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Contains fulltext : 50045.pdf (Publisher’s version ) (Closed access) Sialuria is an inborn error of metabolism characterized by coarse face, hepatomegaly and recurrent respiratory tract infections. The genetic defect in this disorder results in a loss of feedback control of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine-kinase by CMP-N-acetylneuraminic acid (CMP-NeuAc) resulting in a substantial overproduction of cytoplasmic free sialic acid. This study addresses fibroblast CMP-NeuAc levels and N- and O-glycan sialylation of serum proteins from Sialuria patients. CMP-NeuAc levels were measured with HPLC in fibroblasts. Isoelectric focusing (IEF) of serum transferrin and of apolipoprotein C-III (apoC-III) was performed on serum of three Sialuria patients. Isoforms of these proteins can be used as specific markers for the biosynthesis of N- and core 1 O-glycans. Furthermore, total N- and O-linked glycans from serum proteins were analyzed by HPLC. HPLC showed a clear overproduction of CMP-NeuAc in fibroblasts of a Sialuria patient. Minor changes were found for serum N-glycans and hypersialylation was found for core 1 O-glycans on serum apoC-III and on total serum O-glycans in Sialuria patients. HPLC showed an increased ratio of disialylated over monosialylated core 1 O-glycans. The hypersialylation of core 1 O-glycans is due to the increase of NeuAcalpha2,6-containing structures (mainly NeuAcalpha2-3Galbeta1-3[NeuAcalpha2-6]GalNAc). This may relate to KM differences between GalNAc-alpha2,6-sialyltransferase and alpha2,3-sialyltransferases. This is the first study demonstrating that the genetic defect in Sialuria results in a CMP-NeuAc overproduction. Subsequently, increased amounts of alpha2,6-linked NeuAc were found on serum core 1 O-glycans from Sialuria patients. N-glycosylation of serum proteins seems largely unaffected. Sialuria is the first metabolic disorder presenting with hypersialylated O-glycans.

Contains fulltext : 70559.pdf (Publisher’s version ) (Closed access) BACKGROUND: Sepiapterin reductase (SR) deficiency is a rare inherited disorder of neurotransmitter metabolism; less than 25 cases have been described in the literature so far. METHODS: We describe the clinical history and extensive cerebrospinal fluid (CSF) and urine examination of two Greek siblings with the diagnosis of SR deficiency. The diagnosis was confirmed by enzyme activity measurement in cultured fibroblasts and by mutation analysis. RESULTS: Both patients suffered from a progressive and complex L-dopa responsive movement disorder. Very low concentrations of the neurotransmitter metabolites homovanillic acid (HVA), 5-hydroxyindolacetic acid (5-HIAA) and 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) were observed in CSF. CSF neopterin and biopterin concentrations were abnormal in one case only, whereas in both cases sepiapterin concentrations were abnormally high and 5-hydroxytryptophan was undetectable. Urine concentrations of HVA, 5-HIAA and vanillyl mandelic acid (VMA) were decreased in both cases. Both patients had no detectable SR enzyme activity in primary dermal fibroblasts, and upon analysis of genomic DNA revealed the same homozygous point mutation introducing a premature stop codon into the reading frame of the SPR gene (mutant allele K251X). CONCLUSIONS: Our cases illustrate that, apart from HVA and 5-HIAA analysis, the specific quantification of sepiapterin in CSF, rather than neopterin and biopterin alone, is crucial to the final diagnosis of SR deficiency. In addition, urinary concentrations of neurotransmitter metabolites may be abnormal in SR deficiency and may provide an initial indication of SR deficiency before CSF analysis is performed. The known, impressive beneficial response of SR deficient patients to treatment with L-dopa, is illustrated again in our cases.

Biliary copper excretion was studied in male, bile-cannulated rats of the inbred strains Fischer, Brown Norway, WAG/Rij and Lewis. After intravenous injection of 10, 30 and 50 micrograms copper per 100 gm body weight, two patterns of copper excretion were observed; their profiles varied with the copper dose and the strain of the rats used. The lowest amounts of copper were excreted by Fischer rats, the highest by WAG/Rij rats; this was related to the effect of the copper dose on both patterns. The subcellular distribution of copper in the liver was studied in Fischer and Brown Norway rats after doses of 50, 100, and 200 micrograms per 100 gm body weight. Brown Norway rats accumulated more copper in the liver, although the copper concentration was the same in both strains 1 hr after injection of all doses. Fischer rats accumulated proportionally more copper in lysosomal and nuclear mitochondrial fractions whereas Brown Norway rats accumulated proportionally more copper in the cytosol. Gel filtration of liver supernatants revealed that the amount of copper accumulating in the protein presumed to be metallothionein was 2 to 3 times higher in Brown Norway rats, whereas in the Fischer rats more copper eluted in the void volume fraction. We conclude that both biliary copper excretion and copper distribution in the liver are under genetic control. Because of its low copper excretion and reduced binding of copper to metallothionein the Fischer rat, compared to other strains, may be a suitable model for studying the involvement of the liver in copper intoxication.