Investigation of a wide spectrum of inherited metabolic disorders by 13C NMR spectroscopy.

High-resolution (1)H NMR spectroscopy of body fluids has proved to be very useful in diagnostics of inherited metabolic diseases, whereas (13)C NMR remains almost unexploited. In this paper the application of (13)C NMR spectroscopy of fivefold concentrated urine samples for diagnosis of selected metabolic diseases is reported. Various marker metabolites were identified in test urine samples from 33 patients suffering from 10 different diseases, providing information which could be crucial for their diagnoses. Spectra were accumulated for 2 h or overnight when using spectrometers operating at 9.4 or 4.7 T magnetic fields, respectively. Interpretation of the measurement results was based on a comparison of the peak positions in the measured spectrum with reference data. The paper contains a table with (13)C NMR chemical shifts of 73 standard compounds. The method can be applied individually or as an auxiliary technique to (1)H NMR or any other analytical method.

Absolute configuration of N-acetylaspartate in urine from patients with Canavan disease.

High-resolution 1H and 13C NMR of N-acetylaspartic acid as its 2-(S)-butyl diester allows the two enantiomers to be unambiguously identified. Analysis of urine samples from five patients with Canavan disease (N-aspartoacylase deficiency) showed that more than 95% of the excreted N-acetylaspartate had the S-configuration.

Rapid and sensitive screening for and chemical diagnosis of Canavan disease by gas chromatography-mass spectrometry.

The use of a rapid and sensitive assay for N-acetylaspartate (NAA) in urine or eluates from dried urine on filter paper to make a chemical diagnosis of Canavan disease (CD) is described. It involves a simplified urease pretreatment for sample preparation and gas chromatography-mass spectrometry (EI, scanning mode) with or without stable isotope dilution. Significant improvements in the recovery of NAA and the GC-MS data-handling device made the assay without stable isotope dilution sensitive and quantitative enough to diagnose CD: Its coefficient of variation (CV) was below 12%. The CV obtained with stable isotope dilution was below 9%. One patient with CD had an abnormal NAA level that was more than 6 S.D. above the mean of the age-matched controls. This diagnostic procedure is accurate for screening and for the chemical diagnosis of CD, with a good cost:benefit ratio. The urinary NAA levels of the healthy controls decreased significantly with age. This change should be considered in making a chemical diagnosis of this disease.

Mild elevation of N-acetylaspartic acid and macrocephaly: diagnostic problem.

Patients with slightly increased excretion of N-acetylaspartic acid in urine, together with macrocephaly, present a dignostic dilemma for Canavan's disease. We describe a 13-year-old male patient with macrocephaly, mild developmental delay, increased signal intensity in the basal ganglia bilaterally, partial cortical blindness, and retinitis pigmentosa. Although the clinical course and magnetic resonance imaging findings did not resemble typical Canavan's disease, N-acetylaspartic acid excretion in the patient's urine was slightly elevated, 99.90 +/- 4.00 microg/mg creatinine, whereas the normal control range was < 83 microg/mg creatinine. Cultured skin fibroblasts from the patient showed no aspartoacylase activity. Cloning of genomic DNA isolated from the patient's fibroblasts showed an intronic mutation, specifically deletion of -2A and -3C at the acceptor site of exon 3 and disrupting the normal splicing of the gene. A second mutation was found in exon 6, 863 A-->G in aspartoacylase complementary DNA, causing a tyrosine-to-cysteine (Y288C) amino acid substitution. Expression of the mutation on exon 6 showed normal aspartoacylase activity. These data suggest that expression of the mutation may help to understand the enzyme defect in a patient with slightly increased N-acetylaspartic acid excretion.

Characterisation of the 1H and 13C NMR spectra of N-acetylaspartylglutamate and its detection in urine from patients with Canavan disease.

1H and 13C NMR spectra of N-acetylaspartylglutamate (NAAG) have been recorded and interpreted. The values of the 1H chemical shifts and 1H-(1)H coupling constants at different pH were obtained by iterative computer fitting of 1-D 1H NMR spectra. This provided information on the solution conformation of the investigated molecule. Proton-decoupled high resolution 13C NMR spectra of NAAG have been measured in a series of dilute water solution of various acidity. These data have provided a basis for unequivocal determination of the presence of NAAG in the urine sample of a patient suffering from Canavan disease. NMR spectroscopy provides a possibility of detecting NAAG in body fluids.

N-acetylaspartylglutamate in Canavan disease: an adverse effector?

UNLABELLED: We measured N-acetylaspartate and its precursor/product N-acetylaspartylglutamate (NAAG) in urine of patients with Canavan disease using capillary zone electrophoresis. Abnormal levels of NAAG were found in 32 of 43 patients examined. Elevated NAAG was also present in the CSF of one patient. Given that NAAG may interfere with N-methyl-D-aspartate receptor function, the occurrence of high levels of NAAG in patients' urine conceivably represents a participating factor in the pathogenesis of Canavan disease. CONCLUSION: The biochemical role of N-acetylaspartylglutamate and its relationship to glutamatergic function may be relevant to the pathogenesis of Canavan disease.

[Magnetic resonance tomography and localized proton spectroscopy in 2 siblings with Canavan's disease]. / Magnetresonanztomographie und lokalisierte Protonenspektroskopie bei zwei Geschwisterkindern mit Morbus Canavan.

We present the findings of magnetic resonance imaging (MRI) and localised 1H magnetic resonance spectroscopy (MRS) in two brothers with Canavan's disease, a rare autosomal recessive leukodystrophy. Urine specimens of one child were evaluated by MRS. All examinations were performed in the same whole body 1.5 T superconducting magnet. MRI revealed the typical pattern of leukodystrophy including a more severe demyelination in the older child. The younger brother showed additional high signal lesions in the globi pallidi on T2-weighted images. MRS of the brain had an elevated ratio of N-acetyl-aspartate (NAA)/phosphocreatin + creatin (Cr) while the ratio of Cholin/Cr was reduced. in urine spectroscopy the concentration of NAA was markedly increased. The ratio of NAA/creatin + creatinin was 880 +/- 10% mmol/mol (normal: 5-21 mmol/mol). Diagnosis of Canavan's disease was supported by gas chromatographic urine examination with an 80-100 fold elevation of NAA concentration. Hence, the diagnosis of Canavan's disease could be established by increased ratio of NAA/Cr and decreased ratio of Cho/Cr relation in brain spectroscopy and high NAA concentration in urine spectroscopy.