N-acetylated metabolites in urine: proton nuclear magnetic resonance spectroscopic study on patients with inborn errors of metabolism.

BACKGROUND: There is no comprehensive analytical technique to analyze N-acetylated metabolites in urine. Many of these compounds are involved in inborn errors of metabolism. In the present study, we examined the potential of proton nuclear magnetic resonance ((1)H-NMR) spectroscopy as a tool to identify and quantify N-acetylated metabolites in urine of patients with various inborn errors of metabolism. METHODS: We performed (1)H-NMR spectroscopy on a 500 MHz spectrometer. Using a combination of one- and two-dimensional correlation spectroscopy (COSY) (1)H-NMR spectra, we were able to assign and quantify resonances of characteristic N-acetylated compounds products in urine of patients with 13 inborn errors of metabolism. RESULTS: The disease-specific N-acetylated metabolites were excreted at concentrations >100 micromol/mmol of creatinine in the patients' urine. In control urine samples, the concentration of individual N-acetyl-containing compounds was <40 micromol/mmol of creatinine. The combination of one- and two-dimensional COSY NMR spectroscopy led to the correct diagnosis of nine different inborn errors of metabolism. No abnormalities were observed in the spectra of urine from patients with G(M1)- or G(M2)-gangliosidosis. We also determined the (1)H-NMR characteristics of N-acetylated metabolites that may be relevant to human metabolism. CONCLUSION: (1)H-NMR spectroscopy may be used to identify and quantify N-acetylated metabolites of diagnostic importance for the field of inborn errors of metabolism.

Late-Onset visceral presentation with cardiomyopathy and without neurological symptoms of adult Sanfilippo A syndrome.

Sanfilippo A syndrome, mucopolysaccharidosis type IIIA, is caused by a deficiency of heparan sulphamidase activity, and usually presents in childhood with neurodegeneration leading to death in teenage years. Visceral symptoms are limited to coarsening and diarrhea. We now describe an adult patient who presented with cardiomyopathy. At age 45 years she had hypertension, and the next year she developed a progressively worsening cardiomyopathy with prominent apical hypertrophy and atrial fibrillation. At age 53, she had severe concentric hypertrophic nonobstructive cardiomyopathy in both ventricles. There was no coarsening of features. Neurologic function, skeleton, cornea, liver, and spleen were normal. Percutaneous endomyocardial biopsy showed ballooned cardiomyocytes with storage vacuoles, containing acid mucopolysaccharides. Leucocytes, uterus, and brain biopsy did not show this storage material. There was a slight increase in total urine mucopolysaccharides, with an increased proportion of heparan sulfates. Heparan sulphamidase activity was deficient in leukocytes and heparan sulphamidase protein and activity were reduced in cultured fibroblasts. No mutations were identified after sequencing of the heparan sulphamidase gene at the cDNA and the genomic level. This new clinical presentation expands the clinical spectrum of Sanfilippo A syndrome to include a primary visceral presentation of cardiomyopathy without neurologic symptoms in the adult. The late onset may be related to the residual heparan sulphamidase activity. The genetic basis of this new variant is still unclear. Physicians evaluating adults must remain aware of possible new adult presentations of storage conditions.

Moderate citrullinaemia without hyperammonaemia in a child with mutated and deficient argininosuccinate synthetase.

In a patient with microcephaly, feeding problems and restlessness, moderately increased serum and urine citrulline concentrations were observed. Protein and allopurinol loading did not result in additional indications for a urea cycle defect. The diagnosis of citrullinaemia was made at both the enzyme and DNA level, resulting from a novel mutation in the argininosuccinate synthetase gene. The fact that the patient has not suffered from severe deterioration, and that there were only minor abnormalities in metabolite concentrations, suggests that the argininosuccinate synthetase capacity was less affected in vivo than in vitro. In vitro nuclear magnetic resonance investigation suggested an active acetylation mechanism for citrulline. This case illustrates the importance of performing extensive biochemical and molecular investigations in order to reach a definitive diagnosis, particularly in instances of moderate citrullinaemia.

Muscle uridine diphosphate-hexosamines do not decrease despite correction of hyperglycemia-induced insulin resistance in type 2 diabetes.

Animal studies suggest that overactivity of the hexosamine pathway, resulting in increased UDP-hexosamines [UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylgalactosamine (UDP-GalNAc)] is an important mechanism by which hyperglycemia causes insulin resistance. This study was performed to test this hypothesis in patients with type 2 diabetes mellitus (DM). Eight obese patients with uncontrolled DM type 2 and severe insulin resistance were treated with iv insulin for 28 +/- 6 d aimed at euglycemia. Before and after iv insulin treatment, insulin sensitivity was measured using a hyperinsulinemic euglycemic clamp, and a muscle biopsy was taken for measurement of UDP-GlcNAc, UDP-GalNAc, UDP-glucose, and UDP-galactose levels. Also, isoelectric focusing patterns of serum transferrin and the urinary excretion of glycosaminoglycans as measures of final products of the hexosamine pathway were examined. After euglycemia, insulin resistance improved, as demonstrated by an increase in the glucose infusion rate during the clamp from 12.7 +/- 5.6 to 22.4 +/- 8.8 micro mol/kg.min (P < 0.0005) and a decrease in insulin requirement from 1.7 +/- 0.9 to 1.1 +/- 0.6 U/kg.d (P < 0.005), whereas metabolic control improved. Surprisingly, both UDP-GlcNAc, from 8.81 +/- 1.21 to 12.31 +/- 2.52 nmol/g tissue (P < 0.005), and UDP-GalNAc concentrations, from 4.49 +/- 0.85 to 5.89 +/- 1.55 nmol/g tissue (P < 0.05) increased. Isoelectric focusing patterns of serum transferrin and excretion of glycosaminoglycans were similar before and after euglycemia. In conclusion, after amelioration of hyperglycemia- induced insulin resistance, UDP-hexosamines increased in skeletal muscle of patients with type 2 DM. These results do not support the hypothesis that accumulation of products of the hexosamine pathway plays a major role in hyperglycemia-induced insulin resistance.

Defects in degradation of blood group A and B glycosphingolipids in Schindler and Fabry diseases.

Skin fibroblast cultures from patients with inherited lysosomal enzymopathies, alpha-N-acetylgalactosaminidase (alpha-NAGA) and alpha-galactosidase A deficiencies (Schindler and Fabry disease, respectively), and from normal controls were used to study in situ degradation of blood group A and B glycosphingolipids. Glycosphingolipids A-6-2 (GalNAc (alpha 1-->3)[Fuc alpha 1-->2]Gal(beta1-->4)GlcNAc(beta 1-->3)Gal(beta 1--> 4)Glc (beta 1-->1')Cer, IV(2)-alpha-fucosyl-IV(3)-alpha-N-acetylgalactosaminylneolactotetraosylceramide), B-6-2 (Gal(alpha 1-->3)[Fuc alpha 1--> 2] Gal (beta 1-->4)GlcNAc(beta 1-->3)Gal(beta 1-->4)Glc(beta 1-->1')Cer, IV(2)- alpha-fucosyl-IV(3)-alpha-galactosylneolactotetraosylceramide), and globoside (GalNAc(beta 1-->3)Gal(alpha 1-->4)Gal(beta 1-->4)Glc(beta 1-->1') Cer, globotetraosylceramide) were tritium labeled in their ceramide moiety and used as natural substrates. The degradation rate of glycolipid A-6-2 was very low in fibroblasts of all the alpha-NAGA-deficient patients (less than 7% of controls), despite very heterogeneous clinical pictures, ruling out different residual enzyme activities as an explanation for the clinical heterogeneity. Strongly elevated urinary excretion of blood group A glycolipids was detected in one patient with blood group A, secretor status (five times higher than upper limit of controls), in support of the notion that blood group A-active glycolipids may contribute as storage compounds in blood group A patients. When glycolipid B-6-2 was fed to alpha-galactosidase A-deficient cells, the degradation rate was surprisingly high (50% of controls), while that of globotriaosylceramide was reduced to less than 15% of control average, presumably reflecting differences in the lysosomal enzymology of polar glycolipids versus less-polar ones. Relatively high-degree degradation of substrates with alpha-D-Galactosyl moieties hints at a possible contribution of other enzymes.