High-throughput capillary electrophoretic method for determination of total aminothiols in plasma and urine.
Increased interest in the analysis of aminothiols in body fluids during the last years results in a request for high-throughput analytical methods for their determination. We report here a novel, high-throughput method for the determination of total concentrations of biogenous aminothiols - homocysteine, cysteine, glutathione, cysteinylglycine, gamma-glutamylcysteine, and of penicilamine, mercaptopropionylglycine, and cysteamine, three compounds used to treat disorders of aminothiol metabolism in plasma and urine. Samples were reduced with tris(carboxyethyl)phosphine and labeled with 5-(bromomethyl)fluorescein. Capillary electrophoretic separations were performed in 60 mmol/L borate - 15 mmol/L sodium dodecyl sulfate - 2-amino-2-methyl-1-propanol, pH 10.0, with laser-induced fluorescence detection. Analysis time was less than 2 min. The assay is linear (r > 0.999) up to 500 micromol/L. Reproducibilities of migration times (coefficient of variation, CV) were < 0.5%. Interassay repeatabilities (CV, n = 10) were 5.08% and 6.09% for 5 micromol/L addition of homocysteine and 0.60% and 3.78% for 100 micromol/L addition of cysteine in plasma and urine, respectively. Recovery values were within 94-106% and sensitivity was better than 0.19 micromol/L for all analyzed compounds. Results agreed well with a standard high-performance liquid chromatography (HPLC) method. The diagnostic usefulness of the method has been proven on 79 samples of cystinuric patients and 12 samples of homocystinuric patients. We report here a novel method for the determination of aminothiols in body fluids by capillary electrophoresis (CE). Determination is fast and sensitive enough for diagnostic purposes.
Lochman
Pavel
P
Laboratory for Inherited Metabolic Disorders, Department of Clinical Biochemistry, Medical Hospital Olomouc, Czech Republic.
Adam
Tomás
T
Friedecký
David
D
Hlídková
Eva
E
Skopková
Zuzana
Z
D000293:Adolescent; D000328:Adult; D000603:Amino Acids, Sulfur / Q000097:blood / Q000302:isolation & purification* / Q000652:urine; D001826:Body Fluids / Q000737:chemistry; D002648:Child; D002675:Child, Preschool; D019075:Electrophoresis, Capillary / Q000379:methods*; D005260:Female; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008297:Male; D008875:Middle Aged; D015203:Reproducibility of Results; D013997:Time Factors
D000603:Amino Acids, Sulfur