Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase deficiency: clinical course and description of causal mutations in two patients.

Hereditary deficiency of mitochondrial HMG-CoA synthase (mHS, OMIM 600234) is a poorly defined, treatable, probably underdiagnosed condition that can cause episodes of severe hypoketotic hypoglycemia. We present clinical follow-up and molecular analysis of the two known mHS-deficient patients. The diagnosis of mHS deficiency is challenging because the symptoms and metabolite pattern are not specific. Moreover, enzyme analysis is technically difficult and requires sampling of an expressing organ such as liver. The patients, now aged 16 and 6 y, have normal development and have had no further decompensations since diagnosis. Patient 1 is homozygous for a phenylalanine-to-leucine substitution at codon 174 (F174L). Interestingly, although the F174 residue is conserved in vertebrate mHS and cytoplasmic HS isozymes, a Leu residue is predicted in the corresponding position of HS-like sequences from Caenorhabditis elegans, Arabidopsis thaliana, and Brassica juncea. Bacterial expression of human F174L-mHS produces a low level of mHS polypeptide with no detectable activity. Similarly, in purified cytoplasmic HS, which in contrast to purified human mHS is stable and can be studied in detail, the corresponding F-->L substitution causes a 10,000-fold decrease in V(max) and a 5-fold reduction in thermal stability. Patient 2 is a genetic compound of a premature termination mutation, R424X, and an as-yet uncharacterized mutant allele that is distinguishable by intragenic single nucleotide polymorphisms that we describe. Molecular studies of mHS are useful in patients with a suggestive clinical presentation.

In vivo disposal of phenylalanine in phenylketonuria: a study of two siblings.

Mutation at the phenylalanine hydroxylase (PAH) locus is a cause of hyperphenylalaninaemia. Genotype-phenotype correlation relative to the predicted PAH activity may differ at the metabolite level and at the IQ level in untreated phenylketonuria. Discordant metabolic phenotypes have been noted in siblings; influences on transport and metabolism of phenylalanine determining homeostasis may account for differing metabolic phenotypes. We report two siblings of different sex and identical genotype at the PAH locus who demonstrate a difference in phenylalanine disposal. A stable isotope infusion of [2H5]phenylalanine was used to measure protein turnover, phenylalanine hydroxylation and excretion of phenylalanine transamination metabolites. The siblings were observed to have identical hydroxylation rates under the experimental conditions of the study while manifesting differences in renal excretion rates of phenylalanine transamination metabolites and protein accretion.

Pyridoxine-responsive gyrate atrophy of the choroid and retina: clinical and biochemical correlates of the mutation A226V.

We discovered the missense mutation, A226V, in the ornithine-delta-aminotransferase (OAT) genes of two unrelated patients with gyrate atrophy of the choroid and retina (GA). One patient, who was a compound for A226V and for the premature termination allele R398ter, showed a significant (P < .01) decrease in mean plasma ornithine levels, following pyridoxine supplementation with a constant protein intake: 826 +/- 128 microM (n = 5; no pyridoxine supplementation) versus 504 +/- 112 microM (n = 6; 500 mg pyridoxine/d) and 546 +/- 19 microM (n = 6; 1,000 mg pyridoxine/d). In extracts of fibroblasts from a second GA patient homozygous for A226V and from Chinese hamster ovary cells expressing an OAT-cDNA-containing A226V, we found that OAT activity increased from undetectable levels to approximately 10% of normal when the concentration of pyridoxal phosphate was increased from 50 to 600 microM. A226V is the fourth disease-causing pyridoxine-responsive human mutation to be reported.

A topoisomerase II cleavage site is associated with a novel mitochondrial DNA deletion.

Mitochondrial myopathies and encephalopathies can be caused by nucleotide substitutions, deletions or duplications of the mitochondrial DNA (mtDNA). In one such disorder, Kearns-Sayre Syndrome (KSS), large-scale heteroplasmic mtDNA deletions are often found. We describe a 14-year-old boy with clinical features of KSS, plus some additional features. Analysis of the entire mitochondrial genome by the polymerase chain reaction and Southern blotting revealed a 7864-bp mtDNA deletion, heteroplasmic in its tissue distribution. DNA sequencing established that the deletion was between nucleotides 6238 and 14,103, and flanked by a 4-bp (TCCT) direct repeat sequence. Deletions between direct repeats have been hypothesised to occur by a slipped-mismatching or illegitimate recombination event, or following the DNA cleavage action of topoisomerase II. Analysis of the gene sequence in the region surrounding the mtDNA deletion breakpoint in this patient revealed the presence of putative vertebrate topoisomerase II sites. We suggest that direct repeat sequences, together with putative topoisomerase II sites, may predispose certain regions of the mitochondrial genome to deletions.

Contribution of odd-chain fatty acid oxidation to propionate production in disorders of propionate metabolism.

Whole-body propionate and protein kinetics and energy substrate metabolism were studied in five metronidazole-treated patients with propionic or methylmalonic acidemias by the use of a primed, 4-h constant infusion of [1-13C]propionate and L-[O-2H5]phenylalanine combined with indirect calorimetry. Measurements were performed during fasting and carbohydrate feeding, successively, to assess the contribution of odd-chain fatty acid oxidation to total propionate production. Fat oxidation decreased from 490 +/- 179 to 57 +/- 49 mumol.kg-1.h-1 (P < 0.05) as a result of feeding. Propionate appearance rate was 38.6 +/- 8 mumol.kg-1.h-1 during fasting and decreased to 22.6 +/- 5 mumol.kg-1.h-1 (P < 0.05) on the carbohydrate diet. Precursor amino acid catabolism did not change significantly (22 +/- 5 vs 21.2 +/- 5 mumol.kg-1.h-1), suggesting that the 41% reduction in propionate production observed in response to feeding was related to the suppression of fatty acid oxidation. Therefore, significant therapeutic gains may be expected from the use of diets aimed at reducing lipid oxidation.

Production and disposal of medium-chain fatty acids in children with medium-chain acyl-CoA dehydrogenase deficiency.

The effect of fasting on plasma concentrations of fatty acids has been determined in four children with medium-chain acyl-CoA dehydrogenase (MCAD) deficiency. In addition, the in vivo rate of octanoate oxidation was measured, using [1-13C]octanoate. In the three older children (1.5-11.2 years), fasting for up to 18 h stimulated lipolysis, as reflected by the increasing concentration of free fatty acids, but with little rise in concentrations of medium-chain fatty acids, octanoate, decanoate and cis-4-decenoate. In an infant (0.5 year), lipolysis was greater and was accompanied by rising concentrations of medium-chain fatty acids. After 13.5 h there was a rapid increase in the concentration of decanoate and cis-4-decenoate. The calculated in vivo rate of octanoate oxidation was substantial in all patients studied (6.4-13.1 mumol/kg per h) despite very low MCAD activity in vitro. It is concluded that under basal conditions the in vivo oxidation rate of medium-chain fatty acids is near normal in the four children studied with MCAD deficiency.

A novel mtDNA deletion in an infant with Pearson syndrome.

Pearson syndrome is a multisystem mitochondrial disorder of infancy that is associated with deletions in the mitochondrial DNA (mtDNA) genome. We report a study on a male infant with Pearson syndrome. Assessment of oxidative phosphorylation activity indicated combined respiratory-chain defects in muscle, liver and fibroblasts; in particular, activity of complex I was reduced. Analysis of the patient's mtDNA identified a novel heteroplasmic 2.461 kb deletion, present at levels greater than 50% of the total mtDNA in the tissues examined. The deletion spanned nucleotides 10368 to 12828 and was flanked by a 3 bp GCC direct repeat sequence. Gene sequences affected are subunits 3, 4, 4L and 5 of complex I, and tRNAs for arginine, histidine, serine and leucine. Our findings correlate with the multiorgan involvement observed in Pearson syndrome.

A fluorimetric assay for succinic semialdehyde dehydrogenase activity suitable for prenatal diagnosis of the enzyme deficiency.

Succinic semialdehyde dehydrogenase (SSAD) is an enzyme involved in the turnover of the neurotransmitter 4-aminobutyrate (GABA). Deficiency of SSAD results in developmental delay, ataxia, seizures and 4-hydroxybutyric aciduria. We have developed a simple fluorimetric assay for the enzyme and applied it to measurement of SSAD activity in a range of cell types often used for prenatal and postnatal diagnosis of enzyme defects. Lymphocytes from children with SSAD deficiency were found to have < 5% of the activity found in lymphocytes from normal children. Heterozygotes are asymptomatic and have intermediate enzyme activities. Although SSAD activity has been detected previously in uncultured chorionic villi, we found that SSAD was not expressed in cultured chorionic villus cells nor in some fibroblast-like amniocytes from control fetuses. Lymphocytes from fetal blood and non-fibroblastic amniocytes have high SSAD activities, and should be suitable for prenatal diagnosis of SSAD deficiency.

Protein turnover in pregnancy.

Whole-body protein metabolism was studied at 13, 24 and 35 weeks gestation in six healthy women using a continuous infusion [13C]leucine technique. Values were expressed in terms of fat-free body weight (FFM) calculated using literature standards for changes in total body potassium during pregnancy. Mean protein synthesis increased from 5.3 (+/- 0.6 SD) g/kg FFM/24h at 13 weeks to 5.9 +/- 0.5 (P less than 0.1) at 24 weeks and 6.1 +/- 0.6 at 35 weeks (P less than 0.05 vs 3 weeks). Similar increases were noted in catabolism so that net loss changed little. Protein synthesis at 24 and 35 weeks gestations was significantly greater than that in 17 healthy, non-pregnant women (4.9 +/- 0.6, P less than 0.001). These data indicate that there are substantial increases in protein turnover during pregnancy.

Maternal tyrosinaemia II: management and successful outcome.

A 25-year-old woman with tyrosinaemia type II was treated from the 5th week of pregnancy with a protein-restricted diet supplemented with a tyrosine/phenylalanine-free amino acid mixture. Tyrosine concentrations were maintained in the range 100-200 mumol/l by restricting natural protein intake to 0.16 g/kg per 24 h in early pregnancy, with increases up to 0.38 g/kg per 24 h in the last trimester. This treatment maintained plasma phenylalanine concentrations in the range 20-40 mumol/l. Maternal weight gain and fetal growth were normal, and the mother remained asymptomatic throughout the pregnancy. A normal infant was born at term with length, weight and head circumference between the 25-50th per centiles.

Pregnancy in phenylketonuria: dietary treatment aimed at normalising maternal plasma phenylalanine concentration.

The transport characteristics of the placenta, which favour higher phenylalanine concentrations in the fetus than in the mother, and regression data of head circumference at birth against phenylalanine concentration at conception in maternal phenylketonuria (PKU), suggest that treatment of maternal PKU should ideally aim to maintain plasma phenylalanine concentration within the normal range throughout pregnancy. A patient with classical PKU was treated from before conception by aiming to maintain plasma phenylalanine concentration within the range 50-150 mumol/l and tyrosine within the range 60-90 mumol/l. The diet was supplemented with phenylalanine-free amino acids (100-180 g/day) and tyrosine (0-5 g/day). Plasma amino acid concentrations were monitored weekly by amino acid analyser. Dietary phenylalanine intake ranged from 6 mg/kg/day at conception to 30 mg/kg/day at delivery. Normal weight gain and fetal growth were maintained throughout the pregnancy. A normal baby was born at term with a head circumference of 35.5 cm; at 1 year of age no abnormality is detectable. These results show that with careful monitoring and compliance it is possible, and may be advisable, to maintain plasma phenylalanine concentration within the normal range in the management of PKU pregnancy.

Acute illness in maple syrup urine disease: dynamics of protein metabolism and implications for management.

Acute metabolic decompensation in maple syrup urine disease (MSUD) during otherwise minor illnesses has generally been presumed to result from massive release of leucine from protein catabolism. A stable isotope method based on the continuous infusion of (2H5)phenylalanine was used to measure protein metabolism in vivo in two children with MSUD during acute illness and when well. Net protein catabolism was greater in the unwell state (0.51 and 0.40 gm/kg per 24 hours in each child, respectively) than in the basal state (0.34 and 0.32). This rate of release of leucine from protein is compatible only with a slow (several days) rather than a dramatic rise in plasma leucine levels during acute illness in MSUD. Poor oral intake leading to a relative increase in time spent in the fasting state appears to be a more important determinant of increasing leucine levels than the catabolic effect of infection in itself. These factors suggested that branched-chain amino acid restriction should be commenced at the start of minor illness in children with MSUD, and that intake of other nutrients should be maintained or increased throughout the illness. A regimen based on these concepts was used during nine episodes of minor illness in two children with MSUD. Plasma branched-chain amino acid levels remained acceptable (less than 700 mumol/L) throughout each of these episodes. Dietary supplementation of this type may reduce the risk of metabolic decompensation during acute illnesses in children with MSUD.

Continuous venovenous hemofiltration in the management of acute decompensation in inborn errors of metabolism.

Continuous venovenous hemofiltration was used to treat two neonates, one with maple syrup urine disease and the other with an inborn error of long-chain fatty acid oxidation. The latter infant had hypoglycemia, hyperammonemia and lactic acidosis. In both cases, acceptable biochemical control was achieved within 8 hours. This therapy offers the potential to overcome acute crises rapidly in a wide range of inborn errors of intermediary metabolism.

Measurement of whole-body protein turnover in insulin-dependent (type 1) diabetic patients during insulin withdrawal and infusion: comparison of [13C]leucine and [2H5]phenylalanine methodologies.

1. The aims of this study were twofold: (i) to investigate the ability of a recently described [2H5]phenylalanine method for quantifying whole-body protein turnover during acute physiological perturbation; (ii) to determine specifically whether the previously observed increase in protein synthesis on insulin withdrawal in insulin-dependent (type 1) diabetic patients seen when employing the [13C]leucine technique could be corroborated by using [2H5]phenylalanine. 2. Whole-body protein turnover was measured by both the [2H5]phenylalanine and [13C]leucine primed continuous infusion methods applied simultaneously to six type I post-absorptive diabetic patients during insulin withdrawal and infusion. 3. Values were determined by the [13C]leucine method by measuring either [13C]leucine (primary pool) or alpha-[13C] ketoisocaproic acid (reciprocal pool) enrichment in plasma. 4. Values of whole-body protein breakdown during insulin withdrawal derived from the [2H5]phenylalanine and primary and reciprocal pool [13C]leucine models respectively were 3.54 +/- 0.43, 3.85 +/- 0.41 and 4.62 +/- 0.44 g day-1 kg-1 (means +/- SD). Insulin infusion resulted in a significant reduction (P less than 0.02) to 3.07 +/- 0.34, 3.05 +/- 0.26 and 3.82 +/- 0.4 g day-1 kg-1, respectively. Synthesis values fell significantly but by a smaller amount than breakdown, resulting in increased (P less than 0.05) net protein deposition, regardless of the model used. 5. These data demonstrate that the [2H5]phenylalanine and [13C]leucine methods generate similar results both in absolute and relative terms in response to short-term insulin infusion. 6. The confirmation of increased whole-body protein synthesis during insulin withdrawal by two independent methods supports the validity of this observation.

Techniques for studying hepatic metabolism in vivo.

Techniques for studying metabolic events in vivo in patients with inborn errors are reviewed. Loading or provocation tests that have been used widely are insensitive and frequently non-specific. Compounds labelled with stable isotopes can be used to study enzyme kinetics and substrate turnover, providing more detailed and specific information. Intracellular events may be studied using nuclear magnetic resonance spectroscopy. The results using these techniques to study patients with selected inborn errors are discussed, namely phenylketonuria, glycogen storage disease type I and propionic acidaemia.

Sources of propionate in inborn errors of propionate metabolism.

Amino acids are widely regarded as the most important sources of propionate in disorders of propionate metabolism. Propionate production was measured in the fasting state by continuous infusion of sodium [1-13C]propionate in three children with methylmalonic acidemia (MMA) and three with propionic acidemia (PA). The contribution of isoleucine, valine, threonine, and methionine catabolism to total propionate production was estimated by extrapolation from the hydroxylation of phenylalanine determined by a continuous-infusion [2H5]phenylalanine technique. The contribution of gut bacterial propionate production was determined by measuring total propionate production before and after treatment with oral metronidazole (10 to 20 mg/kg/d for 1 week). Amino acid catabolism accounted for a mean of 51.7% (range, 24.5% to 66.4%) of total propionate production. The mean decrease in propionate production after metronidazole was 22.2% +/- 8.5 (P less than .02); this percentage is likely to represent the minimum propionate production attributable to gut bacteria. Approximately 30% of total propionate production was unaccounted for, and is likely to arise primarily from odd-chain fatty acid catabolism in the fasting state. These results indicate that sources of propionate other than from protein catabolism are important in disorders of propionate metabolism, and explain the generally disappointing response to dietary protein restriction.

Protein metabolism in phenylketonuria and Lesch-Nyhan syndrome.

Animal and in vitro studies have implicated decreased protein synthesis in the pathogenesis of tissue damage in phenylketonuria (PKU) and of growth failure in Lesch-Nyhan syndrome. Protein turnover was measured in vivo in ten young adult subjects with classical PKU, two subjects with hyperphenylalaninemia, and three children with Lesch-Nyhan syndrome using techniques based on continuous infusions of [13C]leucine and, in Lesch-Nyhan subjects, [2H5]phenylalanine. The PKU subjects had various degrees of dietary phenylalanine restriction and plasma phenylalanine levels at the time of study ranged from 450-1540 mumol/L (mean 1106). Plasma phenylalanine in the two hyperphenylalaninemic subjects was 533 and 402 mumol/L. Rates of protein synthesis in all PKU subjects (mean 3.71 g/kg/24 h, range 2.68-5.10, [13C]leucine as tracer) were in a range similar to or above control values (mean 2.97, range 2.78-3.22, n = 6), as were rates of protein catabolism (PKU mean 4.23 g/kg/24 h, range 3.15-5.45; controls 3.64, 3.50-3.91). Protein turnover values in hyperphenylalaninemia were also similar to those in controls. With [13C]leucine as tracer, both mean protein synthesis and catabolism values in Lesch-Nyhan subjects (mean 4.80 and 5.64 g/kg/24 h, respectively) were higher than values in control children matched for protein intake (synthesis 4.32 +/- 0.74 (SD) and catabolism 4.85 +/- 0.57 (g/kg/24 h, n = 5). Similar results were obtained in Lesch-Nyhan subjects using [2H5]phenylalanine as tracer. These results suggest that protein turnover is not decreased in either PKU or Lesch-Nyhan syndrome. This conclusion is inconsistent with the hypothesis that tissue damage in PKU results from impaired protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)