Biotinidase determination in serum and dried blood spots--high sensitivity fluorimetric ultramicro-assay.
A miniaturized quantitative biotinidase assay has been developed using biotin 6-amidoquinoline as substrate and the 100-fold enhanced fluorescence of 6-amidoquinoline measured using apolar solvents. Amidoquinoline is measured after deproteinization by ethanol/acetone using individual standardisation and solvent resistant microtiter plates. The assay was optimized for end point determinations of biotinidase activities in serum and for newborn screening using dried blood spots. Serum activities obtained are closely correlated with values obtained using a quantitative validation method (r=0.96). Analytical sensitivity is around 2% of the mean activity (7.01+/-1.92 nmol/min/ml, mean+/-SD) of a healthy control population. With dried blood spots, a close correlation with values obtained using the Wallac-test kit (r=0.92) was found. Biotinidase activities of a healthy population of 651 newborns amount to 0.2429+/-0.07 nmol/min/ml blood. The analytical sensitivity is close to 1% of the mean activity.
Broda
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E
Institute of Biochemistry, Medical Faculty, Friedrich Schiller University, D-07740 Jena, Germany.
Baumgartner
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ER
Scholl
S
S
Stopsack
M
M
Horn
A
A
Rhode
H
H
D000293:Adolescent; D000328:Adult; D000368:Aged; D000581:Amidohydrolases / Q000097:blood*; D001710:Biotin; D043523:Biotinidase; D003365:Costs and Cost Analysis; D003890:Desiccation; D005260:Female; D005456:Fluorescent Dyes; D006801:Humans; D007202:Indicators and Reagents; D007700:Kinetics; D008297:Male; D008875:Middle Aged; D036103:Nanotechnology; D012015:Reference Standards; D015203:Reproducibility of Results; D012997:Solvents; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D012997:Solvents; D001710:Biotin; D000581:Amidohydrolases; D043523:Biotinidase