J. R. Bonham

Biochemical, clinical and molecular findings in LCHAD and general mitochondrial trifunctional protein deficiency

SummaryGeneral mitochondrial trifunctional protein (TFP) deficiency leads to a wide clinical spectrum of disease ranging from severe neonatal/infantile cardiomyopathy and early death to mild chronic progressive sensorimotor poly-neuropathy with episodic rhabdomyolysis. Isolated long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency resulting from the common Glu510Gln mutation usually gives rise to a moderately severe phenotype with multiorgan involvement with high morbidity and mortality. However, isolated LCHAD deficiency can also be consistent with long-term survival in patients identified and treated from an early age. We present biochemical, clinical and mutation data in 9 patients spanning the full spectrum of disease. Fibroblast acylcarnitine profiling shows good correlation with clinical phenotype using the ratio C18(OH)/(C14(OH)+C12(OH)). This ratio shows a gradation of values, from high in four patients with severe neonatal disease (2.5±0.8), to low in two neuromyopathic patients (0.35, 0.2). Fibroblast fatty acid oxidation flux assays also show correlation with the patient phenotype, when expressed either as percentage residual activity with palmitate or as a ratio of percentage activity of myristate/oleate (M/O ratio). Fibroblasts from four patients with severe neonatal disease gave an M/O ratio of 4.0±0.6 compared to 1.97 and 1.62 in two neuromyopathic patients. Specific enzyme assay of LCHAD and long-chain 3-ketothiolase activity in patient cells shows lack of correlation with phenotype. These results show that measurements in intact cells, which allow all determinative and modifying cellular factors to be present, better reflect patient phenotype. Mutation analysis reveals a number of α- and β-subunit mutations. Peripheral sensorimotor polyneuropathy, often as the initial major presenting feature but usually later accompanied by episodic rhabdomyolysis, is a manifestation of mild TFP protein deficiency. The mild clinical presentation and relative difficulty in diagnosis suggest that this form of TFP is probably underdiagnosed.

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

Recommended approaches for the laboratory measurement of homocysteine in the diagnosis and monitoring of patients with hyperhomocysteinaemia.

Several recent studies have indicated that an increased concentration of plasma homocysteine is an independent risk factor for the premature development of vascular disease. These important findings emphasize the need for careful selection of an appropriate analytical approach to diagnose and treat individuals who may be at risk. We compared the results obtained from the measurement of plasma total homocysteine (free + protein-bound fractions) by high-performance liquid chromatography (HPLC) with the measurement of plasma free homocystine (free fraction) by conventional ion-exchange chromatography in 10 patients with inherited defects of homocysteine metabolism and 13 obligate heterozygote individuals. This study can be used to formulate recommendations on the appropriate use of these assays in different clinical circumstances. Our results show that the concentration of total plasma homocysteine must exceed 60 μmol/L before plasma free homocystine becomes detectable by conventional ion-exchange chromatography. Similarly, assessment of the urinary excretion of homocysteine in these patients indicates that it may not become consistently detectable by conventional ion-exchange chromatography or HPLC until plasma total homocysteine exceeds 150 μmol/L. On this basis, while most patients with classical homocystinuria would be detected by analysis of plasma using conventional ion-exchange chromatography or by measurement of of the urinary homocysteine excretion, occasional patients would be missed. When monitoring patients receiving treatment for classical homocystinuria, in whom metabolic control is good, and when investigating individuals with a suspected inherited defect of cobalamin or folate metabolism, a method which measures plasma total homocysteine should be used. The identification of moderate hyperhomocysteinaemia of undefined cause investigated in relation to a history of early vacsular disease can only be identified by this approach.

The allopurinol load test lacks specificity for primary urea cycle defects but may indicate unrecognized mitochondrial disease.

Thirty-three children ranging from 2 weeks to 12 years of age were selected for allopurinol loading, 16 on the basis of an increased urinary orotate excretion detected by routine organic acid analysis (group A), and 17 for clinical reasons suggesting a urea cycle defect (group B). The allopurinol load test proved positive in 13 of 16 patients from group A, mean peak orotate 64.0μmol/mmol creatinine (upper limit of reference range, 13.2) and 11 of 17 patients from group B, mean peak orotate 41.0μmol/mmol creatinine (upper limit of reference range, 13.2). Thorough investigation of these patients including urinary and plasma amino acid analysis and, in 17 cases, liver biopsy for histology and measurement of ornithine carbamyltransferase (OCT) and carbamyl-phosphate synthetase (CPS) activity failed to identify any evidence of a urea cycle disorder. However, muscle biopsies performed in 11 patients showed some evidence of mitochondrial disease in four cases, two defined on the basis of reduced respiratory chain enzyme activity and two on the basis of mtDNA abnormalities. These findings indicate that an increased excretion of orotate in sick children may not be uncommon and that a positive allopurinol load test result may not indicate a specific inherited urea cycle defect. In addition, these results raise the interesting possibility that defective ureagenesis may be a feature of mitochondrial disease in some individuals.

Quality assessment of urinary organic acid analysis

The number of known inherited metabolic disorders resulting in an organic aciduria has increased steadily over the past two decades. Prompt and reliable detection is both clinically and technically demanding but is essential if appropriate treatment is to be undertaken. This is the first study of laboratory performance in the detection of these disorders to be undertaken in the UK. Some conditions were accurately identified by most laboratories: for example for maple syrup urine disease, 12 of 14 laboratories provided an appropriate response and medium chain acyl-CoA dehydrogenase deficiency was correctly identified by 15 of 17 laboratories. However, accuracy of detection was poorer for other conditions: for example, only eight of 17 laboratories detected tyrosinaemia type 1 and nine of 18 laboratories detected 4-hydroxybutyric aciduria. The strongest correlation with good performance was obtained by comparison with the extent of peak identification: r=0·62, P=0·002. The need for regular attendance at scientific symposia was also supported by a weaker positive correlation with the average score achieved, P=0·08. Evidence also suggested that some of the laboratories with a low workload performed less well. No significant difference in performance could be demonstrated between the 17 laboratories who used gas chromatography -mass spectrometry and the six participants who used gas chromatography alone.

Systematic review and meta-analysis to estimate the birth prevalence of five inherited metabolic diseases

Many newborn screening programmes now use tandem mass spectrometry in order to screen for a variety of diseases. However, countries have embraced this technology with a differing pace of change and for different conditions. This has been facilitated by the ability of this diagnostic method to limit analysis to specific metabolites of interest, enabling targeted screening for particular conditions. MS/MS was introduced in 2009 in England to implement newborn bloodspot screening for medium chain acyl-CoA dehydrogenase deficiency (MCADD) raising the possibility of screening for other inherited metabolic disorders. Recently, a pilot screening programme was conducted in order to evaluate the health and economic consequences of screening for five additional inherited metabolic disorders in England. As part of this study we conducted a systematic review and meta-analysis to estimate the birth prevalence of these conditions: maple syrup urine disease, homocystinuria (pyridoxine unresponsive), glutaric aciduria type I, isovaleric acidaemia and long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency including trifunctional protein deficiency. We identified a total of 99 studies that were able to provide information on the prevalence of one or more of the disorders. The vast majority of studies were of screening programmes with some reporting on clinically detected cases.

Pitfalls in the measurement of some intermediary metabolites

Lactate, pyruvate, 3-hydroxybutyrate, acetoacetate and non-esterified fatty acids are intermediary metabolites that normally occur in blood and all have a vital role in energy metabolism. Their relative concentrations are an expression of nutritional balance, providing a snapshot of the metabolic disturbances arising in a patient. They are therefore invaluable tools to investigate intermediary metabolism in health and disease, particularly in the fields of diabetes and inherited metabolic disease. Although the analysis of these key metabolites would appear to be straightforward, with apparently simple assays widely available, there are many pitfalls in their measurement. To compound this difficulty there is limited advice available for the optimum pre-analytical and analytical aspects of their measurement and also for the interpretation of results. In this personal view, we aim to highlight a number of these problems, such as sample stability, assay interference and availability of reference ranges, with the aim of producing guidelines for the measurement and interpretation of these metabolites.

Problems in the detection of fatty acid oxidation defects: Experience of a quality assurance programme for qualitative urinary organic acid analysis

An external quality assurance scheme for qualitative urinary organic acid analysis was started in 1992 with 23 participants, mostly from the United Kingdom (Bonham et al 1994), and has steadily grown under the ERNDIM (European Research Network for Evaluation and Improvement of Screening, Diagnosis and Treatment of Inherited Metabolic Disorders) initiative to the current 111 participants from 28 countries worldwide. Eighty-nine per cent of participants use GC-MS, 10% use GC only and there is one HPLC user. Ether and/or ethyl acetate extraction with trimethylsilyl derivatization is used by > 90% of laboratories. Twelve samples are circulated annually. Each sample comprises 2 ml heat-treated urine (not spiked but sometimes pooled) from children who have a known disorder or who are normal. Brief clinical details are supplied and participants are asked to report in the usual way used in that laboratory and to include an annotated chromatogram. Urine samples from patients with 26 different disorders have been circulated to date and there has been an improvement in the rate of recognition of some of these conditions, supporting the educational value of this scheme. However, the identification of fatty acid oxidation defects is a continuing problem for some laboratories, from both an analytical and an interpretational standpoint.

Vitreous humour and cerebrospinal fluid hypoxanthine concentration as a marker of pre-mortem hypoxia in SIDS.

AIMS--To assess the rate at which premortem hypoxia occurs in sudden infant death syndrome (SIDS) when compared with death in early childhood. METHODS--The hypoxanthine concentration was measured as a marker of premortem hypoxia in vitreous humour and cerebrospinal fluid samples obtained at necropsy from 119 children whose ages ranged from 1 week to 2 years. RESULTS--Increasing interval between death and necropsy was accompanied by an increase in the hypoxanthine concentration of vitreous humour for the first 24 hours, at a rate of 8.3 mumol/l/hour. Thereafter, there was little change with time, and the results wer corrected to 24 hours according to a regression equation. Cerebrospinal fluid concentrations showed no significant change with time following death. Patients were divided into three groups according to the cause of death: SIDS, cardiac or pulmonary disease, and others. Median values for the cerebrospinal fluid hypoxanthine concentrations were not significantly different among the groups and no difference could be shown between the vitreous humour hypoxanthine concentration in cases of SIDS and those children dying from other causes. Patients with established cardiac or pulmonary disease had a significantly reduced vitreous humour hypoxanthine concentration which may have reflected the premortem use of artificial ventilation. CONCLUSIONS--The results of this study do not support the view that pre-mortem hypoxia is a common feature in SIDS when compared with other causes of death.

Normal acylcarnitines in maternal urine during a pregnancy affected by glutaric aciduria type II

Normal acylcarnitines in maternal urine during a pregnancy a†ected by glutaric aciduria type II N. J. Manning1*, J. R. Bonham1, M. Downing1, R. G. Edwards1, S. E. Olpin1, R. J. Pollitt1, M. Pourfarzam3, M. J. Sharrard2 and M. S. T anner2 1 Department of Chemical Pathology and Neonatal Screening, 2 Department of Paediatrics, The ChildrenÏs Hospital, Sheffield ; 3 Department of Child Health, The Royal Victoria InÐrmary, Newcastle upon Tyne, UK * Correspondence : Department of Chemical Pathology and Neonatal Screening, The ChildrenÏs Hospital, Western Bank, Sheffield S10 2TH, UK