurology, gastroenterology, cardiology, and genetics.

Hypoxanthine and xanthine concentrations determined by high performance liquid chromatography in biological fluids from patients with xanthinuria.

In the present paper, we report the biochemical features of six cases of xanthinuria. For these studies, the concentrations of hypoxanthine and xanthine have been measured in urine, plasma and also erythrocyte samples by a rapid, sensitive high performance liquid chromatographic (HPLC) method. The analyses of plasma and erythrocyte samples require a very sensitive method relative to physiological concentrations and rigorous sampling conditions in order to achieve accurate results. In the cases reported in the literature, total oxypurine levels (hypoxanthine + xanthine) have been generally measured in plasma and urine by an enzymatic spectrophotometric method. In our studies, using HPLC, we found that xanthine is the major oxypurine compound in plasma and urine samples from patients with xanthinuria. In erythrocytes, a biological sample which has not been analysed up to now, we found that xanthine is present at high concentrations whereas it is not detectable in erythrocytes from healthy subjects.

Purification of electron transfer flavoprotein from pig liver mitochondria and its application to the diagnosis of deficiencies of acyl-CoA dehydrogenases in human fibroblasts.

Medium chain acyl-CoA dehydrogenase (MCAD) and long chain acyl-CoA dehydrogenase (LCAD) deficiency are defects of mitochondrial beta-oxidation. The method of choice to measure specifically acyl-CoA dehydrogenase activity in human tissues uses purified electron transfer flavoprotein (ETF). We describe a simple and optimized method of purification allowing isolation of ETF with a degree of purity never reported so far. An assay for acyl-CoA dehydrogenase activity in cultured skin fibroblasts was developed using microquantities of electron transfer flavoprotein and substrate. MCAD deficiency was demonstrated in fibroblasts from nine patients and LCAD deficiency in fibroblasts from two patients.

Mitochondrial very-long-chain acyl-coenzyme A dehydrogenase deficiency: clinical characteristics and diagnostic considerations in 30 patients.

Very-long-chain acyl-CoA dehydrogenase (VLCAD) is an enzyme catalyzing the dehydrogenation of long-chain fatty acids in the first step of mitochondrial fatty acid oxidation. Using an ETF (electron transfer flavoprotein, the physiological electron acceptor of VLCAD) reduction assay, we identified VLCAD deficiency in cultured skin fibroblasts or liver tissue from 30 patients in 27 families. They clinically presented two phenotypes: a 'severe' presentation characterized by an early onset of symptoms, with hypertrophic cardiomyopathy and a high incidence of death, and a 'mild' form with hypoketotic hypoglycaemia, resembling MCAD (medium-chain acyl-CoA dehydrogenase) deficiency. Cells isolated from patients who develop cardiomyopathy characteristically accumulate longer-chain length acylcarnitines (hexadecanoylcarnitine and tetradecanoylcarnitine) when incubated with palmitate. However, cells from patients with the hypoglycaemic presentation produced relatively shorter-chain-length intermediates (mainly dodecanoylcarnitine). Inhibition of carnitine palmitoyl transferase I, in vitro, eliminated these intermediates with cells from both phenotypes indicating their intramitochondrial origin. Although the explanation for these distinct biochemical findings is not obvious, the correlation with the two phenotypes provides an opportunity for accurate prognosis and early implementation of appropriate treatment. Prenatal diagnosis of this life-threatening disorder was successfully performed in seven pregnancies in six of those families by assay of trophoblasts or amniocytes. In an at risk family, diagnosis of an affected fetus by measurement of VLCAD activity in noncultured chorionic villi allowed termination of the pregnancy before 13 weeks of gestation.

General (medium-chain) acyl-CoA dehydrogenase deficiency (non-ketotic dicarboxylic aciduria): quantitative urinary excretion pattern of 23 biologically significant organic acids in three cases.

Urinary analysis of the pattern of 23 organic acid metabolites derived from fatty acids in three patients with general (medium-chain) acyl-CoA dehydrogenase deficiency was performed. Although there exist quantitative differences in the excreted amounts of the different metabolites in the three patients the qualitative picture was the same. The excretion of adipic, suberic and sebacic acids was substantial, whereas that of dodecanedioic acid was within or just above control limit. The monounsaturated C6-C10-dicarboxylic acid excretion was only marginally or not increased. 5-OH-hexanoic acid and hexanoylglycine were excreted in excessive amounts, whereas 7-OH-octanoic acid, 9-OH-decanoic acid, octanoylglycine and decanoylglycine were excreted in limited amounts. The excreted amounts of 6-OH-hexanoic, 8-OH-octanoic and 10-OH-decanoic acids were not or only marginally elevated compared to controls. In one of the patients the excretion of ethylmalonic and methylsuccinic acids was enhanced, whereas the excretion of these two acids in the two other patients was comparable to that in controls. The urinary excretion of hexanoic, octanoic, decanoic and dodecanoic acids was just a little above the control limit, whereas the esterified hexanoic and octanoic acids were excreted in appreciable amounts. It is argued that the microsomal omega- and omega-1-oxidation systems are involved in the dicarboxylic and omega-1-OH-monocarboxylic acids formation at C10 and C12 level and that the C8-C6-dicarboxylic and omega-1-OH-monocarboxylic acids are formed from higher chained acids by beta-oxidation in both mitochondria and peroxisomes.

Acylcarnitine removal in a patient with acyl-CoA beta-oxidation deficiency disorder: effect of L-carnitine therapy and starvation.

Carnitine levels and acylcarnitine profiles in a patient with mild multiple acyl-CoA dehydrogenase deficient beta-oxidation were compared with control results. Whereas blood and urine total carnitine levels were moderately decreased, blood esterified carnitine levels in the patient were about 2-fold higher than in controls. Urinary acylcarnitine profiles presented with a larger variety of carnitine esters than in controls and included propionylcarnitine, butyrylcarnitine, 2-methylbutyrylcarnitine, hexanoylcarnitine and octanolycarnitine. Total carnitine levels in body fluids were similarly affected by chronic oral L-carnitine administration in patient and controls. By contrast, esterified carnitine level increase was 2-fold more important in controls than in patient. Whereas no qualitative changes in urinary acylcarnitine profiles were induced by L-carnitine therapy in controls, several alterations of these profiles were observed in the patient. The effect of starvation on metabolites was also studied, especially beta-oxidation rates assessed by free fatty acids to 3-hydroxybutyric acid ratios in blood from the patient in the untreated and L-carnitine treated states. In the L-carnitine-supplemented patient, the effect of starvation on the time course of carnitine levels and acylcarnitine profiles could also be documented. The ability of chronic oral L-carnitine administration to remove relatively less important amounts of acylcarnitines in the patient than in controls is further discussed, as well as qualitative alterations of acylcarnitine profiles induced by this therapy in the pathological condition.

Plasma free fatty acids in mitochondrial fatty acid oxidation defects.

Plasma free fatty acid profiles from patients suffering from various mitochondrial beta-oxidation deficiencies were analyzed by gas chromatography-mass spectrometry. cis-4-Decenoic acid (10:1n-6) in medium-chain acyl-CoA dehydrogenase deficiency and cis-5-tetradecenoic acid (14:1n-9) in very-long-chain and 3-hydroxy-long chain acyl-CoA dehydrogenase deficiencies are characteristic of these diseases. In addition, patients with 3-hydroxy-long chain acyl-CoA dehydrogenase deficiency showed a specific increase of 3-hydroxy-long chain fatty acids. The study of plasma free fatty acids is an easy and useful methodology for the diagnostic approach of some mitochondrial beta-oxidation deficiencies, allowing us to establish a quick differentiation between medium- and long-chain defects.

Metabolic studies in a patient with severe carnitine palmitoyltransferase type II deficiency.

Here we report on a patient with severe ("non-classic") carnitine palmitoyltransferase type II (CPT II) deficiency. Hypoglycemia prompted by an infectious episode and associated with non-ketotic dicarboxylic aciduria orientated diagnosis towards beta-oxidation deficiency disorders. Blood carnitine levels revealed a secondary carnitine deficiency that was responsive to oral L-carnitine supplementation. Blood acylcarnitine profiles were abnormal and included acetyl (C2:0), butyryl/isobutyryl (C4:0), isovaleryl/2-methylbutyryl (C5:0), hexanoyl (C6:0), myristoyl (C14:0), palmitoyl (C16:0), hexadecenoyl (C16:1), oleyl (C18:1) and stearoyl (C18:0) carnitine. In urine, excess excretion of dicarboxylylcarnitines, mainly dodecanedioylcarnitine, was noticed. Upon carnitine supplementation, C8 to C12 fatty acylcarnitines, with decanoylcarnitine as well as C10 to C14 dicarboxylylcarnitines being prominent, were observed in urine. Biochemical measurements disclosed a severe reduction of mitochondrial CPT II activity (7% of normal values). Correlations of metabolic findings in the patient and physiological roles of CPT II are briefly discussed.

Urinary excretion of deuterated metabolites in patients with tyrosinemia type I after oral loading with deuterated L-tyrosine.

1. The metabolic fate of orally given deuterated L-tyrosine, 50 mg/kg body weight, was investigated in seven patients with tyrosinemia type I in order to obtain evidence that the primary defect is at the level of fumarylacetoacetase. 2. The absence of fumarylacetoacetase could be proved in liver biopsy specimens obtained from four patients. 3. All patients excreted deuterated succinylacetoacetate and deuterated succinylacetone was detected in six out of seven. The total amount of these compounds was rather low; maximal 8.3% of the dose. The peak of the excretion occurred 3-6 h after loading, indicating an endogenous formation of the metabolites. 4. All patients excreted deuterated 4-hydroxyphenyl acids, probably reflecting secondary 4-hydroxyphenylpyruvate dioxygenase deficiency connected with liver damage. 5. No evidence for other secondary routes of tyrosine metabolism was found.

Mevalonic aciduria in 3 siblings: a new recognizable metabolic encephalopathy.

Mevalonic aciduria, due to mevalonate kinase deficiency, is the first recognized defect in the biosynthesis of cholesterol and isoprenoids. Very few patients with this disorder have been reported. Three siblings born from consanguineous parents are reported. Several clinical signs were present in all 3 children, including failure-to-thrive, susceptibility to infections, hepatosplenomegaly, cataract, and psychomotor retardation. Dysmorphic features were more apparent in the two older siblings. Urinary organic acid analysis by gas chromatography/mass spectrometry invariably revealed a high urinary excretion rate of mevalonic acid. Mevalonate kinase activity assayed in fibroblasts was very low. Diagnosis of this very rare disease may be suspected simply on clinical evidence; it is confirmed by abnormal excretion of mevalonic acid.

[Biological diagnosis of hereditary metabolic diseases. From selective screening to the mutant-cell bank]. / Diagnostic biologique des maladies héréditaires du métabolisme. Du dépistage orienté à la banque de cellules mutantes.

The experience of a specialized laboratory for the biological diagnosis of inborn errors of metabolism in selected pediatrics patients is reported. The strategy starts with a wide testing of blood and urine, as many inborn errors of metabolism can be detected through testing of blood and urine for increased concentration of specific metabolites known to be associated with the genetic defect. Then enzymatic or DNA studies are performed to confirm the diagnosis. The mutant cells mostly fibroblasts are stored in a cell bank and available for other research.

[Inborn errors of lysine metabolism]. / Erreurs innées du métabolisme de la lysine.

A wide variety of inborn errors of lysine catabolism have been described. They include: abnormalities of the mitochrondrial catabolism via the "saccharopine pathway", abnormalities of the peroxisomal catabolism via the pipecolic pathway, and "lysine intolerance with periodic ammonia intoxication" linked to urea cycle pathway. These inborn errors are associated with a wide variety of clinical symptoms and biochemical profiles as persistent hyperlysinemia, saccharopinuria, "amino and ketoadipic aciduria, and glutaric aciduria.

[Lysine metabolism in man]. / Le métabolisme de la lysine chez l'homme.

Lysine is an important amino acid: it is incorporated to collagene, one of the most important component of conjonctive tissue and, though, necessary for growth. It is also important for carnitine synthesis. Its intestinal absorption requires two transport systems that lysine shares with ornithine, arginine and cystine for the former and with ornithine and arginine for the latter. The same transport systems are present in the hepatocytes, were lysine is extensively catabolized, and in renal tubular cells. The main catabolic pathway for lysine, via saccharopine, is a mitochondrial pathway leading to acetyl-CoA. The peroxisomal pathway, via pipecolic acid is of less physiological importance and is mainly active in brain. The existence of the two minor pathways, the acetyllysine pathway and the lysine-urea cycle, remains to be demonstrated.

[Hyperglycinemia without ketosis. Biochemical and enzymatic study]. / Hyperglycinémie sans cétose. Etude biochimique et enzymatique.

We report a biochemical and enzymatic study of two neonatal cases of non ketotic hyperglycinemia. We report the comparative evolution of glycine level in plasma and CSF during a restrictive diet excluding glycine and serine. The high levels of glycine found in CSF and brain are likely to reflect the brain damage. After autopsy, the glycine synthase activity determination shows a significative partial deficiency in the liver and a total deficiency in the brain. Glycine synthase affinity for glycine is similar for controls and patients and this lead use to think that the deficiency is due to a diminished biosynthesis of the enzyme molecule.

[Study of plasma acylcarnitines using tandem mass spectrometry. Application to the diagnosis of metabolism hereditary diseases]. / Etude des acylcarnitines plasmatiques par spectrométrie de masse en tandem. Application au diagnostic des maladies héréditaires du métabolisme.

BACKGROUND: L-carnitine is known to transport long chain fatty acids through the mitochondrial membrane but also to export accumulated acyl-CoA's as acylcarnitine esters. Acylcarnitine identification in body fluids allows the diagnosis of mitochondrial inborn errors especially fatty oxidation defects. Tandem mass spectrometry represents a new method for isolation and identification of acylcarnitines in plasma or in blood spotted onto filter paper (Guthrie cards). MATERIAL AND METHODS: In order to validate our method, we studied 30 plasmas from children affected with 15 different inborn errors of metabolism and five amniotic fluids from fetuses affected with several organic acidurias. Fourty-six samples from children at risk for mitochondrial fatty oxidation disorders have been analyzed. We developed a method of tandem mass spectrometry with liquid secondary ion mass spectrometry using deuterated acylcarnitines as internal standards. RESULTS: This method is very sensitive (detection limit = 2 microM). In all affected patients specific acylcarnitine signals corresponding to the metabolic block were constantly found. This confirms the diagnosis and validates the method. Among the 46 at risk children, four defects of long chain fatty acid oxidation were identified. CONCLUSION: This new method is of great interest especially for the long chain fatty acid oxidation defects. These defects are very difficult to diagnose with classical methods as urinary organic acid profiling. A small amount of plasma (100 microL) or blood spotted onto paper is required. The acylcarnitine profile allows a rapid diagnosis if a dedicated apparatus is available.

[Hereditary diseases causing kidney calculi]. / Les maladies héréditaires responsables de lithiase rénale.

Urolithiasis and/or nephrocalcinosis due to hereditary diseases are a rare event which must be kept in mind of physicians who take care of children (10 to 40% of all causes of lithiases) as well as of adults (less than 15% of all causes of lithiases) since a specific management is usually required. The most frequent inborn disorders are idiopathic hypercalciuria, distal tubular acidosis, cystinuria and hyperoxaluria. Stone formation is always secondary to an increased urine concentration of promotors, i.e. calcium, oxalate, phosphate, cystine, xanthine. One of the most informative diagnosis investigation is infrared spectrophotometry which can identify stone composition. When such a technique is not available, biochemical investigations should be adapted to both personal and family history. In addition to high fluid intake (2 to 3 L/m2/24 h) sometimes associated with alcalinisation, the management of hereditary stone disease requires specific procedure. In all cases, the long-term renal prognosis is related to both primary disease and therapeutic compliance.