Measurement of free and total sialic acid by isotopic dilution liquid chromatography tandem mass spectrometry method.

The measurement of urine sialic acid (N Acetylneuraminic Acid: Neu5Ac) is useful for screening sialic acid storage disorders. We developed a new LC MS/MS method for the determination of a sialic acid. Urine samples were analyzed, after an HCl n-Butanol derivatization step, by a reverse phase based high-performance liquid chromatography method using 1,2,3-(13)C(3) N-Acetyl-D-neuraminic Acid ((13)C-Neu5Ac) as an internal standard. Selective detection was performed by tandem mass spectrometry using an electrospray source operating in positive ionization mode employing multiple reactions monitoring to monitor N-Acetylneuraminic Acid and the internal standard. The transitions m/z 366→330 and 369→333 for Neu5Ac and (13)C-Neu5Ac were respectively monitored. The limit of the method quantification was 1.40 µM of N-Acetylneuraminic Acid and the calibration curve showed a good linearity up to 1000 µM. The within assay precision and accuracy of the method ranged from 3.22 to 5.95% and 98.69 to 109.18%, respectively and the between assay precision and accuracy ranged, respectively, from 5.15 to 7.65% and 96.14 to 102.30%. The method can be applied for the determination of N-Acetylneuraminic Acid concentrations in urine and other biological fluids (e.g., amniotic and peritoneal fluids).

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.

Free sialic acid storage disease without sialuria.

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.

Quantification of free and total sialic acid excretion by LC-MS/MS.

BACKGROUND: The main purpose for measuring urinary free sialic acid (FSA) is to diagnose sialic acid (SA) storage diseases. Elevated amounts of conjugated sialic acid (CSA) are observed in several diseases indicating the need to quantify CSA as well. A LC-MS/MS method for quantification of FSA and total sialic acid (TSA) in urine is developed and validated. METHODS: FSA is analyzed directly after filtration of urine samples. For determination of TSA an enzymatic (neuraminidase) and a chemical (acid) hydrolysis were compared. 13C3-sialic acid was used as internal standard. LC-MS/MS was performed in negative electrospray ionisation mode with multiple reaction monitoring of transitions m/z 308.2-->87.0 (SA) and m/z 311.2-->90.0 (13C3-SA). CSA was calculated by subtracting FSA from TSA. RESULTS: Limit of detection for FSA and TSA was 0.3 and 1.7 micromol/L, respectively. Limit of quantification for FSA and TSA was 1.0 and 5.0 micromol/L. Intra- and inter-assay variations of FSA were 4.6% and 6.6% (n=10) for FSA and 6.5% and 3.6% (n=10) for TSA. Linearity was tested till 7800 micromol/L (r2=0.9998). Values of SA analyzed after neuraminidase- or acid hydrolysis treatment were comparable. Urine samples from patients with inborn errors of SA (related) metabolism were analyzed and compared with age-related reference values. CONCLUSION: A method has been developed for routine determination of urinary FSA and TSA. The method is rapid, specific, robust and sensitive. Age-related reference values for FSA, TSA and CSA were determined and improved diagnostic efficacy.

Clinical, biochemical, and cytochemical studies on a Japanese Salla disease case associated with a renal disorder.

We report the first Japanese case of Salla disease. A 5-year-old male patient developed unique proteinuria with other clinical manifestations, including coarse facies, dysostosis multiplex, mild mitral valve regurgitation, umbilical and inguinal herniation, and mild developmental delay. Pathological analysis of biopsied kidney tissues showed marked vacuolation of podocytes, mesangial cells, capillary endothelial cells, and tubular cells. Biochemical studies involving thin-layer chromatography and mass spectrometry revealed increased excretion of free sialic acid (N-acetylneuraminic acid) into the patient's urine. Immuno- and lectin staining of the patient's cells demonstrated the accumulation of sialyl and asialyl glycoconjugates in lysosomes and late endosomes. A defect in sialyl glycoconjugate metabolism is thought to have occurred in the patient's cells, besides impairment of the lysosomal transport of free sialic acid residues. A renal disorder should be considered as an important manifestation, not only in infantile free sialic acid storage disease but also in Salla disease.

Phenotypic spectrum of Salla disease, a free sialic acid storage disorder.

Salla disease (MIM 269920) represents the mildest phenotype among recessively inherited lysosomal-free sialic acid storage disorders. Although the vast majority of Salla disease patients in Finland share the same founder mutation, R39C in the SLC17A5 gene, there still is a wide clinical variation among mentally retarded, ataxic patients. We evaluated neurologic and neurocognitive findings of Salla disease in a cross-sectional study of 41 Finnish patients who were 11 months to 63 years of age (median = 19.5 years). The phenotype of Salla disease could be classified into two main categories. The majority of patients (90%) had so-called conventional phenotype, including a subgroup of seven patients with relatively mild symptoms. All but two patients with conventional phenotype were homozygous for the Finnish founder mutation. Four severely disabled, profoundly mentally retarded patients, 15-28 years of age, clearly could be clinically delineated as a separate group, likely reflecting the underlying compound heterozygous genotype. A typical developmental pattern could be outlined in the conventional type of the disease, emphasizing a strong motor handicap in Salla disease. The cognitive profile consisted of better verbal ability, especially speech comprehension, compared with nonverbal functioning in all patients. Our results indicate a partial genotype-phenotype correlation, although factors other than the molecular background are also involved in the phenotypic manifestation of Salla disease.