RESUMO
BACKGROUND: Analysis of sialic acid (SA) metabolites in cerebrospinal fluid (CSF) is important for clinical diagnosis. In the present study, a high-performance liquid chromatography-tandem mass spectrometry (HPLC/MS/MS) method for free sialic acid (FSA) and total sialic acid (TSA) in human CSF was validated. METHODS: The method utilized a simple sample-preparation procedure of protein precipitation for FSA and acid hydrolysis for TSA. Negative electrospray ionisation was used to monitor the transitions m/z 308.2-->87.0 (SA) and m/z 311.2--> 90.0 ((13)C(3)-SA). Conjugated sialic acid (CSA) was calculated by subtracting FSA from TSA. We established reference intervals for FSA, TSA and CSA in CSF in 217 control subjects. The method has been applied to patients' samples with known differences in SA metabolites like meningitis (n=6), brain tumour (n=2), leukaemia (n=5), and Salla disease (n=1). RESULTS: Limit of detection (LOD) was 0.54 microM for FSA and 0.45 mM for TSA. Intra- and inter-assay variation for FSA (21.8 microM) were 4.8% (n=10) and 10.4% (n=40) respectively. Intra- and inter-assay variation for TSA (35.6 microM) were 9.7% (n=10) and 12.8% (n=40) respectively. Tested patients showed values of TSA above established reference value. CONCLUSION: The validated method allows sensitive and specific measurement of SA metabolites in CSF and can be applied for clinical diagnoses.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Ácido N-Acetilneuramínico/líquido cefalorraquidiano , Espectrometria de Massas em Tandem/métodos , Neoplasias Encefálicas/líquido cefalorraquidiano , Humanos , Leucemia/líquido cefalorraquidiano , Meningite/líquido cefalorraquidiano , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Doença do Armazenamento de Ácido Siálico/líquido cefalorraquidianoRESUMO
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.
Assuntos
Doenças Desmielinizantes/líquido cefalorraquidiano , Dipeptídeos/líquido cefalorraquidiano , Transportadores de Ânions Orgânicos/genética , Doença do Armazenamento de Ácido Siálico/líquido cefalorraquidiano , Doença do Armazenamento de Ácido Siálico/diagnóstico , Simportadores/genética , Criança , Pré-Escolar , Estudos Transversais , Doenças Desmielinizantes/etiologia , Doenças Desmielinizantes/urina , Feminino , Testes Genéticos , Genótipo , Humanos , Lactente , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Masculino , Mutação , Ácido N-Acetilneuramínico/líquido cefalorraquidiano , Ácido N-Acetilneuramínico/urina , Doença do Armazenamento de Ácido Siálico/complicações , Doença do Armazenamento de Ácido Siálico/genética , Doença do Armazenamento de Ácido Siálico/urina , Adulto JovemRESUMO
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.
