P. Divry

The inborn errors of mitochondrial fatty acid oxidation.

To date, seven inborn errors of mitochondrial fatty acid oxidation have been identified. A total of about 100 patients in the world have been reported. Clinically the β-oxidation defects are more often characterized by episodic hypoglycaemia leading to a coma mimicking Reyes syndrome. The hypoglycaemia is non-ketotic since the synthesis of ketone bodies is deficient. Periods of decompensation occur when carbohydrate supply is poor, e.g. prolonged fasting, vomiting, or increased caloric requirements, as and when lipid stores are used. Defects in β-oxidation have also been reported to be one cause of sudden infant death syndrome. The diagnosis of these inborn errors is by biochemical investigation since where symptoms suggest such a defect, the precise aetiology cannot be assessed. The biochemical diagnosis is based firstly on identification of abnormal plasma and of urinary metabolites during acute attacks. Derivatives of the ω-oxidation and ω-1-oxidation of medium chain fatty acids have been identified, as well as acylglycine and acylcarnitine conjugates. These metabolites are nearly always absent when patients are in good clinical condition. Secondly, the diagnosis must be based on the identification of the enzymatic defects: this involves global assays which allow a localization of the ‘level’ of the defect (i.e. the oxidation of long, medium or short chain fatty acids) and specific measurement of enzyme activities (acyl-CoA dehydrogenases and electron carriers: ETF and ETF-DH). The diagnosis of these disorders is of prime importance because of the severity of the clinical symptoms. These can be prevented, in some cases, by an appropriate diet (a high carbohydrate, low fat diet, sometimes supplemented withl-carnitine). In other cases, genetic counselling can be offered.

Diagnosis of inborn errors of metabolism by acylcarnitine profiling in blood using tandem mass spectrometry.

L-Carnitine is known to transport long-chain fatty acids through the mitochondrial membrane and also to export accumulated acyl-CoAs as acylcarnitine esters. Acylcarnitine identification in body fluids using tandem mass spectrometry was developed in the late 1980s, allowing the diagnosis of most inborn errors of mitochondrial metabolism and especially fatty acid oxidation defects (reviewed by Millington and Chace, 1992). We developed a technique of LSIMS/MS (liquid secondary-ion tandem mass spectrometry) for acylcarnitine profiling in blood.

The mutational spectrum in very long-chain acyl-CoA dehydrogenase deficiency

ConclusionWe have shown that all of seven unrelated patients with defective palmitoyl-CoA dehydrogenation have mutations in VLCAD, indicating that they suffer from VLCAD deficiency.Our study illustrations that there is a large mutational heterogeneity in VLCAD deficiency. It is not at present possible to judge whether or to what extent this large mutational heterogeneity may explain the varying clinical picture observed in this disease.

A new patient with 4-hydroxybutyric aciduria, a possible defect of 4-aminobutyrate metabolism

Abstract A new patient with 4-hydroxybutyric aciduria is described, adding further evidence that 4-hydroxybutyric aciduria is a clinical entity. Main clinical symptoms were: motor and mental retardation, muscular hypotonia and ataxia. 4-Hydroxybutyric acid was increased in urine, plasma and cerebrospinal fluid. Glutamic acid was shown to be a precursor and on valproate therapy concentrations in plasma and urine seemed to be enhanced.

Carnitine palmitoyl transferase I deficiency presenting as a Reye-like syndrome without hypoglycaemia

An apparently healthy girl aged 2 years 9 months developed a coma with hepatomegaly within 24h after an influenza-like infection. Plasma glucose and urinary organic acid profile were normal but plasma and urinary carnitine concentrations were increased. Despite symptomatic therapy, she died 11 days later. Oxidation of [1-14C] palmitic acid in the patients fibroblasts was severely decreased (13% of controls). Further investigations revealed a deficiency of carnitine palmitoyl transferase I (CPTI) in the patients fibroblasts (15% of controls) whereas CPT II activity was normal. Only four patients with CPT I deficiency have been reported so far. The subtle clinical and biochemical, presentation of this disorder, which may account for the small number of cases diagnosed, is discussed.

Stroke, hemiparesis and deficient mitochondrial β-oxidation

We describe on a 3-year-old child referred for evaluation and therapy of a cerebral vascular accident with residual hemiplegia and partial epilepsy. Metabolic investigations initially showed normal urinary organic acids as well as normal blood and urinary amino acids. Blood carnitine fractions had been pathological and a secondary carnitine deficiency was diagnosed and treated by oral L-carnitine supplementation. During carnitine treatment, abnormal urinary acylcarnitine profiles were noticed with excessive amounts of several carnitine esters including propionylcarnitine, butyryl-and/or isobutyryl-carnitine, isovaleryl- and/or 2-methylbutyryl-carnitine, hexanoylcarnitine and octanoyl-carnitine. Subsequently, an urinary organic acid profile suggestive of glutaric aciduria type 11 was recorded during a clinical decompensation crisis. Morphological and biochemical studies on skeletal muscle and skin fibroblasts were performed and confirmed the existence of a defect of the mitochondrial β-oxidadation pathways with lipidic myopathy, reduced palmitate and octanoate oxidation rates in cultured fibroblasts. Glutaric aciduria type 11 increases the list of metabolic disorders characterized by hemiplegia and other sequelae of brain ischaemia such as stroke-like episode, seizures, aphasia, ataxia and myoclonia, similar to those seen in MELAS.

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.

Infantile form of carnitine palmitoyltransferase II deficiency in a girl with rapid fatal onset.

confirmed that the patient had severe heart failure due to pulmonary hypertension. The administration of various drugs was ineffective and the patient died on day 7 after admission. Autopsy was not performed. There are only three reported cases of pulmonary hypertension occurring in patients with GSD I (Pizzo 1980; Hamaoka et al 1990). The characteristics of these patients (including our case) were as follows: (1) they developed overt pulmonary hypertension in their second decade of life or later; (2) their serum lipid, lactate and uric acid levels had seemed to be high for a long time; and (3) the outcome was fatal. Although the aetiology of pulmonary hypertension in GSD I is unknown, the development of this complication should be considered in the evaluation and treatment of all post-childhood cases of GSD I.

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.

Acylcarnitine removal in a patient with acyl-CoA β-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.