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...

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Bibliographic Details
Published in:	Clinica chimica acta Vol. 314; no. 1; pp. 175 - 185
Main Authors:	Broda, Elke, Baumgartner, E.Regula, Scholl, Sabine, Stopsack, Marina, Horn, Anton, Rhode, Heidrun
Format:	Journal Article
Language:	English
Published:	Shannon Elsevier B.V 01-12-2001 Elsevier
Subjects:	Adolescent Adult Aged Amidohydrolases - blood Biological and medical sciences Biotin Biotinidase Biotinidase deficiency Costs and Cost Analysis Desiccation Female Fluorescent Dyes Fluorimetry Humans Indicators and Reagents Investigative techniques, diagnostic techniques (general aspects) Kinetics Male Medical sciences Metabolic diseases Middle Aged Nanotechnology Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques Reference Standards Reproducibility of Results Screening Solvents Spectrometry, Fluorescence Biotinidase deficiency AQ Screening MOPS BAQ HEPES MES Fluorimetry Bis/Tris Tris FU Human EC 3.5.1.12 Biochemical analysis Biotinidase Enzyme Deficiency Metabolic diseases Medical screening Enzymopathy Genetic disease Newborn Fluorescent labelling Clinical biology Microequipment Hydrolases Miniaturization Technique Aminoacid disorder Quantitative analysis
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Summary:	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.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0009-8981 1873-3492
DOI:	10.1016/S0009-8981(01)00690-8