[Lactic acidosis and accumulation of glutamate in the blood of neonates following treatment with calcium levulinate for hypocalcaemia]. / Lactaatacidose en glutamaatophoping in het bloed van neonaten na behandeling met calciumlevulaat wegens hypocalciëmie.

The data from 5 clinics concerning 8 infants, who had developed severe lactic acidosis and hyperglutamic acidaemia were reviewed. Blood-lactate levels were up to 15 mmol/l (reference level: < 2) and plasma-glutamate levels up to 1632 pmol/l (reference level: 14-78), and there was no concomitant hyperglutaminaemia (levels up to 1032 micromol/l (reference level: 333-809)). A positive correlation between the amount of calcium levulinate administered and the degree of hyperglutamic acidaemia was found. Replacement of the calcium levulinate by another calcium salt caused a reversal of the biochemical abnormalities of the patients. Two of the infants had a 22q11 microdeletion. This development of severe acidosis in infants who had been given a calcium supplement in the form of calcium levulinate may be related to genetic predisposition. The paradoxal hyperketonaemia and generalized aminoaciduria in 4 other patients suggested disturbed function ofthe mitochondrial respiratory chain. The hypothesis of the occurrence of an underlying defect of the mitochondrial respiratory chain was tested in the muscle tissue of one 22q11 patient, but this showed no abnormalities. Excessive accumulation of glutamate because of dysfunction ofglutamine synthetase, which forms glutamate from glutamine seems unlikely because of the relatively low values of plasma glutamate compared to the glutamine plasma levels. Calcium levulinate should no longer be used in neonates as it may lead to lactic acidosis.

Albumin and whole-body protein synthesis respond differently to intraperitoneal and oral amino acids.

Patients with peritoneal dialysis are at risk for malnutrition and hypoalbuminemia, which are indicators of poor outcome. Recently, it was shown that dialysis solutions containing amino acids (AAs) and glucose improve protein anabolism in peritoneal dialysis patients. We determined if the same solutions could increase the fractional synthesis rate of albumin along with whole-body protein synthesis. Changes in the fractional albumin synthetic rate reflect acute change in hepatic albumin synthesis. A random-order cross-over study compared the effects of Nutrineal (AA source) plus Physioneal (glucose) dialysate with Physioneal alone dialysate. Eight patients in the overnight fasting state were compared to 12 patients in the daytime-fed state. Fractional albumin synthetic rate and whole-body protein synthesis were determined simultaneously using a primed-continuous infusion of L-[1-(13)C]-leucine. Fractional albumin synthesis on AAs plus glucose dialysis did not differ significantly from that on glucose alone in the fasting or the fed state. Protein intake by itself (fed versus fasting) failed to induce a significant increase in the fractional synthetic rate of albumin. Conversely, the oral protein brought about a significant stimulation of whole-body protein synthesis. Our findings show that the supply of AAs has different effects on whole-body protein synthesis and the fractional synthetic rate of albumin.

Peritoneal protein losses and cytokine generation in automated peritoneal dialysis with combined amino acids and glucose solutions.

OBJECTIVES: Protein-energy malnutrition as a consequence of deficient protein intake frequently occurs in peritoneal dialysis (PD) patients. Previously, we showed that peritoneal dialysate containing a mixture of amino acids (AA) and glucose has anabolic effects. However AA-dialysate has been reported to increase intraperitoneal protein and AA losses and the release of proinflammatory cytokines (interleukine-6 (IL-6) and tumor necrosis factor alpha (TNFalpha)). We investigated the effect of AA plus glucose (AAG) solutions on peritoneal protein losses and cytokine generation. METHODS: In 6 patients on standard automated peritoneal dialysis (APD) 12 APD sessions of 6 cycles each were performed during the night using dialysate containing 1.1% AA plus glucose or glucose alone as control. Protein losses and TNFalpha and IL-6 concentrations were measured in dialysates separately collected from nightly cycling and daytime dwell. RESULTS: The 24 hour-protein losses with AAG (median 6.7 g, range 4.7-9.4 g) were similar to control dialysate (median 6.0 g, range 4.2-9.2 g). Daytime dialysate IL-6 levels were higher after nightly AAG dialysis than after control dialysis (142 pg/ml and 82 pg/ml, respectively, P<.05). TNFalpha concentrations were very low. CONCLUSION: Nightly APD with amino acids containing dialysate was associated with an increase in peritoneal IL-6 generation during the day. The addition of AA to standard glucose dialysis solutions did not induce a significant increase of peritoneal protein losses.

Brain abnormalities in a case of malonyl-CoA decarboxylase deficiency.

Malonyl-CoA decarboxylase (MCD) deficiency is an extremely rare inborn error of metabolism that presents with metabolic acidosis, hypoglycemia, and/or cardiomyopathy. Patients also show neurological signs and symptoms that have been infrequently reported. We describe a girl with MCD deficiency, whose brain MRI shows white matter abnormalities and additionally diffuse pachygyria and periventricular heterotopia, consistent with a malformation of cortical development. MLYCD-gene sequence analysis shows normal genomic sequence but no messenger product, suggesting an abnormality of transcription regulation. Our patient has strikingly low appetite, which is interesting in the light of the proposed role of malonyl-CoA in the regulation of feeding control, but this remains to be confirmed in other patients. Considering the incomplete understanding of the role of metabolic pathways in brain development, patients with MCD deficiency should be evaluated with brain MRI and unexplained malformations of cortical development should be reason for metabolic screening.

Two novel mutations in SLC6A8 cause creatine transporter defect and distinctive X-linked mental retardation in two unrelated Dutch families.

Four Dutch male patients, two brothers from unrelated families were referred for investigation of psychomotor and severe language/speech delay. All four patients showed growth deficiency over the years. Facial features and poor body habitus were quite similar in the patients and in their mothers. Brain MRI showed nonspecific periventricular white matter lesions. In all the patients neuropsychological tests revealed moderate mental retardation, attention deficit and hyperactivity with impulsivity, a semantic-pragmatic language disorder, and oral dyspraxia. This specific cognitive profile is different from other children with mental retardation syndromes and seems to be unique. Excretion of creatine to creatinine ratio in urine of the four boys was increased compared to controls and their creatine uptake in fibroblasts was deficient. In the two brothers from the first pedigree, DNA sequence analysis revealed a novel mutation in the splice donor site in intron 10 (IVS10 + 5G>C, c.1495 + 5G>C) of the SLC6A8 gene leading to skipping of exon 10. In the other sib pair a novel missense mutation (c. 1361C>T; p.Pro544Leu) was found. These are the first families reported, in which the clinical suspicion of a creatine transporter disorder was raised on clinical grounds, before a brain 1H-MRS suggested the diagnosis. Screening of apparently X-linked mental retarded patients with this somatic and behavioral phenotype by the biochemical assay of creatine to creatinine ratio in the urine or DNA sequence analysis of SLC6A8 is worthwhile even when 1H-MRS is not available.

[Diagnosis of vitamin B12 deficiency revised]. / De diagnostiek van vitamine-B12-deficiëntie herzien.

Vitamin B12 (cobalamin) deficiency is a common disorder with potential irreversible haematological and neurological consequences. Currently used diagnostic tests such as the evaluation of serum vitamin B12 and the Schilling test are insufficient, e.g. the positive predictive value of a low serum vitamin B12 level for actual vitamin B12 deficiency (i.e. tissue deficiency) is low. Insufficient availability of vitamin B12 will lead to the accumulation of methylmalonic acid and homocysteine in the body. Nearly all patients with vitamin B12 deficiency also have substantially increased levels of methylmalonic acid and homocysteine. New tests of serum methylmalonic acid and homocysteine are highly sensitive for vitamin B12 deficiency and may obviate the need for the somewhat cumbersome Schilling test.

Clinical, enzymatic, and molecular genetic characterization of a biochemical variant type of argininosuccinic aciduria: prenatal and postnatal diagnosis in five unrelated families.

A biochemical variant of argininosuccinate lyase deficiency, found in five individuals, is introduced. In comparison to classical patients, the variant cases of argininosuccinate lyase deficiency were characterized by residual enzyme activity as measured by the incorporation of [14C]citrulline into proteins. The five patients of different ethnic backgrounds presented with relatively mild clinical symptoms, variable age of onset, marked argininosuccinic aciduria and severe, but not complete, deficiency of argininosuccinate lyase. [14C]Citrulline incorporation into proteins, which is completely blocked in classical argininosuccinic aciduria, was only partially reduced in fibroblasts of these patients. Further investigation showed that previous standard conditions of the assay were not optimal. Higher concentrations of citrulline in the incubation medium strongly stimulated 14C incorporation in normal cells, but not in the patients; as a result, the relative incorporation level in the patients dropped to 6-28% compared to 18-75% of normal in the original procedure. Prenatal diagnosis was successfully performed in three of the families. Affected pregnancies were indicated by (partial) deficiency of [14C]citrulline incorporation in chorionic villi and/or increased levels of argininosuccinate in amniotic fluid. Analysis of the ASL gene in the five patients revealed a considerable allelic heterogeneity. Three novel mutations--R385C (2 patients), V178M and R379C--were detected in homozygous states, whereas one patient was compound heterozygous for the known mutations R193Q and Q286R. In conclusion, there are patients of different ethnic backgrounds who are characterized by residual activity of argininosuccinate lyase and who present with less severe clinical courses. In addition, we present an improved biochemical assay for accurate prenatal and postnatal diagnosis.

Prenatal diagnosis of the Hunter syndrome and the introduction of a new fluorimetric enzyme assay.

Prenatal diagnosis of the Hunter syndrome (mucopolysaccharidosis type II; MPS II) is preferably achieved by the assay of iduronate-2-sulphate sulphatase (IDS) in uncultured chorionic villi (CV) as this allows early (12th week), rapid (2-3 days) and reliable results. We summarize the results of 174 prenatal analyses in the past 30 years, using various methods such as radiolabelled sulphate incorporation in amniotic fluid (AF) cells, glycosaminoglycan (GAG)-electrophoresis in AF and IDS assay in CV, CV-cells, AF and AF-cells. Twenty-seven fetuses with MPS II were diagnosed after finding clearly abnormal results in pregnancies with a male fetus; very low IDS activity has also been measured in some pregnancies with a (heterozygous) female fetus, emphasizing the need to combine enzyme assay with fetal sex determination. IDS activity has until recently been assessed by a cumbersome radioactive enzyme assay. Here we describe the use of a novel fluorigenic 4-methylumbelliferyl substrate, which allows a sensitive, rapid and convenient assay of IDS activity and reliable early prenatal diagnosis. This novel IDS assay was validated in retrospective analyses of 14 CV, CV-cell, AF and AF-cell samples from affected pregnancies in addition to prospective prenatal diagnosis in eight pregnancies at risk with one MPS II-affected fetus.

Pitfalls in the diagnosis of multiple sulfatase deficiency.

Multiple sulfatase deficiency (MSD, OMIM 272200) is an autosomal recessive leukodystrophy associated with the deficiency of several, in total seven, sulfatases. The disorder is clinically and biochemically variable. The clinical picture combines features of mucopolysaccharidosis and metachromatic leukodystrophy (MLD, OMIM 250100) in a variable spectrum. Here we report a 3-year old Iranian girl with an MLD-like presentation of MSD. Arylsulfatase A deficiency and sulfatide excretion were found. Differently from what was previously reported in the literature, this girl never showed abnormal mucopolysaccharide excretion in the urine. There were no additional visceral or skeletal signs. She was originally diagnosed as having MLD. Only when she developed ichthyosis were seven additional sulfatases measured. In leukocytes, arylsulfatase A, steroid sulfatase and N-acetylglucosamine-6 sulfatase were profoundly deficient, while iduronate-2 sulfatase and arylsulfatase B were moderately reduced. In fibroblasts, N-acetylglucosamine-6 sulfatase was deficient, while arylsulfatase A was moderately reduced. This case illustrates the possible pitfalls in the clinical and laboratory diagnosis of MSD.

Functional hyperactivity of hepatic glutamate dehydrogenase as a cause of the hyperinsulinism/hyperammonemia syndrome: effect of treatment.

OBJECTIVE: The combination of persistent hyperammonemia and hypoketotic hypoglycemia in infancy presents a diagnostic challenge. Investigation of the possible causes and regulators of the ammonia and glucose disposal may result in a true diagnosis and predict an optimum treatment. PATIENT: Since the neonatal period, a white girl had been treated for hyperammonemia and postprandial hypoglycemia with intermittent hyperinsulinism. Her blood level of ammonia varied from 100 to 300 micromol/L and was independent of the protein intake. METHODS: Enzymes of the urea cycle as well as glutamine synthetase and glutamate dehydrogenase (GDH) were assayed in liver tissue and/or lymphocytes. RESULTS: The activity of hepatic GDH was 874 nmol/(min.mg protein) (controls: 472-938). Half-maximum inhibition by guanosine triphosphate was reached at a concentration of 3.9 micromol/L (mean control values:.32). The ratio of plasma glutamine/blood ammonia was unusually low. Oral supplements with N-carbamylglutamate resulted in a moderate decrease of the blood level of ammonia. The hyperinsulinism was successfully treated with diazoxide. CONCLUSION: A continuous conversion of glutamate to 2-oxoglutarate causes a depletion of glutamate needed for the synthesis of N-acetylglutamate, the catalyst of the urea synthesis starting with ammonia. In addition, the shortage of glutamate may lead to an insufficient formation of glutamine by glutamine synthetase. As GDH stimulates the release of insulin, the concomitant hyperinsulinism can be explained. This disorder should be considered in every patient with postprandial hypoglycemia and diet-independent hyperammonemia.

Genomic organization of the human phosphomannose isomerase (MPI) gene and mutation analysis in patients with congenital disorders of glycosylation type Ib (CDG-Ib).

CDG-Ib is the "gastro-intestinal" type of the congenital disorders of glycosylation (CDG) and a potentially treatable disorder. It has been described in patients presenting with congenital hepatic fibrosis and protein losing enteropathy. The symptoms result from hypoglycosylation of serum- and other glycoproteins. CDG-Ib is caused by a deficiency of mannose-6-phosphate isomerase (synonym: phosphomannose isomerase, EC 5.3.1.8), due to mutations in the MPI gene. We determined the genomic structure of the MPI gene in order to simplify mutation detection. The gene is composed of 8 exons and spans only 5 kb. Eight (7 novel) different mutations were found in seven patients with a confirmed phosphomannose isomerase deficiency, analyzed in the context of this study: six missense mutations, a splice mutation and one insertion. In the last, the mutation resulted in an unstable transcript, and was hardly detectable at the mRNA level. This emphasizes the importance of mutation analysis at the genomic DNA level.

Characterization of a novel mitochondrial DNA deletion in a patient with a variant of the Pearson marrow-pancreas syndrome.

We have recently diagnosed a patient with anaemia, severe tubulopathy, and diabetes mellitus. As the clinical characteristics resembled Pearson marrow-pancreas syndrome, despite the absence of malfunctioning of the exocrine pancreas in this patient, we have performed DNA analysis to seek for deletions in mtDNA. DNA analysis showed a novel heteroplasmic deletion in mtDNA of 8034bp in length, with high proportions of deleted mtDNA in leukocytes, liver, kidney, and muscle. No deletion could be detected in mtDNA of leukocytes from her mother and young brother, indicating the sporadic occurrence of this deletion. During culture, skin fibroblasts exhibited a rapid decrease of heteroplasmy indicating a selection against the deletion in proliferating cells. We estimate that per cell division heteroplasmy levels decrease by 0.8%. By techniques of fluorescent in situ hybridisation (FISH) and mitochondria-mediated transformation of rho(o) cells we could show inter- as well as intracellular variation in the distribution of deleted mtDNA in a cell population of cultured skin fibroblasts. Furthermore, we studied the mitochondrial translation capacity in cybrid cells containing various proportions of deleted mtDNA. This result revealed a sharp threshold, around 80%, in the proportion of deleted mtDNA, above which there was strong depression of overall mitochondrial translation, and below which there was complementation of the deleted mtDNA by the wild-type DNA. Moreover, catastrophic loss of mtDNA occurred in cybrid cells containing 80% deleted mtDNA.

First-trimester diagnosis of Morquio disease type A.

Since the introduction in 1990 of a novel fluorogenic substrate for galactose-6-sulphate sulphatase we have used this substrate for prospective prenatal diagnosis in 10 pregnancies at risk for Morquio disease type A. Chorionic villi were analysed in five cases. The results indicated an affected fetus in one pregnancy which represents the first case of first-trimester diagnosis of this disorder; heterozygosity was demonstrated in two cases. Following amniocentesis, two affected fetuses and one heterozygote were diagnosed. The results of the present prospective prenatal analyses confirm our previous retrospective studies and demonstrate the reliability and convenience of the 4-methylumbelliferyl substrate.

Type I sialidosis: a clinical, biochemical and neuroradiological study.

We report biochemical, morphological and neuroradiological findings in a 40-year-old woman affected with type I sialidosis. The clinical symptoms, consisting of a cerebellar syndrome, were first noted at the age of 17 years. The macular cherry-red spot was first observed after 23 years of disease. A CT scan performed at 21 years of age showed enlargement of the fourth ventricle. Nuclear magnetic resonance imaging of the brain performed at the age of 40 showed severe atrophy of the cerebellum and pontine region; atrophy of cerebral hemispheres and of the corpus callosum was also observed. We emphasize the prolonged course of illness in this patient, observed over a long period of time. Of particular interest is the neuroradiological study showing our findings both at the beginning of the disease and after 20 years.

Juvenile hyaline fibromatosis: clinical heterogeneity in three patients.

BACKGROUND: Systemic hyalinoses are genetic generalized fibromatoses characterized by an accumulation of hyalin in the dermis. Two distinctive syndromes are recognized in the literature: infantile systemic hyalinosis (ISH) and juvenile hyaline fibromatosis (JHF). ISH and JHF are sometimes difficult to separate since they show significant overlap. OBSERVATIONS: We report on 3 children from two unrelated families suffering from JHF. The first child is severely handicapped by joint contracture, massive hyperplasia of the gingivae, diffuse skin papules and subcutaneous nodules occupying the scalp, face, perianal area, palms, soles and chest. At the same age, the second child only shows pearly skin papules on the face, groin and perianal area and gingival hyperplasia without joint stiffness or any other subjective complaint. The third patient, a brother of the second child, developed mild skin abnormalities by the end of the first year. The occurrence in siblings and consanguinity in the second family suggests autosomal recessive inheritance. Histological skin examination in the 3 cases showed hyaline deposition in the dermis and abnormal ultrastructure of fibroblasts. Biochemical findings showed mucopolysaccharide abnormalities in both families. CONCLUSION: Our patients do not only illustrate the different expressions of JHF but also show some overlap with ISH, suggesting a common cause for both disorders. Genetic studies will finally answer this question.

Clinical, biochemical and molecular findings in a two-generation Morquio A family.

Mucopolysaccharidosis type IVA (Morquio A) is caused by a deficiency of N-acetylgalactosamine-6-sulfatase (GALNS), an enzyme capable of cleaving the sulfate group from both N-acetylgalactosamine-6-sulfate and galactose-6-sulfate. We describe here a two-generation Morquio A family with two distinct clinical phenotypes. The two probands from the second generation showed intermediate signs of the disease whereas their affected mother, aunt and two uncles had only very mild symptoms. Galactose-6-sulfatase (GALS) activity in leukocytes and fibroblasts of the affected family members was clearly deficient. Molecular genetic analysis of the GALNS gene revealed that two different point mutations segregate in the family, which correlated well with the clinical phenotype. The probands with intermediate symptoms were compound heterozygotes for the mutations R259Q and R94G, the latter one being inherited from the unaffected father. The mother and her affected siblings with the unusually mild phenotype were proven to be homozygous for the novel missense point mutation R259Q.

L-2-hydroxyglutaric aciduria: clinical heterogeneity versus biochemical homogeneity in a sibship.

Three out of four sibs in a North-African family were affected with L-2-hydroxyglutaric aciduria. The youngest sib was most severely handicapped: she was diagnosed at 2.5 years of age, whereas the then 7- and 10-year-old siblings had a less pronounced psychomotor retardation. All patients had an increased head circumference in contrast to the healthy, non-affected sibling. Urine and plasma levels of L-2-hydroxyglutaric acid in the three sibs were similar and showed only a small variation. Magnetic resonance imaging (MRI) of the brain in the eldest sib showed hyperintense signal on T2-weighted images of the basal ganglia, dentate nucleus and subcortical white matter. The youngest sib showed identical white matter abnormalities of the corpus medullare cerebelli. These abnormalities were consistent with demyelination and/or spongiosis. On two occasions cerebrospinal fluid amino acid chromatography in the youngest sib showed an increased concentration of lysine and a decreased level of glutamine. Plasma lysine was normal. It is concluded that L-2-hydroxyglutaric aciduria is almost invariably associated with neurological disease; the severity of the symptoms does not seem to be completely dependent on the extent of the biochemical abnormalities and may even be variable within a family.