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Contains fulltext : 290862.pdf (Publisher’s version ) (Open Access) Congenital disorders of glycosylation (CDG) are a clinically and biochemically heterogeneous subgroup of inherited metabolic disorders. Most CDG with abnormal N-glycosylation can be detected by transferrin screening, however, MOGS-CDG escapes this routine screening. Combined with the clinical heterogeneity of reported cases, diagnosing MOGS-CDG can be challenging. Here, we clinically characterize ten MOGS-CDG cases including six previously unreported individuals, showing a phenotype characterized by dysmorphic features, global developmental delay, muscular hypotonia, and seizures in all patients and in a minority vision problems and hypogammaglobulinemia. Glycomics confirmed accumulation of a Glc(3) Man(7) GlcNAc(2) glycan in plasma. For quantification of the diagnostic Glcα1-3Glcα1-3Glcα1-2Man tetrasaccharide in urine, we developed and validated a liquid chromatography-mass spectrometry method of 2-aminobenzoic acid (2AA) labeled urinary glycans. As an internal standard, isotopically labeled (13) C(6) -2AA Glc(3) Man was used, while labeling efficiency was controlled by use of (12) C(6) -2AA and (13) C(6) -2AA labeled laminaritetraose. Recovery, linearity, intra- and interassay coefficients of variability of these labeled compounds were determined. Furthermore, Glc(3) Man was specifically identified by retention time matching against authentic MOGS-CDG urine and compared with Pompe urine. Glc(3) Man was increased in all six analyzed cases, ranging from 34.1 to 618.0 μmol/mmol creatinine (reference

Hyperprolinemia type II (HPII) is an inborn error of metabolism due to genetic variants in ALDH4A1, leading to a deficiency in Δ-1-pyrroline-5-carboxylate (P5C) dehydrogenase. This leads to an accumulation of toxic levels of P5C, an intermediate in proline catabolism. The accumulating P5C spontaneously reacts with, and inactivates, pyridoxal 5’-phosphate, a crucial cofactor for many enzymatic processes, which is thought to be the pathophysiological mechanism for HPII. Here, we describe the use of a combination of LC-QTOF untargeted metabolomics, NMR spectroscopy and infrared ion spectroscopy (IRIS) to identify and characterize biomarkers for HPII that result of the spontaneous reaction of P5C with malonic acid and acetoacetic acid. We show that these biomarkers can differentiate between HPI, caused by a deficiency of proline oxidase activity, and HPII. The elucidation of their molecular structures yields insights into the disease pathophysiology of HPII.

Contains fulltext : 237508.pdf (Publisher’s version ) (Open Access) The current diagnostic work-up of inborn errors of metabolism (IEM) is rapidly moving toward integrative analytical approaches. We aimed to develop an innovative, targeted urine metabolomics (TUM) screening procedure to accelerate the diagnosis of patients with IEM. Urinary samples, spiked with three stable isotope-labeled internal standards, were analyzed for 258 diagnostic metabolites with an ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) configuration run in positive and negative ESI modes. The software automatically annotated peaks, corrected for peak overloading, and reported peak quality and shifting. Robustness and reproducibility were satisfactory for most metabolites. Z-scores were calculated against four age-group-matched control cohorts. Disease phenotypes were scored based on database metabolite matching. Graphical reports comprised a needle plot, annotating abnormal metabolites, and a heatmap showing the prioritized disease phenotypes. In the clinical validation, we analyzed samples of 289 patients covering 78 OMIM phenotypes from 12 of the 15 society for the study of inborn errors of metabolism (SSIEM) disease groups. The disease groups include disorders in the metabolism of amino acids, fatty acids, ketones, purines and pyrimidines, carbohydrates, porphyrias, neurotransmitters, vitamins, cofactors, and creatine. The reporting tool easily and correctly diagnosed most samples. Even subtle aberrant metabolite patterns as seen in mild multiple acyl-CoA dehydrogenase deficiency (GAII) and maple syrup urine disease (MSUD) were correctly called without difficulty. Others, like creatine transporter deficiency, are illustrative of IEM that remain difficult to diagnose. We present TUM as a powerful diagnostic screening tool that merges most urinary diagnostic assays expediting the diagnostics for patients suspected of an IEM.

Untargeted liquid chromatography-mass spectrometry (LC-MS)-based metabolomics strategies are being increasingly applied in metabolite screening for a wide variety of medical conditions. The long-standing "grand challenge" in the utilization of this approach is metabolite identification-confidently determining the chemical structures of m/z-detected unknowns. Here, we use a novel workflow based on the detection of molecular features of interest by high-throughput untargeted LC-MS analysis of patient body fluids combined with targeted molecular identification of those features using infrared ion spectroscopy (IRIS), effectively providing diagnostic IR fingerprints for mass-isolated targets. A significant advantage of this approach is that in silico-predicted IR spectra of candidate chemical structures can be used to suggest the molecular structure of unknown features, thus mitigating the need for the synthesis of a broad range of physical reference standards. Pyridoxine-dependent epilepsy (PDE-ALDH7A1) is an inborn error of lysine metabolism, resulting from a mutation in the ALDH7A1 gene that leads to an accumulation of toxic levels of alpha-aminoadipic semialdehyde (alpha-AASA), piperideine-6-carboxylate (P6C), and pipecolic acid in body fluids. While alpha-AASA and P6C are known biomarkers for PDE in urine, their instability makes them poor candidates for diagnostic analysis from blood, which would be required for application in newborn screening protocols. Here, we use combined untargeted metabolomics-IRIS to identify several new biomarkers for PDE-ALDH7A1 that can be used for diagnostic analysis in urine, plasma, and cerebrospinal fluids and that are compatible with analysis in dried blood spots for newborn screening. The identification of these novel metabolites has directly provided novel insights into the pathophysiology of PDE-ALDH7A1.

Liquid chromatography-mass spectrometry (LC-MS) is, due to its high sensitivity and selectivity, currently the method of choice in (bio)analytical studies involving the (comprehensive) profiling of metabolites in body fluids. However, as closely related isomers are often hard to distinguish on the basis of LC-MS(MS) and identification is often dependent on the availability of reference standards, the identification of the chemical structures of detected mass spectral features remains the primary limitation. Infrared ion spectroscopy (IRIS) aids identification of MS-detected ions by providing an infrared (IR) spectrum containing structural information for a detected MS-feature. Moreover, IR spectra can be routinely and reliably predicted for many types of molecular structures using quantum-chemical calculations, potentially avoiding the need for reference standards. In this work, we demonstrate a workflow for reference-free metabolite identification that combines experiments based on high-pressure liquid chromatography (HPLC), MS and IRIS with quantum-chemical calculations that efficiently generate IR spectra and give the potential to enable reference-standard free metabolite identification. Additionally, a scoring procedure is employed which shows the potential for automated structure assignment of unknowns. Via a simple, illustrative example where we identify lysine in the plasma of a hyperlysinemia patient, we show that this approach allows the efficient assignment of a database-derived molecular structure to an unknown.

The identification of molecular biomarkers is critical for diagnosing and treating patients and for establishing a fundamental understanding of the pathophysiology and underlying biochemistry of inborn errors of metabolism. Currently, liquid chromatography/high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy are the principle methods used for biomarker research and for structural elucidation of small molecules in patient body fluids. While both are powerful techniques, several limitations exist that often make the identification of unknown compounds challenging. Here, we describe how infrared ion spectroscopy has the potential to be a valuable orthogonal technique that provides highly-specific molecular structure information while maintaining ultra-high sensitivity. Here, we characterize and distinguish two well-known biomarkers of inborn errors of metabolism, glutaric acid for glutaric aciduria and ethylmalonic acid for short-chain acyl-CoA dehydrogenase deficiency, using infrared ion spectroscopy. In contrast to tandem mass spectra, in which ion fragments can hardly be predicted, we show that the prediction of an IR spectrum allows reference-free identification in the case that standard compounds are either commercially or synthetically unavailable. Finally, we illustrate how functional group information can be obtained from an IR spectrum for an unknown and how this is valuable information to, for example, narrow down a list of candidate structures resulting from a database query. Early diagnosis in inborn errors of metabolism is crucial for enabling treatment and depends on the identification of biomarkers specific for the disorder. Infrared ion spectroscopy has the potential to play a pivotal role in the identification of challenging biomarkers.

Lithium, given to bipolar disorder patients, causes nephrogenic diabetes insipidus (Li-NDI), a urinary-concentrating defect. Li-NDI occurs due to downregulation of principal cell AQP2 expression, which coincides with principal cell proliferation. The metabolic effect of lithium on principal cells, however, is unknown and investigated here. In earlier studies, we showed that the carbonic anhydrase (CA) inhibitor acetazolamide attenuated Li-induced downregulation in mouse-collecting duct (mpkCCD) cells. Of the eight CAs present in mpkCCD cells, siRNA and drug treatments showed that downregulation of CA9 and to some extent CA12 attenuated Li-induced AQP2 downregulation. Moreover, lithium induced cell proliferation and increased the secretion of lactate. Lithium also increased urinary lactate levels in wild-type mice that developed Li-NDI but not in lithium-treated mice lacking ENaC, the principal cell entry site for lithium. Inhibition of aerobic glycolysis with 2-deoxyglucose (2DG) attenuated lithium-induced AQP2 downregulation in mpkCCD cells but did not attenuate Li-NDI in mice. Interestingly, NMR analysis demonstrated that lithium also increased the urinary succinate, fumarate, citrate, and NH4+levels, which were, in contrast to lactate, not decreased by 2DG. Together, our data reveal that lithium induces aerobic glycolysis and glutaminolysis in principal cells and that inhibition of aerobic glycolysis, but not the glutaminolysis, does not attenuate Li-NDI.

Exploratory metabolomics studies of cerebrospinal fluid (CSF), using proton nuclear magnetic resonance (1H NMR) spectroscopy, hold major potential application in neurodiagnostics. Such studies, however, rely upon established databases of known metabolites. Here we address the 'unknowns' in the 1H NMR spectra of CSF from treated pediatric meningitis cases. Through knowledge of the clinical information given by the pediatrician and analytical application of 1H NMR spectroscopy on pure reference compounds of the medication used, we identified four of the previously unknown compounds in the 1H NMR CSF spectra - the drugs pyrazinamide, isoniazid, acyclovir, and sulfamethoxazole. We report on the one- and two-dimensional 1H NMR spectral data and chemical information of these four compounds. By expanding our knowledge of 1H NMR CSF spectra from treated meningitis cases, we are able to bring 1H NMR closer to the forefront of neurodiagnostics.

The -omics technologies are becoming increasingly important in health care and are expected to contribute to personalized health care. In a typical experiment, cases and controls are compared as a two-class classification problem. This approach is often unsuitable, for example, because the classes are not well defined due to associated populations being biologically too heterogeneous. Recently, statistical health monitoring (SHM) was introduced as a complementary approach to allow for predictions at the individual level. This approach could be of use in all sorts of applications such as diagnosis of rare diseases, analysis of individual patterns in disease manifestation, disease monitoring, or personalized therapy.SHM uses the framework of statistical process monitoring (SPM) in a clinical setting. The method essentially combines estimation of Mahalanobis distances (MD) with principal component analysis (PCA) to evaluate the difference in the -omics data of an individual subject to a normal reference range (normal operating conditions). It is well known from SPM, however, that reliable identification of the variables primarily responsible for this difference is hampered by the smearing effect, which is a result of the PCA step. To avoid this problem, we propose to combine estimation of the MD with variable selection via an 11-norm penalty instead of using dimension reduction. This way a sparse MD metric is obtained.The effectiveness of this method is illustrated by several simulation studies and its application to urine H-1-NMR metabolomics data for diagnosis of multiple inborn errors of metabolism.

The goal of metabolomics is to measure as many metabolites as possible in order to capture biomarkers that may indicate disease mechanisms. Variable selection in chemometric methods can be divided into the following two groups: (1) sparse methods that find the minimal set of variables to discriminate between groups and (2) methods that find all variables important for discrimination. Such important variables can be summarized into metabolic pathways using pathway analysis tools like Mummichog. As a test case, we studied the metabolic effects of treatment with nicotinamide riboside, a form of vitamin B3, in a cohort of patients with ataxia&ndash, telangiectasia. Vitamin B3 is an important co-factor for many enzymatic reactions in the human body. Thus, the variable selection method was expected to find vitamin B3 metabolites and also other secondary metabolic changes during treatment. However, sparse methods did not select any vitamin B3 metabolites despite the fact that these metabolites showed a large difference when comparing intensity before and during treatment. Univariate analysis or significance multivariate correlation (sMC) in combination with pathway analysis using Mummichog were able to select vitamin B3 metabolites. Moreover, sMC analysis found additional metabolites. Therefore, in our comparative study, sMC displayed the best performance for selection of relevant variables.

Contains fulltext : 152310.pdf (Publisher’s version ) (Closed access) A unique, unsolved O-mannosyl glycan on alpha-dystroglycan is essential for its interaction with protein ligands in the extracellular matrix. Defective O-mannosylation leads to a group of muscular dystrophies, called dystroglycanopathies. Mutations in isoprenoid synthase domain containing (ISPD) represent the second most common cause of these disorders, however, its molecular function remains uncharacterized. The human ISPD (hISPD) crystal structure showed a canonical N-terminal cytidyltransferase domain linked to a C-terminal domain that is absent in cytidyltransferase homologs. Functional studies demonstrated cytosolic localization of hISPD, and cytidyltransferase activity toward pentose phosphates, including ribulose 5-phosphate, ribose 5-phosphate, and ribitol 5-phosphate. Identity of the CDP sugars was confirmed by liquid chromatography quadrupole time-of-flight mass spectrometry and two-dimensional nuclear magnetic resonance spectroscopy. Our combined results indicate that hISPD is a cytidyltransferase, suggesting the presence of a novel human nucleotide sugar essential for functional alpha-dystroglycan O-mannosylation in muscle and brain. Thereby, ISPD deficiency can be added to the growing list of tertiary dystroglycanopathies.

Contains fulltext : 153789.pdf (Publisher’s version ) (Open Access) Serum free light chain (sFLC) assays are well established in the diagnosis and monitoring of plasma cell disorders. However, current FLC immunoassays are subject to several analytical issues, which results in a lack of harmonized results. To facilitate sFLC standardization, we investigated the strengths and limitations of mass spectrometry as a novel technological platform for sFLC quantification. Stable isotope labeled reference peptides are added to serum samples for quantitation by selected reaction monitoring (SRM). The use of redundant peptide sets allows for quality control measures during data analysis. Measurements on serum provide information on intact immunoglobulins, but depletion of these intact molecules from the sera during sample processing permits the quantitation of sFLC. sFLC concentrations measured with SRM were comparable to those obtained by nephelometry and showed excellent linearity (r(2) > 0.99). In samples with high levels of sFLC, SRM data was more consistent with serum protein electrophoresis than nephelometric data and SRM is unaffected by antigen excess. The lower limits of quantitation were 3.8 and 2.7 mg/L for kappa and lambda sFLC. Errors due to polymorphic sequences were prevented by comparison of redundant peptide pairs. The application of stable isotope labeling combined with SRM can overcome many of the current potential analytical issues of sFLC analysis. We describe which hurdles still need to be taken to make SRM a robust and more accurate method for sFLC measurements.

The diagnosis of respiratory chain deficiencies (RCDs) is complicated and the need for a diagnostic biomarker or biosignature has been widely expressed. In this study, the metabolic profile of a selected group of 29 RCD patients, with a predominantly muscle disease phenotype, and 22 controls were investigated using targeted and untargeted analyses of three sub-sections of the human metabolome, including urinary organic acids and amino acids [measured by gas chromatography–mass spectrometry (GC–MS)], as well as acylcarnitines (measured by electrospray ionization tandem MS). Although MS technologies are highly sensitive and selective, they are restrictive by being applied only to sub-sections of the metabolome; an untargeted nuclear magnetic resonance (NMR) spectroscopy approach was therefore also included. After data reduction and pre-treatment, a biosignature comprising six organic acids (lactic, succinic, 2-hydroxyglutaric, 3-hydroxyisobutyric, 3-hydroxyisovaleric and 3-hydroxy-3-methylglutaric acids), six amino acids (alanine, glycine, glutamic acid, serine, tyrosine and α-aminoadipic acid) and creatine, was constructed from uni- and multivariate statistical analyses and verified by cross-validation. The results presented here provide the first proof-of-concept that the metabolomics approach is capable of defining a biosignature for RCDs. We postulate that the composite of organic acids ≈ amino acids > creatine > betaine > carnitines represents the basic biosignature for RCDs. Validated through a prospective study, this could offer an improved ability to assign individual patients to a group with defined RCD characteristics and improve case selection for biopsy procedures, especially in infants and children.

Mutations in the SLC17A5 gene encoding the lysosomal transporter sialin are associated with the free sialic acid storage diseases (SASD): Salla disease (or the Finnish type of sialuria), the more severe infantile free sialic acid storage disease (ISSD), and intermediate phenotypes with clinical findings of both Salla disease and ISSD.1 SASD are characterized by the abnormal retention of free sialic acid in the lysosome (Online Mendelian Inheritance in Man 604369 and 269920). Patients with SASD usually present with nystagmus, progressive cerebellar ataxia, spasticity, and severe psychomotor delay. These symptoms are associated with diffuse supratentorial hypomyelination, thin corpus callosum, and cortical and cerebellar atrophy. The increase of free sialic acid in urine has been considered the biochemical hallmark of the disease.1 However, we have recently reported that patients with SASD can present without sialuria.2 The mechanisms leading to hypomyelination in SASD remain largely unknown. Here, we report the increase of N- acetylaspartylglutamate (NAAG) in the CSF of patients with SASD, which may provide new insight into the pathophysiology of the disease.

An unassigned and prominent resonance in the region from delta 2.0-2.1 ppm has frequently been found in the in vivo MR spectra of cancer patients. We demonstrated the presence of this resonance with in vivo MRS in the cyst fluid of a patient with an ovarian tumor. (1)H-NMRS on the aspirated cyst fluid of this patient confirmed the observation. A complex of resonances was observed between 2.0 and 2.1 ppm. It was also present in 11 additional ovarian cyst fluid samples randomly chosen from our biobank. The resonance complex was significantly more prominent in samples from mucinous tumors than in samples from other histological subtypes. A macromolecule (>10 kDa) was found responsible for this complex of resonances. A correlation spectroscopy (COSY) experiment revealed cross peaks of two different types of bound sialic acid suggesting that N-glycans from glycoproteins and/or glycolipids cause this resonance complex. In the literature, plasma alpha-1 acid glycoprotein (AGP), known for its high content of N-linked glycans, has been suggested to contribute to the delta 2.0-2.1 spectral region. The AGP cyst fluid concentration did not correlate significantly with the peak height of the delta 2.0-2.1 resonance complex in our study. AGP may be partly responsible for the resonance complex but other N-acetylated glycoproteins and/or glycolipids also contribute. After deproteinization of the cyst fluid, N-acetyl-L-aspartic acid (NAA) was found to contribute significantly to the signal in this spectral region in three of the 12 samples. GC-MS independently confirmed the presence of NAA in high concentration in the three samples, which all derived from benign serous tumors. We conclude that both NAA and N-acetyl groups from glycoproteins and/or glycolipids may contribute to the delta 2.0-2.1 ppm resonance complex in ovarian cyst fluid. This spectral region seems to contain resonances from biomarkers that provide relevant clinical information on the type of ovarian tumor.

In patients with guanidinoacetate methyltransferase (GAMT) deficiency several parameters may point towards the diagnosis of GAMT deficiency. These include the low levels of creatine and creatinine in urine, the high concentration of guanidinoacetic acid (GAA) in urine and the low levels of creatine and creatinine in the cerebrospinal fluid (CSF). In this study, body fluids from 10 GAMT deficient patients were analysed using (1)H NMR spectroscopy. The urine 1D (1)H NMR spectra of all the patients showed a doublet resonance at 3.98 ppm (pH 2.50) derived from GAA present in high concentration. For this compound, a good recovery and good correlation was found between an LC-MS/MS method and (1)H NMR spectroscopy. In CSF NMR spectra of these patients, the singlet resonances of creatine and creatinine (3.05 and 3.13 ppm, respectively) were absent (normally always present in (1)H NMR spectra of CSF). Due to overlap by other resonances, the doublet of GAA could not be observed. Our data demonstrate that (1)H NMR spectroscopy of urine and CSF can be used to diagnose patients with GAMT deficiency. ispartof: NMR in Biomedicine vol:22 issue:5 pages:538-544 ispartof: location:England status: published

Contains fulltext : 80646.pdf (Publisher’s version ) (Closed access) We performed high-resolution in vitro proton nuclear magnetic resonance spectroscopy on cerebrospinal fluid and urine samples of 44 patients with leukodystrophies of unknown cause. Free sialic acid concentration was increased in cerebrospinal fluid of two siblings with mental retardation and mild hypomyelination. By contrast, urinary excretion of free sialic acid in urine was normal on repeated testing by two independent methods. Both patients were homozygous for the K136E mutation in SLC17A5, the gene responsible for the free sialic acid storage diseases. Our findings demonstrate that mutations in the SLC17A5 gene have to be considered in patients with hypomyelination, even in the absence of sialuria.

Contains fulltext : 71077.pdf (Publisher’s version ) (Closed access) The most common mutation in the nephropathic cystinosis (CTNS) gene is a homozygous 57-kb deletion that also includes an adjacent gene carbohydrate kinase-like (CARKL). The latter gene encodes a protein that is predicted to function as a carbohydrate kinase. Cystinosis patients with the common 57-kb deletion had strongly elevated urinary concentrations of sedoheptulose (28-451 mmol/mol creatinine; controls and other cystinosis patients sedoheptulose-7-phosphate + ADP and that deletion of CARKL causes urinary accumulation of sedoheptulose and erythritol.

Contains fulltext : 50017.pdf (Publisher’s version ) (Closed access) N-terminal acetylation of proteins is a widespread and highly conserved process. Aminoacylase 1 (ACY1; EC 3.5.14) is the most abundant of the aminoacylases, a class of enzymes involved in hydrolysis of N-acetylated proteins. Here, we present four children with genetic deficiency of ACY1. They were identified through organic acid analyses using gas chromatography-mass spectrometry, revealing increased urinary excretion of several N-acetylated amino acids, including the derivatives of methionine, glutamic acid, alanine, leucine, glycine, valine, and isoleucine. Nuclear magnetic resonance spectroscopy analysis of urine samples detected a distinct pattern of N-acetylated metabolites, consistent with ACY1 dysfunction. Functional analyses of patients' lymphoblasts demonstrated ACY1 deficiency. Mutation analysis uncovered recessive loss-of-function or missense ACY1 mutations in all four individuals affected. We conclude that ACY1 mutations in these children led to functional ACY1 deficiency and excretion of N-acetylated amino acids. Questions remain, however, as to the clinical significance of ACY1 deficiency. The ACY1-deficient individuals were ascertained through urine metabolic screening because of unspecific psychomotor delay (one subject), psychomotor delay with atrophy of the vermis and syringomyelia (one subject), marked muscular hypotonia (one subject), and follow-up for early treated biotinidase deficiency and normal clinical findings (one subject). Because ACY1 is evolutionarily conserved in fish, frog, mouse, and human and is expressed in the central nervous system (CNS) in human, a role in CNS function or development is conceivable but has yet to be demonstrated. Thus, at this point, we cannot state whether ACY1 deficiency has pathogenic significance with pleiotropic clinical expression or is simply a biochemical variant. Awareness of this new genetic entity may help both in delineating its clinical significance and in avoiding erroneous diagnoses.

Contains fulltext : 50036.pdf (Publisher’s version ) (Closed access) A diagnosis of 3-methylglutaconic aciduria type I (OMIM: 250950) based on elevated urinary excretion of 3-methylglutaconic acid (3MGA), 3-methylglutaric acid (3MG) and 3-hydroxyisovaleric acid (3HIVA) was made in a 61-year-old female patient presenting with leukoencephalopathy slowly progressing over more than 30 years. The diagnosis was confirmed at the enzymatic and molecular level. In vivo brain MR spectroscopic imaging (MRSI) was performed at 3.0 T, and one-dimensional and two-dimensional in vitro NMR spectroscopy of body fluids of the patient was performed at 11.7 T. Additionally, we measured 1D (1)H-NMR spectra of urine of seven patients with a total of four different inborn errors of leucine metabolism. Increased concentrations of 3HIVA, 3MGA (cis and trans) and 3MG were observed in the NMR spectra of the patient's urine. In the cerebrospinal fluid, the 3HIVA concentration was 10 times higher than in the plasma of the patient and only the cis isomer of 3MGA was observed. In vivo brain MRSI showed an abnormal resonance at 1.28 ppm that may be caused by 3HIVA. Comparison of (1)H-NMR spectra of urine samples from all eight patients studied, representing five different inborn errors of leucine metabolism, showed that each disease has typical NMR characteristics. Our leukoencephalopathy patient suffers from a late-onset form of 3-methylglutaconic aciduria type I. In the literature, only very few adult patients with this conditions have been described, and 3HIVA accumulation in white matter in the brain has not been presented before in these patients. Our data demonstrate that (1)H-NMR spectroscopy of urine can easily discriminate between the known inborn errors of leucine metabolism and provide the correct diagnosis.

Contains fulltext : 58433.pdf (Publisher’s version ) (Closed access) 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).

Item does not contain fulltext There is no definitive treatment protocol for alkaptonuria. A patient with alkaptonuria was treated with ascorbic acid (0.5 g/day) from the age of 4 years. He developed episodes of severe recurrent joint pain at 9.5 years of age after which a protein-restricted diet (1.3 g/kg/day) was started. Protein restriction in combination with ascorbic acid therapy (1 g/day in two divided doses) resulted in a significant decrease but not a normalization of the urinary homogentisic acid excretion. Joint pain resolved and the radiological evidence of 'moth-eaten' irregularities on the articular surface in both knees disappeared. He is currently well, growing normally and in nitrogen balance. Our findings document a reversal of bone abnormalities and clinical symptoms in a case of alkaptonuria. The results should be confirmed in a larger study. We suggest that protein restriction should be applied in combination with ascorbic acid in affected patients as soon as joint pain occurs.

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.

Three urine samples from two prolidase-deficient patients were analysed using 1H NMR spectroscopy. One-dimensional 1H NMR spectra showed a characteristic pattern of overlapping resonances of the proline and hydroxyproline protons of the imidodipeptides. The model compounds Ala-Pro, Gly-Pro, Phe-Pro, Leu-Pro, Val-Pro, Gly-Hyp and Pro-Hyp were measured as well. The non-proline resonances of Val-Pro, Ala-Pro and Gly-Pro could be assigned in the urine spectra. These resonances could then be used for quantification of the corresponding imidodipeptids. The presence of Leu-Pro in the patients' urine was demonstrated by the results of COSY experiments. However, this imidodipeptide could not be quantified owing to overlap of the resonaces in the one-dimensional 1H NMR spectrum of the patients' urine. Phe-Pro, Pro-Hyp and Gly-Hyp could not be assigned in the spectrum of the patient's urine. The characteristic resonances in the urine from a prolidase-deficient patient, i.e. Ala-Pro, Val-Pro, Gly-Pro, and resonances of the (hydroxy)proline part of the imidodipeptides can be used to diagnose this disease.

1H NMR spectroscopy of body fluids has been used in the diagnosis of many inborn errors of metabolism (Lehnert and Hunkler 1986; Iles and Chalmers 1988). To our knowledge there is no systematic study available of body fluids from patients with inborn errors in the purine or pyrimidine metabolism. The main advantage of the technique over others that are used diagnostically in the screening for inborn errors of metabolism is the minimal sample pretreatment required for NMR spectroscopy. Fractionation, extraction or derivatization of metabolites is not required. A further advantage is the overall view of proton-containing metabolites. Quantification of metabolites is possible. An obvious disadvantage is the substantial cost of high-field NMR spectrometers that are required for this work. This paper demonstrates that NMR spectroscopy can be used in diagnosing inborn errors in purine and pyrimidine metabolism. Examples are given of NMR spectra of body fluids from patients with dihydropyrimidine dehydrogenase deficiency (McKusick 274270) and with dihydropyrimidinase deficiency (McKusick 222748).

Contains fulltext : 57529.pdf (Publisher’s version ) (Closed access) 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.

Contains fulltext : 88083.pdf (Publisher’s version ) (Closed access) 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. 01 juni 2010

Contains fulltext : 49036.pdf (Publisher’s version ) (Closed access) (1)H-NMR spectroscopy at 500 MHz was used to confirm that a previously unidentified singlet resonance at 3.14 ppm in the spectra of cerebrospinal fluid and plasma samples corresponds to dimethyl sulfone (DMSO(2)). A triple resonance inverse cryogenic NMR probe, with pre-amplifier and the RF-coils cooled to low temperature, was used to obtain an (1)H-(13)C HSQC spectrum of CSF containing 8 microM (753 ng/ml) DMSO(2). The (1)H-(13)C correlation signal for DMSO(2) was assigned by comparison with the spectrum from an authentic reference sample. In plasma and CSF from healthy controls, the concentration of DMSO(2) ranged between 0 and 25 micromol/l. The concentration of DMSO(2) in plasma from three of four patients with severe methionine adenosyltransferase I/III (MAT I/III) deficiency was about twice the maximum observed for controls. Thus, DMSO(2) occurs as a regular metabolite at low micromolar concentrations in cerebrospinal fluid and plasma. It derives from dietary sources, from intestinal bacterial metabolism and from human endogenous methanethiol 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.

Item does not contain fulltext The heterogeneous group of 3-methylglutaconic aciduria (3-MGA-uria) syndromes includes several inborn errors of metabolism biochemically characterized by increased urinary excretion of 3-methylglutaconic acid. Five distinct types have been recognized: 3-methylglutaconic aciduria type I is an inborn error of leucine catabolism; the additional four types all affect mitochondrial function through different pathomechanisms. We provide an overview of the expanding clinical spectrum of the 3-MGA-uria types and provide the newest insights into the underlying pathomechanisms. A diagnostic approach to the patient with 3-MGA-uria is presented, and we search for the connection between urinary 3-MGA excretion and mitochondrial dysfunction. 01 januari 2012

Dimethylglycine dehydrogenase (DMGDH) (E.C. number 1.5.99.2) is a mitochondrial matrix enzyme involved in the metabolism of choline, converting dimethylglycine to sarcosine. Sarcosine is then transformed to glycine by sarcosine dehydrogenase (E.C. number 1.5.99.1). Both enzymes use flavin adenine dinucleotide and folate in their reaction mechanisms. We have identified a 38-year-old man who has a lifelong condition of fishlike body odor and chronic muscle fatigue, accompanied by elevated levels of the muscle form of creatine kinase in serum. Biochemical analysis of the patient's serum and urine, using (1)H-nuclear magnetic resonance NMR spectroscopy, revealed that his levels of dimethylglycine were much higher than control values. The cDNA and the genomic DNA for human DMGDH (hDMGDH) were then cloned, and a homozygous A--G substitution (326 A--G) was identified in both the cDNA and genomic DNA of the patient. This mutation changes a His to an Arg (H109R). Expression analysis of the mutant cDNA indicates that this mutation inactivates the enzyme. We therefore confirm that the patient described here represents the first reported case of a new inborn error of metabolism, DMGDH deficiency.