A survey of natural protein intake in Dutch phenylketonuria patients: insight into estimation or measurement of dietary intake.

This study investigated which methods patients and parents used to determine phenylalanine (Phe) intake and the relationship between the methods applied, age, and blood Phe concentration, as this practice had not been studied before in relation to metabolic control. A questionnaire was sent to 327 Dutch phenylketonuria patients (age 0-29 years) to investigate the method used to determine Phe intake (either by estimation, exact measurement, or a combination of both). Mean blood Phe concentration of each individual patient was related to the method reported to be used. Three different age groups (<10 years, > or =10-15 years, and > or =16 years) were distinguished. The response rate for the questionnaires was 73%. In these 188 patients, data for both Phe concentrations and questionnaires could be used. Of these, 75 used exact measurement, 75 used estimation, and 38 used both methods. The number of patients that estimated Phe intake clearly increased with age. Whatever method was used, an increase in Phe concentrations was seen with age. During childhood, exact measurement was used more frequently, and from adolescence on estimation was used more frequently. The method (exact measurement and/or estimation) did not result in statistically different Phe concentrations in any of the three age groups, although blood Phe concentration tended to be lower in adolescence using exact measurement. Data suggest that estimation and exact measurement of Phe intake are both reliable methods. Therefore, in addition to exact measurement, patients should be instructed in both methods at an early age, so that both methods can be used adequately.

Deficiency or inhibition of lysophosphatidic acid receptor 1 protects against hyperoxia-induced lung injury in neonatal rats.

AIM: Blocking of lysophosphatidic acid (LPA) receptor (LPAR) 1 may be a novel therapeutic option for bronchopulmonary dysplasia (BPD) by preventing the LPAR1-mediated adverse effects of its ligand (LPA), consisting of lung inflammation, pulmonary arterial hypertension (PAH) and fibrosis. METHODS: In Wistar rats with experimental BPD, induced by continuous exposure to 100% oxygen for 10 days, we determined the beneficial effects of LPAR1 deficiency in neonatal rats with a missense mutation in cytoplasmic helix 8 of LPAR1 and of LPAR1 and -3 blocking with Ki16425. Parameters investigated included survival, lung and heart histopathology, fibrin and collagen deposition, vascular leakage and differential mRNA expression in the lungs of key genes involved in LPA signalling and BPD pathogenesis. RESULTS: LPAR1-mutant rats were protected against experimental BPD and mortality with reduced alveolar septal thickness, lung inflammation (reduced influx of macrophages and neutrophils, and CINC1 expression) and collagen III deposition. However, LPAR1-mutant rats were not protected against alveolar enlargement, increased medial wall thickness of small arterioles, fibrin deposition and vascular alveolar leakage. Treatment of experimental BPD with Ki16425 confirmed the data observed in LPAR1-mutant rats, but did not reduce the pulmonary influx of neutrophils, CINC1 expression and mortality in rats with experimental BPD. In addition, Ki16425 treatment protected against PAH and right ventricular hypertrophy. CONCLUSION: LPAR1 deficiency attenuates pulmonary injury by reducing pulmonary inflammation and fibrosis, thereby reducing mortality, but does not affect alveolar and vascular development and, unlike Ki16425 treatment, does not prevent PAH in neonatal rats with experimental BPD.

Multiple deficiencies of mitochondrial DNA- and nuclear-encoded subunits of respiratory NADH dehydrogenase detected with peptide- and subunit-specific antibodies in mitochondrial myopathies.

Antibodies have been raised against synthetic peptides corresponding to several computer-predicted epitopes of three mtDNA-encoded subunits, ND4, ND5 and ND6, of the human respiratory chain NADH dehydrogenase (Complex I). Antibodies were characterized by a sensitive immunoblotting assay using proteins from human skeletal muscle mitochondria and by immunoprecipitation of radio-labeled HeLa cell mitochondrial translation products. Only antibodies against two of six selected peptides of the ND4 subunit, i.e., the C-terminal peptide and an internal peptide close to the C-terminus, reacted in both assays with the subunit. Antibodies raised against an internal peptide close to the N-terminus of the ND5 subunit and antibodies raised against an internal epitope of the ND6 subunit also reacted in both the immunoblotting and immunoprecipitation assays. The antibodies described above and other Complex I subunit- or holoenzyme-specific antibodies were used to investigate the subunit deficiencies of the respiratory NADH dehydrogenase in the skeletal muscle of patients affected by mitochondrial myopathies associated with Complex I defects. The reduction in enzyme activity correlated in an immunoblot assay with a decrease of four mtDNA-encoded subunits of the enzyme, as well as with a decrease of other subunits of Complex I encoded in the nDNA. The present work provides the first evidence of a decrease in NADH dehydrogenase subunits encoded in the mitochondrial genome in myopathy patients.

Successful treatment of pure myopathy, associated with complex I deficiency, with riboflavin and carnitine.

We describe a 6-year-old boy who presented with progressive muscle weakness. Additional investigations revealed the existence of a myopathy and a pure motor neuropathy. Biochemical studies in muscle tissue showed a defect of NADH dehydrogenase (complex I). The patient dramatically improved on treatment with riboflavin and L-carnitine. Seven months after the start of the treatment, complex I activity was determined again and appeared to be normalized. Normalization of the enzymatic defect at this level has not been reported before. We provide a survey of nine patients with pure myopathy, associated with complex I deficiency and onset of symptoms in childhood.

Progressive infantile poliodystrophy. Association with disturbed pyruvate oxidation in muscle and liver.

Progressive infantile poliodystrophy (Alpers' disease) is associated with abnormalities in pyruvate metabolism or in cell mitochondria. A 3-year-old-boy had a severe and rapidly progressive neurologic disorder characterized by psycho-motor retardation, tetraparesis, ataxia, and myoclonic jerks, the illness being exacerbated during periods of infection. Lactate concentration in CSF was elevated. Histopathologic studies revealed lipid storage in liver and muscle. Autopsy showed a progressive infantile poliodystrophy. Mitochondrial abnormalities were found in heart muscle. Biochemical studies of muscle and liver tissue suggested a disturbance in nicotinamide adenine dinucleotide (reduced form) oxidation.

Spinal muscular atrophy-like picture, cardiomyopathy, and cytochrome c oxidase deficiency.

The authors report a child with a spinal muscular atrophy (SMA)-like picture, cardiomyopathy, and cytochrome c oxidase (COX) deficiency. Electromyography and muscle biopsy showed findings typical of SMA. However, COX staining of the muscle was negative. DNA analysis did not detect deletions in the survival motor neuron (SMN) gene. The lactate and lactate-to-pyruvate ratios were increased in blood and CSF. COX activity was decreased in muscle and fibroblasts. Western blot analysis showed reduced contents for all COX subunits. Patients with clinical features resembling SMA but with an intact SMN gene should be screened for a mitochondrial disorder.

Mitochondrial creatine kinase containing crystals, creatine content and mitochondrial creatine kinase activity in chronic progressive external ophthalmoplegia.

Mitochondrial crystals containing mitochondrial creatine kinase (Mi-CK) protein were described recently. From in vitro studies it has been suggested that alterations in creatine concentration are connected to the occurrence of these crystals. In the present study free, phosphorylated and total creatine concentrations as well as Mi-CK activity were determined in muscle samples of six patients with chronic progressive external ophthalmoplegia (CPEO). Two of them showed Mi-CK containing mitochondrial crystals. The activity of Mi-CK was found to be clearly enhanced in those muscle samples in which mitochondrial crystals were present. No relationship was found between the concentration of total, free or phosphorylated creatine and the occurrence of mitochondrial crystals. An up to now unknown mechanism seems to cause the formation of Mi-CK containing crystals in human muscle mitochondria.

Abnormal expression of intermediate filament proteins in X-linked myotubular myopathy is not reproduced in vitro.

Expression patterns of the intermediate filament proteins (IFPs) desmin and vimentin, in biopsy material taken from a 1 day old boy with fatal neonatal X-linked myotubular myopathy (XLMTM) were compared with the expression of these proteins in cultured myotubes, from the same patient. Immunohistochemical studies revealed the persistence of high levels of desmin in virtually all, and vimentin in most, of the myofibres within the patient's biopsy. Analysis of intermediate filament expression in differentiating, cultured muscle cells did not reveal overt differences between XLMTM cultures and cultures of control muscle. Titin distribution patterns indicated a normal process of myofibrillogenesis in XLMTM myotubes. We conclude that the failure to properly regulate IFP-expression is not intrinsic to XLMTM muscle fibres. The possibility that this failure is due to a defective external, possibly neural factor, is discussed.

Postnatal centralization of muscle fibre nuclei in centronuclear myopathy.

Postnatal centralization of muscle fibre nuclei, which were previously located subsarcolemmally, is described in a case of centronuclear myopathy (CNM) in a male patient with generalized muscle weakness since birth. A muscle biopsy was taken at the age of 11 months; no particular abnormalities were observed at this stage apart from an unusual variation in fibre size. A distinctly below average muscle fibre diameter, increased endomysial connective tissue, and features typical for CNM were found in a biopsy taken 9 yr later. Immunohistochemical studies using antibodies to desmin, vimentin, laminin and type IV collagen revealed altered staining patterns compared with normal fibres. The abnormalities in the patterns of cytoskeletal proteins point to a defective regulation of the composition and organization of the cytoskeletal network during development, paralleled by abnormalities in the extracellular matrix.

Myopathology and a mitochondrial DNA deletion in the Pearson marrow and pancreas syndrome.

A patient with the Pearson marrow and pancreas syndrome is presented. She showed an anaemia with neutropenia and thrombopenia, failure to thrive, diarrhoea, disturbed glucose homeostasis and lactic acidosis. An exocrine pancreatic insufficiency was lacking. The disease followed a fatal course. Biochemical investigations of skeletal muscle revealed a disturbed mitochondrial energy metabolism, while many ultrastructural abnormal features were observed in the muscle tissue. Molecular genetic studies showed a de novo deletion in the mitochondrial DNA (mtDNA), different in size from the already published deletions and flanked by two 4 bp direct repeats, interspaced by 4-5 non-repeated nucleotides. mtDNA from 12 other tissues showed the same deletion in different percentages. No obvious relation between these percentages and tissue dysfunction was found. In spite of an open reading frame of 74 codons, only little transcription product of the genomic region resulting from the deletion was found.

Features of a syndrome with congenital cataract and hypertrophic cardiomyopathy.

We studied 12 patients from six unrelated families with a syndrome that has an autosomal recessive pattern of inheritance and can be diagnosed from clinical, histologic, and biochemical characteristics. The four major symptoms are congenital cataract, hypertrophic cardiomyopathy, mitochondrial myopathy of voluntary muscles, and exercise-related lactic acidosis. The patients had bilateral and total cataract in the first weeks of life, underwent cataract surgery, and developed nystagmus and strabismus. Corrected visual acuity was lower than 20/40 in aphakic eyes. Patients were mentally normal, and at school age they visited a school for blind and visually impaired children. The majority of the patients developed axial myopia with myopic fundus changes; aphakic refraction usually was lower than 10.0 diopters after the first decade. The cardiac myopathy was progressive and the cause of premature death. Three of the 12 patients died in the neonatal period and six patients died in early adulthood.

White matter abnormalities in congenital muscular dystrophy.

Central nervous system (CNS) characteristics were examined in seventeen patients with autosomal recessive classic or "pure" congenital muscular dystrophy (CMD). In three patients, neuroradiological examination (CT/MRI) indicated hypodense white matter areas. Two out of these three patients had epilepsy (seizures and epileptic discharges on their EEG). Only two of the remaining patients had epileptic EEG discharges, but without clinical seizures. By comparing our results to data in the literature, we could conclude that the classic or "pure" form of CMD can be subdivided into two subtypes, i.e. those with and those without white matter hypodensities. A mild form of epilepsy or an epileptic predisposition on EEG can be part of the subtype with white matter hypodensities.

Dihydropyrimidine dehydrogenase deficiency. Neurological aspects.

A family with dihydropyrimidine dehydrogenase (DPD) deficiency is presented. In 3 persons a complete deficiency, and in 3 others a partial deficiency was detected in cultured fibroblasts. Two homozygote subjects and 1 heterozygote subject suffered from epileptic manifestations, in one of these homozygote subjects also microcephaly was found. DPD deficiency might be an etiological factor in the clinical picture of these patients. An autosomal recessive mode of inheritance of this deficiency was found.

In vitro contraction test for malignant hyperthermia in patients with unexplained recurrent rhabdomyolysis.

A few cases of non-anaesthetic-induced rhabdomyolysis in humans, predisposed to malignant hyperthermia (MH), have been described in literature. We studied a group of 6 consecutive patients with unexplained and recurrent attacks of rhabdomyolysis with the test used to determine susceptibility to MH, the in vitro contraction test (IVCT). The results of the IVCT showed 5 of these 6 patients to be MH susceptible. In cultured muscle cells from one of these patients a disturbed calcium homeostasis could be demonstrated. The relation between MH and recurrent rhabdomyolysis is discussed.

Clinical heterogeneity in respiratory chain complex III deficiency in childhood.

Six children are presented with an isolated complex III deficiency in muscle tissue. More specifically, oxidation rates and ATP+CrP production rates from both pyruvate and succinate as substrates and/or the activity of decylubiquinol:cytochrome c oxidoreductase were all markedly reduced. Complex III deficiency was also present in liver of two patients tested, but could not be demonstrated in cultured fibroblasts of four patients tested. Mitochondrial DNA, extracted from muscle, was analyzed; no deletions or common point mutations were found. Four patients presented with a multi-organ disorder. Among these patients three presented at neonatal age with neurological signs and lactate elevation in blood and CSF, of whom two had severe neonatal Fanconi syndrome. One child, aged seven years, had encephalomyopathy, ophthalmoplegia, retinopathy and Wolff-Parkinson-White syndrome. The remaining two patients exhibited myopathy only, within the first year of life. Thus, like in other respiratory chain disorders, patients with complex III deficiency may present at any age and show variable symptoms and outcome, ranging from neonatal death to failure to thrive only. Apparently there are no clinical findings which are specific for complex III deficiency.

Immunophenotyping of congenital myopathies: disorganization of sarcomeric, cytoskeletal and extracellular matrix proteins.

We have studied the expression and distribution patterns of the intermediate filament proteins desmin and vimentin, the sarcomere components titin, nebulin and myosin, the basement membrane constituents collagen type IV and laminin, and the reticular layer component collagen type VI in skeletal muscle of patients with "classic" congenital myopathies (CM), using indirect immunofluorescence assays. In all biopsy specimens obtained from patients with central core disease (CCD), nemaline myopathy (NM), X-linked myotubular myopathy (XLMTM) and centronuclear myopathy (CNM), disease-specific desmin disturbances were observed. Vimentin was present in immature fibres in severe neonatal NM, and as sarcoplasmic aggregates in one case of CNM, while the amounts of vimentin and embryonic myosin, observed in XLMTM, decreased with age of the patients. Abnormal expression of myosin isoforms was found in several CM biopsies, although the organization of myosin and other sarcomere components was rarely disturbed. Basement membrane and reticular layer proteins were often prominently increased in severe cases of CM. We conclude that (i) desmin is a marker for individual types of CM and might be used for diagnostic purposes; (ii) the expression patterns of the differentiation markers desmin, vimentin and embryonic myosin in XLMTM, point either to a postnatal muscle fibre maturation or to a variable time-point of maturational arrest in individual patients; (iii) the correlation between the distribution patterns of extracellular matrix proteins and clinical presentation points to a role of these proteins in pathophysiology of CM.

L-Ornithine-ketoacid-transaminase deficiency in cultured fibroblasts of a patient with hyperornithinaemia and gyrate atrophy of the choroid and retina.

L-Ornithine-ketoacid-transaminase deficiency was established in cultured fibroblasts obtained from a patient with hyperornithinaemia (mean ornithine level in serum approximately 100 mumol/l) and gyrate atrophy of the choroid and retina. The deficiency was found both the L-ornithine concentrations of 3.0 mM (about twice the KM value) and 12 mM, indicating that the enzymic defect was not due to a decreased affinity for this substrate. The reliability of the colorimetric assay of ornithine-ketoacid-transaminase activity was established radiochemically. Performance of the radiochemical assay revealed the presence of an impurity in the substrate DL-[2-14C]ornithine - HCl being a strong inhibitor of the enzyme. The passage level and the subcultivation time of the fibroblasts did not influence the enzymic activity.

Elevated urine, blood and cerebrospinal fluid levels of uracil and thymine in a child with dihydrothymine dehydrogenase deficiency.

In the urine of a child with unexplained convulsions large amounts of uracil and thymine were detected by gas chromatography. Identification was performed by coupled gas chromatography-mass spectrometry. Quantitation of the urinary excretion by means of a sensitive high-performance liquid chromatographic (HPLC) method revealed a 1000-fold elevation compared to normal. Serum and cerebrospinal fluid levels of the two pyrimidine bases were about a hundred times higher than normal. In fibroblasts the activity of dihydrothymine dehydrogenase was determined by measuring the conversion of radioactive labelled thymine to dihydrothymine with HPLC of the reaction mixture. In the patient's cells a complete deficiency of dihydrothymine dehydrogenase activity was found. Our patient is the first case described with such a proven enzyme deficiency.

Investigation of mitochondrial metabolism in small human skeletal muscle biopsy specimens. Improvement of preparation procedure.

A method is presented which allows the investigation of almost the complete mitochondrial content of small human skeletal muscle biopsy specimens. Thorough mechanical disruption with a chopper apparatus results in the release of about 50% of the mitochondrial content. Subsequent treatment of the 600 x g sediment with trypsin releases another 30% of the total mitochondrial population. The biochemical characteristics of the two mitochondrial fractions obtained in these two successive steps have been compared. No obvious differences could be established. The procedure is well suited for biochemical investigation of muscle biopsy specimens from patients suspected of suffering from a mitochondrial myopathy.

Differential investigation of the capacity of succinate oxidation in human skeletal muscle.

Procedures are described for the estimation of the succinate:ubiquinone oxidoreductase and succinate:phenazine methosulfate oxidoreductase activities in post-nuclear supernatants of human skeletal muscle homogenates using 2,6-dichlorophenol indophenol as the terminal electron acceptor. The influence of ionic strength and of sucrose upon these assays and upon the succinate:cytochrome c oxidoreductase activity has been investigated. Sucrose markedly interferes with the activation of the succinate dehydrogenase complex. Succinate:cytochrome c oxidoreductase activity and succinate:phenazine methosulfate oxidoreductase activity are inhibited by increasing concentrations of ions and of sucrose. Our results lead us to propose the existence of a single acceptor site for phenazine methosulfate at the succinate dehydrogenase complex, not involved in the physiological electron flux across ubiquinone. Estimation of the enzymatic activities mentioned above allows differential investigation of the functional integrity of a large part of the respiratory chain in patients suspected of suffering from a neuromuscular disorder.