N. J. Manning

A comparison of [9,10-3H]palmitic and [9,10-3H]myristic acids for the detection of defects of fatty acid oxidation in intact cultured fibroblasts.

SummaryThe production of tritiated water from [9,10-3H]myristic acid can be used as a screening assay for the detection of medium-chain acyl-CoA dehydrogenase deficiency, multiple acyl-CoA dehydrogenation defects (glutaric aciduria type 2 and ethylmalonic-adipic aciduria types), and some types of hydroxydicarboxylic aciduria. Comparison with the release of tritiated water from [9,10-3H]palmitic acid may give an indication of the chain-length specificity of the metabolic defect. In a case of ethylmalonic-adipic aciduria such a prediction has been confirmed by examination of accumulated intermediates in the affected fibroblasts.

A sterol biosynthetic pathway in Mycobacterium.

The genome sequence of Mycobacterium tuberculosis (and also M. leprae) revealed a significant number of homologies to Saccharomyces cerevisiae sterol biosynthetic enzymes. We addressed the hypothesis of a potential sterol biosynthetic pathway existing in Mycobacterium using cultures of Mycobacterum smegmatis. Non‐saponifiable lipid extracts subjected to analysis by gas chromatography‐mass spectrometry (GC‐MS) showed cholesterol was present. Sterol synthesis by M. smegmatis was confirmed using 14C‐radiolabelled mevalonic acid and incorporation into C4‐desmethyl sterol co‐migrating with authentic cholesterol on TLC. The sterol biosynthetic pathway has provided a rich source of targets for commercially important bioactive molecules and such agents represent new opportunities for Mycobacteria chemotherapy.

Differential diagnosis of hydroxydicarboxylic aciduria based on release of3H2O from [9,10-3H]myristic and [9,10-3H]palmitic acids by intact cultured fibroblasts

SummaryIntact cultured fibroblasts from patients with deficiency of long-chain 3-hydroxyacyl-CoA dehydrogenase release3H2O from [9,10-3H]myristic acid and [9,10-3H]palmitic acid more slowly than normal. The ratio of activity (palmitate/myristate) is also low and the expression (rate with palmitate)2/(rate with myristate) gives good differentiation between affected and unaffected cells. In some patients who have shown hydroxydicarboxylic aciduria when unwell there is reduced3H2O production from [9,10-3H]myristic and [9,10-3H]palmitic acids by intact cultured fibroblasts but normal 3-hydroxyacyl-CoA dehydrogenase activities in disrupted cells. The palmitate/myristate ratio is higher than in long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency. The basic defect in these patients is still unknown but it is suggested that caution be used over the administration of medium-chain triglyceride.

2-Methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency in a 23-year-old man

Abstract2-Methyl-3-hydroxybutyryl-CoA dehydrogenase (EC 1.1.1.178) deficiency is a recently described defect of isoleucine catabolism. The disorder is characterized by normal early development followed by a progressive loss of mental and motor skills. Deterioration may be rapid or may follow a slower decline with a possible stabilization of the disorder on a low-protein diet and appropriate medication. We report a 23-year-old man with 2-methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency with a very mild clinical course. He had apparently normal early development and remained relatively well until the age of 6 years, when he contracted measles. Following this illness, his motor skills and school progress deteriorated. At 15 years he had significant dysarthria, and generalized rigidity with some dystonic and unusual posturing. He was then treated with a low-protein high-carbohydrate diet with a good response in terms of balance and gait. At 18 years he was given benzhexol (Artane), increased slowly from 2 mg to 6 mg daily, resulting in improvement in tremor and dystonia. At 23 years he can dress himself and works in sheltered employment but remains severely dysarthric.

Carnitine-acylcarnitine translocase deficiency - a mild phenotype

A 15-month-old Pakistani child was admitted to hospital following a prolonged chronic convulsion. He had a depressed level of consciousness but physical examination was otherwise unremarkable. Plasma glucose was 1mmol/L and his urine contained no ketones. His condition improved rapidly with intravenous glucose. His parents are first cousins and his two older siblings are healthy. He had suffered two previous prolonged tonic febrile fits, during which his blood glucose had not been measured. On admission, his plasma lactate was 1.7mmol/L. Further investigations showed a plasma total carnitine of 13μmol/L (normal 23–60) and free carnitine of 4μmol/L (normal 15–53). Urine acylcarnitine was high (60μmol/mmol creatinine; normal 1.0–14.0) and urine free carnitine was low at <1.0μmol/mmol creatinine (normal 4.0–29.0). Serum insulin was appropriately low (0.6mU/L). Urine organic acids showed a hypoketotic dicarboxylic aciduria with prominent excretion of adipate and suberate. The sebacate to adipate ratio was <0.2. A small peak of suberylglycine was detected. He was put on a highcarbohydrate, low-fat diet with frequent feeds and L-carnitine 100mg/kg body weight per day, with the recommendation that he should not fast for more than 8h, subsequently amended to 6h. Dietary long-chain fat was set at a limit of 10g/day (estimation of previous long-chain fat intake was 40g/day) with medium-chain triglyceride supplementation and frequent high-carbohydrate feeds. He is now 20 years old and apart from two brief admissions to hospital during intercurrent infections, the most recent following a hypoglycaemic fit, his growth and development are normal. His cardiac function, as measured by ultrasonography, has remained normal throughout. Measurement of β-oxidation rates by the method of Manning and colleagues (1990) gave mean residual activities, as compared to simultaneous controls for [9,10-3H]myristate, [9,10-3H]palmitate and [9,10-3H]oleate, of 19%, 15%, 15% and 18%, 17% 14% for fibroblasts and lymphocytes, respectively. Measurement of carnitine-acylcarnitine translocase (McKusick 212138) activity by the method of Pande and colleagues (1993) gave 0.10 ±0.038 (8 repeats) vs 1.74±0.46 (n=9) nmol/min per kg protein for patient and controls, respectively. There have been six previous reports of carnitine-acylcarnitine translocase deficiency. All of these cases have had a severe phenotype with generally undetectable enzyme activity and very low β-oxidation flux. All of these patient have had a fatal outcome, most in the neonatal period, although one child survived to 3 years with early medical intervention. Our patient gave a mean residual carnitine-acylcarnitine translocase activity of 6% of controls and a mean residual β-oxidation activity in lymphocytes and fibroblasts of 16%. J. Inher. Metab. Dis. 20 (1997) 000–000

Glutaric aciduria type 1 in South Africa-high incidence of glutaryl-CoA dehydrogenase deficiency in black South Africans.

Glutaric Aciduria type 1 (GA 1) is an inherited disorder of lysine and tryptophan catabolism that typically manifests in infants with acute cerebral injury associated with intercurrent illness. We investigated the clinical, biochemical and molecular features in 14 known GA 1 patients in South Africa, most of whom were recently confirmed following the implementation of sensitive urine organic acid screening at our laboratory. Age at diagnosis ranged from 3days to 5years and poor clinical outcome reflected the delay in diagnosis in all but one patient. Twelve patients were unrelated black South Africans of whom all those tested (n=11) were found homozygous for the same A293T mutation in the glutaryl-CoA dehydrogenase (GCDH) gene. Excretion of 3-hydroxyglutarate (3-OHGA) was >30.1μmol/mmol creatinine (reference range <2.5) in all cases but glutarate excretion varied with 5 patients considered low excretors (glutarate <50μmol/mmol creatinine). Fibroblast GCDH activity was very low or absent in all of five cases tested. Heterozygosity for the A293T mutation was found 1 in 36 (95% CI; 1/54 - 1/24) unrelated black South African newborns (n=750) giving a predicted prevalence rate for GA 1 of 1 in 5184 (95% CI; 1/11664 - 1/2304) in this population. GA 1 is a treatable but often missed inherited disorder with a previously unrecognised high carrier frequency of a single mutation in the South African black population.

Sterol 14alpha-demethylase activity in Streptomyces coelicolor A3(2) is associated with an unusual member of the CYP51 gene family.

The annotation of the genome sequence of Streptomyces coelicolor A3(2) revealed a cytochrome P450 (CYP) resembling various sterol 14alpha-demethylases (CYP51). The putative CYP open reading frame (SC7E4.20) was cloned with a tetrahistidine tag appended to the C-terminus and expressed in Escherichia coli. Protein purified to electrophoretic homogeneity was observed to bind the 14-methylated sterols lanosterol and 24-methylene-24,25-dihydrolanosterol (24-MDL). Reconstitution experiments with E. coli reductase partners confirmed activity in 14alpha-demethylation for 24-MDL, but not lanosterol. An S. coelicolor A3(2) mutant containing a transposon insertion in the CYP51 gene, which will abolish synthesis of the functional haemoprotein, was isolated as a viable strain, the first time a CYP51 has been identified as non-essential. The role of this CYP in bacteria is intriguing. No sterol product was detected in non-saponifiable cell extracts of the parent S. coelicolor A3(2) strain or of the mutant. S. coelicolor A3(2) CYP51 contains very few of the conserved CYP51 residues and, even though it can catalyse 14alpha-demethylation, it probably has another function in Streptomyces. We propose that it is a member of a new CYP51 subfamily.

Prenatal diagnosis of Canavan disease- problems and dilemmas

Canavan disease (spongy degeneration of the brain; McKusick 271900) is a severe neurodegenerative disorder for which there is at present no effective treatment, although attempts at gene therapy are currently being investigated (During 1996). The disorder results from a deficiency of the enzyme aspartoacylase, which leads to an accumulation of N-acetylaspartate (NAA) in tissues and body fluids. The disorder is particularly prevalent among Ashkenazi Jews, where a carrier state of 1:45 common mutation (E285A) in the aspartoacylase gene has been identified. Diagnosis is usually established by the demonstration of raised N-acetylaspartate in urine, but confirmatory enzyme studies have proved difficult owing to low and variable enzyme activities, particularly in cultured cells. Consequently, identification of mutations in Ashkenazi Jewish and other patients is especially useful. Prenatal diagnosis by enzyme assay is generally held to be unreliable (Matalon et al 1995 ; Rolland et al 1994). However, measurement of N-acetylaspartate concentration in amniotic fluid by stable-isotope dilution is considered the most reliable approach, although where possible DNA analysis should complement this (Elpeleg et al 1994). We report our experience with prenatal diagnosis in one family where difficulties in interpreting metabolite results have highlighted the importance of complementary mutation analysis.

Nonsterol related resistance in Ustilago maydis to the polyene antifungals, amphotericin B and nystatin.

Two amphotericin B resistant mutants of Ustilago maydis were isolated following direct selection from a wild-type population. Each mutant was demonstrated to be cross-resistant to nystatin yet remained sensitive to azoles. Sterol analysis indicated a sterol profile similar to the parent strain, precluding the involvement of an alteration in ergosterol biosynthesis as the cause of polyene resistance.

Bile acid aspiration in people with cystic fibrosis before and after lung transplantation

Cystic fibrosis (CF) is a genetic condition that is caused by abnormalities in the CF transmembrane conductance regulator (CFTR) gene. People with CF experience life-long morbidity and premature mortality, the vast majority of which is associated with lung disease. Bile acids are detectable in the lower airway in advanced CF lung disease and persist after lung transplantation http://ow.ly/RTvNW