The clinical relevance of novel biomarkers as outcome parameter in adults with phenylketonuria.

Recent studies in PKU patients identified alternative biomarkers in blood using untargeted metabolomics. To test the added clinical value of these novel biomarkers, targeted metabolomics of 11 PKU biomarkers (phenylalanine, glutamyl-phenylalanine, glutamyl-glutamyl-phenylalanine, N-lactoyl-phenylalanine, N-acetyl-phenylalanine, the dipeptides phenylalanyl-phenylalanine and phenylalanyl-leucine, phenylalanine-hexose conjugate, phenyllactate, phenylpyruvate, and phenylacetate) was performed in stored serum samples of the well-defined PKU patient-COBESO cohort and a healthy control group. Serum samples of 35 PKU adults and 20 healthy age- and sex-matched controls were analyzed using ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometry. Group differences were tested using the Mann-Whitney U test. Multiple linear regression analyses were performed with these biomarkers as predictors of (neuro-)cognitive functions working memory, sustained attention, inhibitory control, and mental health. Compared to healthy controls, phenylalanine, glutamyl-phenylalanine, N-lactoyl-phenylalanine, N-acetyl-phenylalanine, phenylalanine-hexose conjugate, phenyllactate, phenylpyruvate, and phenylacetate were significant elevated in PKU adults (p < 0.001). The remaining three were below limit of detection in PKU and controls. Both phenylalanine and N-lactoyl-phenylalanine were associated with DSM-VI Attention deficit/hyperactivity (R2 = 0.195, p = 0.039 and R2 = 0.335, p = 0.002, respectively) of the ASR questionnaire. In addition, N-lactoyl-phenylalanine showed significant associations with ASR DSM-VI avoidant personality (R2 = 0.265, p = 0.010), internalizing (R2 = 0.192, p = 0.046) and externalizing problems (R2 = 0.217, p = 0.029) of the ASR questionnaire and multiple aspects of the MS2D and FI tests, reflecting working memory with R2 between 0.178 (p = 0.048) and 0.204 (p = 0.033). Even though the strength of the models was not considered strong, N-lactoyl-phenylalanine outperformed phenylalanine in its association with working memory and mental health outcomes.

Functional disruption of pyrimidine nucleoside transporter CNT1 results in a novel inborn error of metabolism with high excretion of uridine and cytidine.

Genetic defects in the pyrimidine nucleoside transporters of the CNT transporter family have not yet been reported. Metabolic investigations in a patient with infantile afebrile tonic-clonic seizures revealed increased urinary uridine and cytidine excretion. Segregation of this metabolic trait in the family showed the same biochemical phenotype in a healthy older brother of the index. Whole exome sequencing revealed biallelic mutations in SLC28A1 encoding the pyrimidine nucleoside transporter CNT1 in the index and his brother. Both parents and unaffected sibs showed the variant in heterozygous state. The transporter is expressed in the kidneys. Compelling evidence is available for the disrupting effect of the mutation on the transport function thus explaining the increased excretion of the pyrimidine nucleosides. The exome analysis also revealed a pathogenic mutation in PRRT2 in the index, explaining the epilepsy phenotype in infancy. At present, both the index (10 years) and his older brother are asymptomatic. Mutations in SLC28A1 cause a novel inborn error of metabolism that can be explained by the disrupted activity of the pyrimidine nucleoside transporter CNT1. This is the first report describing a defect in the family of CNT concentrative pyrimidine nucleoside transporter proteins encoded by the SLC28 gene family. In all likelihood, the epilepsy phenotype in the index is unrelated to the SLC28A1 defect, as this can be fully explained by the pathogenic PRRT2 variant. Clinical data on more patients are required to prove whether pathogenic mutations in SLC28A1 have any clinical consequences or are to be considered a benign metabolic phenotype.

Regularized MANOVA (rMANOVA) in untargeted metabolomics.

Many advanced metabolomics experiments currently lead to data where a large number of response variables were measured while one or several factors were changed. Often the number of response variables vastly exceeds the sample size and well-established techniques such as multivariate analysis of variance (MANOVA) cannot be used to analyze the data. ANOVA simultaneous component analysis (ASCA) is an alternative to MANOVA for analysis of metabolomics data from an experimental design. In this paper, we show that ASCA assumes that none of the metabolites are correlated and that they all have the same variance. Because of these assumptions, ASCA may relate the wrong variables to a factor. This reduces the power of the method and hampers interpretation. We propose an improved model that is essentially a weighted average of the ASCA and MANOVA models. The optimal weight is determined in a data-driven fashion. Compared to ASCA, this method assumes that variables can correlate, leading to a more realistic view of the data. Compared to MANOVA, the model is also applicable when the number of samples is (much) smaller than the number of variables. These advantages are demonstrated by means of simulated and real data examples. The source code of the method is available from the first author upon request, and at the following github repository: https://github.com/JasperE/regularized-MANOVA.

Genotype-specific differences in the tumor metabolite profile of pheochromocytoma and paraganglioma using untargeted and targeted metabolomics.

CONTEXT AND OBJECTIVE: Pheochromocytomas and paragangliomas (PGLs) are neuroendocrine tumors of sympathetic or parasympathetic paraganglia. Nearly 40% of PGLs are caused by germline mutations. The present study investigated the effect of genetic alterations on metabolic networks in PGLs. DESIGN: Homogenates of 32 sporadic PGLs and 48 PGLs from patients with mutations in SDHB, SDHD, SDHAF-2, VHL, RET, and NF-1 were subjected to proton ((1)H) nuclear magnetic resonance (NMR) spectroscopy at 500 MHz for untargeted and HPLC tandem mass spectrometry for targeted metabolite profiling. RESULTS: (1)H NMR spectroscopy identified 28 metabolites in PGLs of which 12 showed genotype-specific differences. Part of these results published earlier reported low complex II activity (P < .0001) and low ATP/ADP/AMP content (P < .001) in SDH-related PGLs compared with sporadics and PGLs of other genotypes. Extending these results, low levels of N-acetylaspartic acid (NAA; P < .05) in SDH tumors and creatine (P < .05) in VHL tumors were observed compared with sporadics and other genotypes. Positive correlation was observed between NAA and ATP/ADP/AMP content (P < .001) and NAA and complex II activity (P < .0001) of PGLs. Targeted purine analysis in PGLs showed low adenine in cluster 1 compared with cluster 2 tumors (SDH P < .0001; VHL P < .05) whereas lower levels (P < .05) of guanosine and hypoxanthine were observed in RET tumors compared with SDH tumors. Principal component analysis (PCA) of metabolites could distinguish PGLs of different genotypes. CONCLUSIONS: The present study gives a comprehensive picture of alterations in energy metabolism in SDH- and VHL-related PGLs and establishes the interrelationship of energy metabolism and amino acid and purine metabolism in PGLs.

3-Methylglutaconic aciduria type I redefined: a syndrome with late-onset leukoencephalopathy.

OBJECTIVE: 3-Methylglutaconic aciduria type I is a rare inborn error of leucine catabolism. It is thought to present in childhood with nonspecific symptoms; it was even speculated to be a nondisease. The natural course of disease is unknown. METHODS: This is a study on 10 patients with 3-methylglutaconic aciduria type I. We present the clinical, neuroradiologic, biochemical, and genetic details on 2 new adult-onset patients and follow-up data on 2 patients from the literature. RESULTS: Two unrelated patients with the characteristic biochemical findings of 3- methylglutaconic aciduria type I presented in adulthood with progressive ataxia. One patient additionally had optic atrophy, the other spasticity and dementia. Three novel mutations were found in conserved regions of the AUH gene. In both patients, MRI revealed extensive white matter disease. Follow-up MRI in a 10-year-old boy, who presented earlier with isolated febrile seizures, showed mild abnormalities in deep white matter. CONCLUSION: We define 3-methylglutaconic aciduria type I as an inborn error of metabolism with slowly progressive leukoencephalopathy clinically presenting in adulthood. In contrast to the nonspecific findings in pediatric cases, the clinical and neuroradiologic pattern in adult patients is highly characteristic. White matter abnormalities may already develop in the first decades of life. The variable features found in affected children may be coincidental. Long-term follow-up in children is essential to learn more about the natural course of this presumably slowly progressive disease. Dietary treatment with leucine restriction may be considered.

In vivo proton MR spectroscopy findings specific for adenylosuccinate lyase deficiency.

Adenylosuccinate lyase (ADSL) deficiency is an inherited metabolic disorder affecting predominantly the central nervous system. The disease is characterized by the accumulation of succinylaminoimidazolecarboxamide riboside and succinyladenosine (S-Ado) in tissue and body fluids. Three children presented with muscular hypotonia, psychomotor delay, behavioral abnormalities, and white matter changes on brain MRI. Two of them were affected by seizures. Screening for inborn errors of metabolism including in vitro high resolution proton MRS revealed an ADSL deficiency that was confirmed genetically in all cases. All patients were studied by in vivo proton MRS. In vitro high resolution proton MRS of patient cerebrospinal fluid showed singlet resonances at 8.27 and 8.29 ppm that correspond to accumulated S-Ado. In vivo proton MRS measurements also revealed a prominent signal at 8.3 ppm in gray and white matter brain regions of all patients. The resonance was undetectable in healthy human brain. In vivo proton MRS provides a conclusive finding in ADSL deficiency and represents a reliable noninvasive diagnostic tool for this neurometabolic disorder.

Elevated CSF N-acetylaspartylglutamate in patients with free sialic acid storage diseases.

OBJECTIVE: To investigate body fluids of patients with undiagnosed leukodystrophies using in vitro (1)H-NMR spectroscopy (H-NMRS). METHODS: We conducted a cross-sectional study using high-resolution in vitro H-NMRS on CSF and urine samples. RESULTS: We found a significant increase of free sialic acid in CSF or urine in 6 of 41 patients presenting with hypomyelination of unknown etiology. Molecular genetic testing revealed pathogenic mutations in the SLC17A5 gene in all 6 patients. H-NMRS revealed an increase of N-acetylaspartylglutamate in the CSF of all patients with SLC17A5 mutation (range 13-114 micromol/L, reference <12 micromol/L). CONCLUSION: In patients with undiagnosed leukodystrophies, increased free sialic acid in CSF or urine is a marker for free sialic acid storage disorder and facilitates the identification of the underlying genetic defect. Because increase of N-acetylaspartylglutamate in CSF has been observed in other hypomyelinating disorders, it can be viewed as a marker of a subgroup of hypomyelinating disorders.

Cerebellar ataxia with elevated cerebrospinal free sialic acid (CAFSA).

In order to identify new metabolic abnormalities in patients with complex neurodegenerative disorders of unknown aetiology, we performed high resolution in vitro proton nuclear magnetic resonance spectroscopy on patient cerebrospinal fluid (CSF) samples. We identified five adult patients, including two sisters, with significantly elevated free sialic acid in the CSF compared to both the cohort of patients with diseases of unknown aetiology (n = 144; P < 0.001) and a control group of patients with well-defined diseases (n = 91; P < 0.001). All five patients displayed cerebellar ataxia, with peripheral neuropathy and cognitive decline or noteworthy behavioural changes. Cerebral MRI showed mild to moderate cerebellar atrophy (5/5) as well as white matter abnormalities in the cerebellum including the peridentate region (4/5), and at the periventricular level (3/5). Two-dimensional gel analyses revealed significant hyposialylation of transferrin in CSF of all patients compared to age-matched controls (P < 0.001)--a finding not present in the CSF of patients with Salla disease, the most common free sialic acid storage disorder. Free sialic acid content was normal in patients' urine and cultured fibroblasts as were plasma glycosylation patterns of transferrin. Analysis of the ganglioside profile in peripheral nerve biopsies of two out of five patients was also normal. Sequencing of four candidate genes in the free sialic acid biosynthetic pathway did not reveal any mutation. We therefore identified a new free sialic acid syndrome in which cerebellar ataxia is the leading symptom. The term CAFSA is suggested (cerebellar ataxia with free sialic acid).

Association of 3-methylglutaconic aciduria with sensori-neural deafness, encephalopathy, and Leigh-like syndrome (MEGDEL association) in four patients with a disorder of the oxidative phosphorylation.

In this paper, we describe a distinct clinical subtype of 3-methylglutaconic aciduria. 3-Methylglutaconic aciduria is a group of different metabolic disorders biochemically characterized by increased urinary excretion of 3-methylglutaconic acid. We performed biochemical and genetic investigations, including urine organic acid analysis, NMR spectroscopy, measurement of 3-methylglutaconyl-CoA hydratase activity, cardiolipin levels, OPA3 gene analysis and measurement of the oxidative phosphorylation in four female patients with 3-methylglutaconic aciduria. 3-Methylglutaconic aciduria type I, Barth syndrome, and Costeff syndrome were excluded as the activity of 3-methylglutaconyl-CoA hydratase, the cardiolipin levels, and molecular analysis of the OPA3 gene, respectively, showed no abnormalities. The children presented with characteristic association of hearing loss and the neuro-radiological evidence of Leigh disease. They also had neonatal hypotonia, recurrent lactic acidemia, episodes with hypoglycemia and severe recurrent infections, feeding difficulties, failure to thrive, developmental delay, and progressive spasticity with extrapyramidal symptoms. Our patients were further biochemically characterized by a mitochondrial dysfunction and persistent urinary excretion of 3-methylglutaconic acid.

Methylsulfonylmethane (MSM) ingestion causes a significant resonance in proton magnetic resonance spectra of brain and cerebrospinal fluid.

The use of methylsulfonylmethane (MSM), available as an "over-the-counter" dietary supplement, led to the occurrence of an abnormal resonance at 3.15 ppm in the in vivo brain proton MR spectrum as well as the in vitro cerebrospinal fluid NMR study of a 4-year-old girl. The concentration of this compound amounted to 1.2 mmol/l in brain tissue and 1.7 mmol/l in cerebrospinal fluid. Our findings illustrate that ingestion of exogenous compounds, e.g., in medication, food or "innocent" supplements, may lead to abnormal resonances in spectroscopy studies that might be difficult to assign.

Aminoacylase I deficiency: a novel inborn error of metabolism.

This is the first report of a patient with aminoacylase I deficiency. High amounts of N-acetylated amino acids were detected by gas chromatography-mass spectrometry in the urine, including the derivatives of serine, glutamic acid, alanine, methionine, glycine, and smaller amounts of threonine, leucine, valine, and isoleucine. NMR spectroscopy confirmed these findings and, in addition, showed the presence of N-acetylglutamine and N-acetylasparagine. In EBV transformed lymphoblasts, aminoacylase I activity was deficient. Loss of activity was due to decreased amounts of aminoacylase I protein. The amount of mRNA for the aminoacylase I was decreased. DNA sequencing of the encoding ACY1 gene showed a homozygous c.1057 C>T transition, predicting a p.Arg353Cys substitution. Both parents were heterozygous for the mutation. The mutation was also detected in 5/161 controls. To exclude the possibility of a genetic polymorphism, protein expression studies were performed showing that the mutant protein had lost catalytic activity.

Follow-up of a child with hypoacetylaspartia.

We provide a 5-year follow-up of a patient previously reported to have no NAA signal on neurospectroscopy. At 8 years this boy was found to have profound neurological dysfunction: he had truncal ataxia, no expressive speech, behaviour abnormalities, secondary microcephaly and cognitive achievements corresponding to less than 12 months of age. He started to have generalized seizures at 5 years 9 months. Although not directly proven we assume an inborn error of NAA metabolism, possibly a defect of the anabolic enzyme L-aspartate N-acetyltransferase (EC 2.3.1.17).

Sedation with 4-hydroxybutyric acid: a potential pitfall in the diagnosis of SSADH deficiency.

Deficiency of succinic semialdehyde dehydrogenase (SSADH) is a rare neurometabolic disorder with accumulation of 4-hydroxybutyric acid (4-HBA) as a biochemical hallmark. We present a boy with an unresolved severe neurological disorder and intermittent elevation of 4-HBA in serum and CSF which was later shown to result from iatrogenic administration of 4-HBA for sedation purposes.

Severe hypomyelination associated with increased levels of N-acetylaspartylglutamate in CSF.

BACKGROUND: Two unrelated girls had early onset of nystagmus and epilepsy, absent psychomotor development, and almost complete absence of myelin on cerebral MRI. The clinical features and MR images of both patients resembled the connatal form of Pelizaeus-Merzbacher disease (PMD), which is an X-linked recessive disorder caused by duplications or mutations of the proteolipid protein gene (PLP). OBJECTIVE: To define a unique neurometabolic disorder with failure of myelination. METHOD: S AND RESULTS: 1H-NMR of CSF in both girls was performed repeatedly, and both showed highly elevated concentrations of N-acetylaspartylglutamate (NAAG). The coding sequence of the gene coding for glutamate carboxypeptidase II, which converts NAAG to N-acetylaspartate (NAA) and glutamate, was entirely sequenced but revealed no mutations. Even though both patients are girls, the authors sequenced the PLP gene and found no abnormality. CONCLUSIONS: NAAG is an abundant peptide neurotransmitter whose exact role is unclear. NAAG is implicated in two cases of unresolved severe CNS disorder. Its elevated concentration in CSF may be the biochemical hallmark for a novel neurometabolic disorder. The cause of its accumulation is still unclear.

Proton nuclear magnetic resonance spectroscopy of body fluids in the field of inborn errors of metabolism.

Proton nuclear magnetic resonance (NMR) spectroscopy of body fluids has been successfully applied to the field of inborn errors of metabolism. This technique has the advantage of minimal sample pretreatment not requiring extraction or derivatization steps. Moreover, the spectrum provides a comprehensive metabolic profile of proton-containing, low-molecular-weight metabolites. The sensitivity limit is in the low micromolar range. This allows diagnosis of many inborn errors of metabolism. This review explains the key features of the NMR spectrum and reviews the available literature on metabolic diseases. Three novel diseases have been delineated with the technique. Relevant parts of the spectra from the urine samples of patients with these diseases are shown. NMR spectroscopy may develop to become a key tool in a metabonomics approach in clinical biochemistry.

beta-Ureidopropionase deficiency: a novel inborn error of metabolism discovered using NMR spectroscopy on urine.

In this work, NMR investigations that led to the discovery of a new inborn error of metabolism, beta-ureidopropionase (UP) deficiency, are reported. 1D (1)H-NMR experiments were performed using a patient's urine. 3-Ureidopropionic acid was observed in elevated concentrations in the urine spectrum. A 1D (1)H-(1)H total correlation spectroscopy (TOCSY) and two heteronuclear 2D NMR techniques (heteronuclear multiple bond correlation (HMBC) and heteronuclear single-quantum correlation (HSQC)) were used to identify the molecular structure of the compound that caused an unknown doublet resonance at 1.13 ppm. Combining the information from the various NMR spectra, this resonance could be assigned to 3-ureidoisobutyric acid. These observations suggested a deficiency of UP. With 1D (1)H-NMR spectroscopy, UP deficiency can be easily diagnosed. The (1)H-NMR spectrum can also be used to diagnose patients suffering from other inborn errors of metabolism in the pyrimidine degradation pathway.

Efficacy of Bach-flower remedies in test anxiety: a double-blind, placebo-controlled, randomized trial with partial crossover.

Bach-flower remedies are a type of alternative medication used increasingly for over-the-counter self-help purposes. We studied the efficacy of a combination of Bach-flower remedies in subjects with test anxiety in a randomized, placebo-controlled, blinded parallel group design, with crossing over the placebo group to remedies after the first phase. Anxiety was measured by a standardized, validated test anxiety questionnaire (the German version of the Test Anxiety Inventory, TAI-G). Fifty-five of 61 subjects with self-reported test anxiety gave valid data. There was no significant difference between the groups, but a significant decrease of test anxiety in all groups was present. We conclude that Bach-flower remedies are an effective placebo for test anxiety and do not have a specific effect.

In vivo and in vitro NMR spectroscopy reveal a putative novel inborn error involving polyol metabolism.

In vivo NMR spectroscopy was performed on the brain of a patient with a leukoencephalopathy, revealing unknown resonances between 3.5 and 4.0 ppm. In addition, urine and CSF of the patient were measured using high-resolution NMR spectroscopy. Also in these in vitro spectra, unknown resonances were observed in the 3.5-4.0 ppm region. Homonuclear (1)H two-dimensional J-resolved spectroscopy (JRES) and (1)H-(1)H correlation spectroscopy (COSY) were performed on the patient's urine for more accurate assignment of resonances. The NMR spectroscopic studies showed that the unknown resonances could be assigned to arabinitol and ribitol. This was confirmed using gas chromatography. The arabinitol was identified as D-arabinitol. The patient is likely to suffer from an as yet unknown inborn error of metabolism affecting D-arabinitol and ribitol metabolism. The primary molecular defect has not been found yet. Urine spectra of patients suffering from diabetes mellitus or galactosemia were recorded for comparison. Resonances outside the 3.2-4.0 ppm region, which are the most easy to recognize in body fluid spectra, allow easy recognition of various sugars and polyols. The paper shows that NMR spectroscopy in body fluids may help identifying unknown resonances observed in in vivo NMR spectra.

Hypoxia in fetal lambs: a study with (1)H-MNR spectroscopy of cerebrospinal fluid.

In fetal lambs, severe hypoxia (SH) will lead to brain damage. Mild hypoxia (MH) is thought to be relatively safe for the fetal brain because compensating mechanisms are activated. We questioned whether MH, leading to mild acidosis, induces changes in cerebral metabolism. Metabolites in cerebrospinal fluid (CSF) samples, as analyzed by proton magnetic resonance spectroscopy, were studied in two groups of seven anesthetized near-term fetal lambs. In group I, SH leading to acidosis with an arterial pH <7.1 was achieved. In group II, MH with an intended pH of 7.23--7.27 was reached [start of MH (SMH)], and maintained during 2 h [end of MH (EMH)]. During SH, choline levels in CSF, a possible indicator of cell membrane damage, were increased. Both during SH and at EMH, CSF levels of lactic acid, alanine, phenylalanine, tyrosine, lysine, branched chain amino acids, and hypoxanthine were increased compared with control values and with SMH, respectively. At EMH, the hypoxanthine CSF-to-blood ratio was increased as compared with SMH. These results indicate that prolonged MH leads to energy degradation in the fetal lamb brain and may not be as safe as assumed.