Cerebral dopamine deficiency, plasma monoamine alterations and neurocognitive deficits in adults with phenylketonuria.

BACKGROUND: Phenylketonuria (PKU), a genetic metabolic disorder that is characterized by the inability to convert phenylalanine to tyrosine, leads to severe intellectual disability and other cerebral complications if left untreated. Dietary treatment, initiated soon after birth, prevents most brain-related complications. A leading hypothesis postulates that a shortage of brain monoamines may be associated with neurocognitive deficits that are observable even in early-treated PKU. However, there is a paucity of evidence as yet for this hypothesis. METHODS: We therefore assessed in vivo striatal dopamine D2/3 receptor (D2/3R) availability and plasma monoamine metabolite levels together with measures of impulsivity and executive functioning in 18 adults with PKU and average intellect (31.2 ± 7.4 years, nine females), most of whom were early and continuously treated. Comparison data from 12 healthy controls that did not differ in gender and age were available. RESULTS: Mean D2/3R availability was significantly higher (13%; p = 0.032) in the PKU group (n = 15) than in the controls, which may reflect reduced synaptic brain dopamine levels in PKU. The PKU group had lower plasma levels of homovanillic acid (p < 0.001) and 3-methoxy-4-hydroxy-phenylglycol (p < 0.0001), the predominant metabolites of dopamine and norepinephrine, respectively. Self-reported impulsivity levels were significantly higher in the PKU group compared with healthy controls (p = 0.033). Within the PKU group, D2/3R availability showed a positive correlation with both impulsivity (r = 0.72, p = 0.003) and the error rate during a cognitive flexibility task (r = 0.59, p = 0.020). CONCLUSIONS: These findings provide further support for the hypothesis that executive functioning deficits in treated adult PKU may be associated with cerebral dopamine deficiency.

Perioperative hyperinsulinaemic normoglycaemic clamp causes hypolipidaemia after coronary artery surgery.

BACKGROUND: Glucose-insulin-potassium (GIK) administration is advocated on the premise of preventing hyperglycaemia and hyperlipidaemia during reperfusion after cardiac interventions. Current research has focused on hyperglycaemia, largely ignoring lipids, or other substrates. The present study examines lipids and other substrates during and after on-pump coronary artery bypass grafting and how they are affected by a hyperinsulinaemic normoglycaemic clamp. METHODS: Forty-four patients were randomized to a control group (n=21) or to a GIK group (n=23) receiving a hyperinsulinaemic normoglycaemic clamp during 26 h. Plasma levels of free fatty acid (FFA), total and lipoprotein (VLDL, HDL, and LDL)-triglycerides (TG), ketone bodies, and lactate were determined. RESULTS: In the control group, mean FFA peaked at 0.76 (sem 0.05) mmol litre(-1) at early reperfusion and decreased to 0.3-0.5 mmol litre(-1) during the remaining part of the study. GIK decreased FFA levels to 0.38 (0.05) mmol litre(-1) at early reperfusion, and to low concentrations of 0.10 (0.01) mmol litre(-1) during the hyperinsulinaemic clamp. GIK reduced the area under the curve (AUC) for FFA by 75% and for TG by 53%. The reduction in total TG was reflected by a reduction in the VLDL (-54% AUC) and HDL (-42% AUC) fraction, but not in the LDL fraction. GIK prevented the increase in ketone bodies after reperfusion (-44 to -47% AUC), but was without effect on lactate levels. CONCLUSIONS: Mild hyperlipidaemia was only observed during early reperfusion (before heparin reversal) and the hyperinsulinaemic normoglycaemic clamp actually resulted in hypolipidaemia during the largest part of reperfusion after cardiac surgery.

3-Methoxytyramine: An independent prognostic biomarker that associates with high-risk disease and poor clinical outcome in neuroblastoma patients.

INTRODUCTION: Prognosis of neuroblastoma patients is very diverse, indicating the need for more accurate prognostic parameters. The excretion of catecholamine metabolites by most neuroblastomas is used for diagnostic purposes, but their correlation with prognosis has hardly been investigated. Therefore, we performed an in-depth analysis of a panel of elevated urinary catecholamine metabolites at diagnosis and their correlation with prognosis. PATIENTS AND METHODS: Retrospective study of eight urinary catecholamine metabolites in a test (n = 96) and validation (n = 205) cohort of patients with neuroblastoma (all stages) at diagnosis. RESULTS: Multivariate analyses, including risk factors such as stage and MYCN amplification, revealed that 3-methoxytyramine (3MT) was an independent risk factor for event-free survival (EFS) and overall survival (OS). Furthermore, only 3MT appeared to be an independent risk factor for both EFS and OS in high-risk patients, which was independent of modern high-risk therapy and immunotherapy. Among high-risk patients, those with elevated 3MT and older than 18 months had an extremely poor prognosis compared to patients with non-elevated 3MT and younger than 18 months (5-year EFS of 14.3% ± 4% and 66.7% ± 18%, respectively, p = 0.001; 5-year OS of 21.8% ± 5% and 87.5% ± 12%, respectively, p < 0.001). CONCLUSIONS: Elevated 3MT at diagnosis was associated with high-risk disease and poor prognosis. For high-risk patients, elevated 3MT at diagnosis was the only significant risk factor for EFS and OS. 3MT was also able to identify subgroups of high-risk patients with favourable and extremely poor prognosis.

Specific genetic deficiencies of the A and B isoenzymes of monoamine oxidase are characterized by distinct neurochemical and clinical phenotypes.

Monoamine oxidase (MAO) exists as two isoenzymes and plays a central role in the metabolism of monoamine neurotransmitters. In this study we compared the neurochemical phenotypes of previously described subjects with genetically determined selective lack of MAO-A or a lack of both MAO-A and MAO-B with those of two subjects with a previously described X chromosome microdeletion in whom we now demonstrate selective MAO-B deficiency. Mapping of the distal deletion breakpoint demonstrates its location in intron 5 of the MAO-B gene, with the deletion extending proximally into the Norrie disease gene. In contrast to the borderline mental retardation and abnormal behavioral phenotype in subjects with selective MAO-A deficiency and the severe mental retardation in patients with combined MAO-A/MAO-B deficiency and Norrie disease, the MAO-B-deficient subjects exhibit neither abnormal behavior nor mental retardation. Distinct neurochemical profiles characterize the three groups of MAO-deficient patients. In MAO-A-deficient subjects, there is a marked decrease in deaminated catecholamine metabolites and a concomitant marked elevation of O-methylated amine metabolites. These neurochemical changes are only slightly exaggerated in patients with combined lack of MAO-A and MAO-B. In contrast, the only biochemical abnormalities detected in subjects with the MAO-B gene deletion are a complete absence of platelet MAO-B activity and an increased urinary excretion of phenylethylamine. The differences in neurochemical profiles indicate that, under normal conditions, MAO-A is considerably more important than MAO-B in the metabolism of biogenic amines, a factor likely to contribute to the different clinical phenotypes.

A homozygous nonsense mutation in the methylmalonyl-CoA epimerase gene (MCEE) results in mild methylmalonic aciduria.

Methylmalonic aciduria (MMA-uria) is an autosomal recessive inborn error of amino acid metabolism, involving valine, threonine, isoleucine, and methionine. This organic aciduria may present in the neonatal period with life-threatening metabolic acidosis, hyperammonemia, feeding difficulties, pancytopenia, and coma. Most affected patients have mutations in the methylmalonyl-coenzyme A (methylmalonyl-CoA) mutase gene. Mildly affected patients may present in childhood with failure to thrive and recurrent attacks of metabolic acidosis. Both a higher residual activity of methylmalonyl-CoA mutase as well as the vitamin B12-responsive defects (cblA and cblB) may form the basis of the mild disorder. A few patients with moderate MMA-uria are known in whom no defect could be identified. Here we present a 16-year-old female patient with persisting moderate MMA-uria (approximately 50 mmol/mol creatinine). She was born to consanguineous Caucasian parents. Her fibroblast mutase activity was normal and no effect of vitamin B12 supplementation could be established. Reduced incorporation of 14C-propionate into macromolecules suggested a defect in the propionate-to-succinate pathway. We found a homozygous nonsense mutation (c.139C>T) in the methylmalonyl-CoA epimerase gene (MCEE), resulting in an early terminating signal (p.R47X). Both parents were heterozygous for this mutation; they were found to excrete normal amounts of methylmalonic acid (MMA). This is the first report of methylmalonyl-CoA epimerase deficiency, thereby unequivocally demonstrating the biochemical role of this enzyme in human metabolism.

A pivotal role for beta-aminoisobutyric acid and oxidative stress in dihydropyrimidine dehydrogenase deficiency?

Dihydropyrimidine dehydrogenase (DPD) constitutes the first step of the pyrimidine degradation pathway in which the pyrimidine bases uracil and thymine are catabolised to beta-alanine and beta-aminoisobutyric acid (beta-AIB), respectively. The mean concentration of beta-AIB was approximately 5- to 8-fold lower in urine of patients with a DPD deficiency, when compared to age-matched controls. Comparable levels of 8-hydroxydeoxyguanosine (8-OHdG) were present in urine from controls and DPD patients at the age <2 year. In contrast, slightly elevated levels of 8-OHdG were detected in urine from DPD patients with an age >2 year, suggesting the presence of increased oxidative stress.

Analysis of pyrimidine synthesis de novo intermediates in urine during crisis of a patient with ornithine transcarbamylase deficiency.

Analysis of pyrimidine synthesis de novo intermediates and pyrimidine degradation products in urine samples from a decompensated patient with an ornithine transcarbamylase deficiency showed a strikingly aberrant metabolic profile. Strongly elevated levels of N-carbamyl-aspartate, orotate and uracil were present whereas the concentration of uridine was only marginally increased. The level of pyrimidine excretion appeared to be independent of the ammonia levels in blood, which were only mildly increased.

Genetic analysis of the first 4 patients with beta-ureidopropionase deficiency.

beta-Ureidopropionase is the third enzyme of the pyrimidine degradation pathway and it catalyses the irreversible hydrolysis of N-carbamyl-ss-aminoisobutyric acid or N-carbamyl-ss-alanine to beta-aminoisobutyric acid or ss-alanine, ammonia, and CO2. Analysis of the beta-ureidopropionase gene (UPB1) of the first 4 patients presenting with a complete enzyme deficiency, revealed the presence of 2 splice-site mutations (IVS1-2A>G and IVS8-1G>A) and one missense mutation (A85E). RT-PCR analysis of the complete beta-ureidopropionase cDNA suggested that both splice-site mutations lead to a variety of alternative splice variants, with deletions of a single or several exons. The alanine at position 85 was not conserved in other eukaryotic beta-ureidopropionase protein sequences.

Pyridoxine responsive epilepsy caused by a novel homozygous PNPO mutation.

We report a patient with anti-epileptic treatment refractory neonatal seizures responsive to pyridoxine. Biochemical analysis revealed normal markers for antiquitin deficiency and also mutation analysis of the ALDH7A1 (Antiquitin) gene was negative. Mutation analysis of the PNPO gene revealed a novel, homozygous, presumed pathogenic mutation (c.481C > T; p.(Arg161Cys)). Measurements of B6 vitamers in a CSF sample after pyridoxine administration revealed elevated pyridoxamine as the only metabolic marker for PNPO deficiency. With pyridoxine monotherapy the patient is seizure free and neurodevelopmental outcome at the age of 14 months is normal.

Eniluracil treatment completely inactivates dihydropyrimidine dehydrogenase in colorectal tumors.

PURPOSE: To determine the effect of eniluracil on colorectal tumor dihydropyrimidine dehydrogenase (DPD) activity. PATIENTS AND METHODS: Patients who were to undergo primary colorectal tumor resection received oral eniluracil 10 mg/m(2) twice daily for 3 days before surgery. Mononuclear cells were obtained before the start of eniluracil and on the morning of surgery, to measure DPD activity, protein, and mRNA. Plasma uracil was also measured at these two time points to assess the effect of eniluracil on pyrimidine accumulation. DPD activity, protein, and mRNA were also assessed in colorectal tumors and adjacent normal mucosa of patients who received eniluracil and untreated control patients. RESULTS: DPD activity in tumors from 10 untreated patients ranged from 30 to 92 pmol/min/mg of protein. In contrast, there was no detectable tumor DPD activity in 10 patients who received eniluracil. A similar pattern was observed in mononuclear cells, where median pretherapy activity was 366.5 pmol/min/mg of protein (range, 265 to 494 pmol/min/mg of protein) and was undetectable immediately before surgery. Plasma uracil changed from a median less than 0.2 micromol/L before therapy to 27.76 micromol/L before surgery. No difference in DPD protein or mRNA was observed between pretherapy and presurgery mononuclear cell samples or between treated and untreated tumor samples. CONCLUSION: This study provides definitive evidence that eniluracil completely inactivates DPD activity in human solid tumors. The increased plasma uracil and decreased DPD activity are consistent with systemic inactivation of the enzyme. The mechanism of inactivation is at the catalytic level, because no changes in DPD protein or mRNA were observed. Treatment with eniluracil will eliminate DPD activity as a source of pharmacokinetic fluorouracil variability or resistance in human colorectal cancer.

A Korean Case of ß-Ureidopropionase Deficiency Presenting with Intractable Seizure, Global Developmental Delay, and Microcephaly.

ß-Ureidopropionase deficiency (OMIM #613161) is a rare autosomal recessive inborn error of metabolism due to mutations in the UPB1 gene, which encodes the third enzyme involved in the pyrimidine degradation pathway. A total of 28 cases have been reported, mainly presenting with seizures, microcephaly, and intellectual disabilities. However, 11 of them were asymptomatic cases (Nakajima et al., J Inherit Metab Dis 37(5):801-812, 2014). We report on a 9-year-old female presenting with intractable epilepsy, microcephaly, and global developmental delay. She was homozygous for p.R326Q (c.977G>A) and heterozygous for p.G31S (c.91G>A) in the UPB1 gene, detected by targeted next-generation sequencing test and subsequently confirmed by biochemical analysis of urine, plasma, and cerebrospinal fluid (CSF) using reversed-phase HPLC, combined with electrospray tandem mass spectrometry. We report a first Korean female case with ß-ureidopropionase deficiency.

Human alpha-N-acetylgalactosaminidase (alpha-NAGA) deficiency: no association with neuroaxonal dystrophy?

Two new individuals with alpha-NAGA deficiency are presented. The index patient, 3 years old, has congenital cataract, slight motor retardation and secondary demyelinisation. Screening of his sibs revealed an alpha-NAGA deficiency in his 7-year-old healthy brother who had no clinical or neurological symptoms. Both sibs are homozygous for the E325K mutation, the same genotype that was found in the most severe form of alpha-NAGA deficiency presenting as infantile neuroaxonal dystrophy. Thus, at the age of 7 years the same genotype of alpha-NAGA may present as a 'non-disease' (present healthy case) and can be associated with the vegetative state (the first two patients described with alpha-NAGA deficiency). The clinical heterogeneity among the 11 known individuals with alpha-NAGA deficiency is extreme, with a 'non-disease' (two cases) and infantile neuroaxonal dystrophy (two cases) at the opposite sides of the clinical spectrum. The broad spectrum is completed by a very heterogeneous group of patients with various degrees of epilepsy/behavioural difficulties/psychomotor retardation (four patients) and a mild phenotype in adults without overt neurological manifestations who have angiokeratoma and clear vacuolisation in various cell types (three cases). These observations are difficult to reconcile with a straightforward genotype-phenotype correlation and suggest that factors or genes other than alpha-NAGA contribute to the clinical heterogeneity of the 11 patients with alpha-NAGA deficiency.

Biochemical monitoring of children with neuroblastoma.

In this article, a short review is given of the biochemical aspects of diagnosis, estimation of prognosis and follow-up of neuroblastoma in children. The importance of determination of patterns of DOPA-metabolites, rather than single metabolite assay, is stressed and illustrated by patient cases. Also the relevance of urinary cystathionine and beta-amino-isobutyric acid is indicated.

Diagnosis and treatment of malignant pheochromocytoma with 131I-meta-iodobenzylguanidine: a case report.

The case of a 67-year-old male patient with metastatic malignant pheochromocytoma is described to illustrate the significant improvement of the diagnosis and treatment of pheochromocytoma since the recent introduction of 131I-meta-iodobenzylguanidine (131I-MIBG). Because of the generally observed tumor heterogeneity as to 131I-MIBG uptake and radiosensitivity in advanced disease, treatment results may be improved further, if 131I-MIBG therapy is initiated when tumor load is still small.

Mild learning difficulties and offending behaviour--is there a link with monoamine oxidase A deficiency?

We have attempted to replicate the findings of Brunner et al., who described a large Dutch kindred where several males were of borderline intelligence and showed characteristically aggressive and sometimes dangerous or extremely antisocial behaviour. The genetic defect for this syndrome was assigned to the p11-p21 region of the X chromosome following linkage analysis in a single kindred. Subsequent sequencing of a candidate gene, monoamine oxidase A (MAO-A), at the position of maximum linkage revealed a causative mutation in the coding region of the MAO-A gene in position 936. In addition to identifying both the phenotype and the associated mutation found by Brunner et al., we also wished to test the hypothesis that mutations elsewhere in the MAO-A gene could cause the low intelligence quotient/personality disorder phenotype associated with low urinary catecholamine degradation products. Fifty-four male subjects similar in clinical characteristics to the affected males in the Dutch kindred were identified within secure mental health facilities in England and Wales. All were assessed using the antisocial personality disorder section of the SCID-II interview instrument, and information about their offending behaviour and family history was obtained from the medical notes. A blood and early-morning urine sample was obtained from each patient. Analysis of urinary excretion patterns of biogenic amines and their metabolites, represented as ratios of normetanefrine to vanillylmandelic acid, revealed two possible cases of MAO-A deficiency, which were found to be negative after resampling.

Fatty acids and homocysteine levels in patients with recurrent depression: an explorative pilot study.

Major depressive disorders (MDD) and cardiovascular disease are mutually associated. They share signs and symptoms of the "metabolic syndrome". Two observations that may be causally related with the metabolic syndrome and therefore with both MDD and cardiovascular disease are a decrease in omega-3 polyunsaturated fatty acids (PUFAs) and a rise in plasma homocysteine (tHcy) levels. Both the rise in tHcy and the decrease in omega-3 PUFAs may be associated with enhanced lipid peroxidation. We exploratively studied 44 randomly chosen patients out of a cohort of 134 patients with the recurrent form of MDD (MDD-R). We measured tHcy levels together with saturated FAs, monounsaturated fatty acids (MUFAs) and PUFAs of the omega-3, omega-6 and omega-9 series in plasma and erythrocytes. Levels were compared with laboratory reference values. The main findings were a decrease in the erythrocytes of C22:5omega-3, C22:6omega-3, C24:1omega-9 and C20:3omega-9 and in the plasma a decrease in C24:1omega-9 and C20:3omega-9. The only significant association we found was between the total of omega-6 fatty acids and plasma tHcy. The FA alterations were found in patients although most of them were clinically recovered, suggesting that the alterations may represent a biological" trait" marker for recurrent depression.

Simultaneous determination of catecholamines and metanephrines in urine by HPLC with fluorometric detection.

A method is described for quantitative analysis of urinary free and conjugated catecholamines and metanephrines. The compounds are isolated from the urine by cation exchange on Amberlite CG 50. Separation is performed by ion-pair reversed-phase high-performance liquid chromatography. For detection of the amines their native fluorescence emitted at 313 nm on excitation at 285 nm was monitored. There was good separation of the compounds of interest, while interference by exogenous compounds other than the catecholamines or metanephrines was minimal. The method is rapid and precise, and it has a broad linear working range for all six DOPA metabolites, making it suitable for clinical analysis. Reference values for children of 0-16 years were established. Examples are shown of excretion patterns of DOPA metabolites from patients with different types of neurogenic tumour.

beta-Aminoisobutyric acid as a marker of thymine catabolism in malignancy.

Urine from untreated patients with various tumours and controls has been examined for the excretion of beta-aminoisobutyric acid and uric acid. The patients were classified into four groups: I, beta-aminoisobutyric acid and uric acid both normal; II, beta-aminoisobutyric acid normal, uric acid elevated; III, beta-aminoisobutyric acid elevated, uric acid normal; IV, beta-aminoisobutyric acid and uric acid both elevated. Uric acid was used as an indicator for tissue-breakdown. Pseudouridine being a specific parameter for t-RNA degradation was estimated for comparison. Increased urinary concentrations of beta-aminoisobutyric acid were frequently found in tumour patients, especially in patients with leukaemia and non-Hodgkin lymphoma. Tissue breakdown being the cause of the beta-aminoisobutyric aciduria could only be considered in part of the patients. Moreover, strongly elevated ratios of beta-aminoisobutyric acid to uric acid were found. Urinary patterns of pyrimidines and purines were determined in order to exclude other abnormalities. The results are discussed in relation to thymine metabolism and renal function.

Monoamine oxidase A deficiency: biogenic amine metabolites in random urine samples.

We have recently described an association between abnormal behaviour and monoamine oxidase A (MAO-A) deficiency in several males from a single large Dutch kindred. A characteristically abnormal excretion pattern of biogenic amine metabolites was present in 24-hour urine of affected males. Because of this strikingly abnormal metabolite pattern observed in 24 hour urine samples of MAO-A deficient males we hypothesized that it should be possible to diagnose this condition by examining random urine samples. We therefore studied multiple urine samples obtained over a two-week study period from two males with selective MAO-A deficiency. The results demonstrate that the characteristic abnormalities in the excretion of biogenic amines and their metabolites were faithfully present in every one of 12 independent samples obtained from the MAO-A deficient males over the two-week study period. We conclude that MAO-A deficiency can be reliably diagnosed by measuring the ratio of normetanephrine (NMN) to VMA (or that of NMN to MHPG) in random urine samples.