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Time-resolved fluoroimmunoassay for equol in plasma and urine
Elke Brouwers1, Rafaëlla L'homme, Nawaf Al-Maharik
1Folkhälsan Research Center, Division of Clinical Chemistry, Institute for Preventive Medicine, Nutrition and Cancer, P.O. Box 63, University of Helsinki, FIN-00014 Helsinki, Finland.
Summary
A new time-resolved fluoroimmunoassay (TR-FIA) method accurately measures equol in plasma and urine. This method aids in understanding equol
Area of Science:
- Biochemistry
- Analytical Chemistry
- Pharmacology
Background:
- Equol is an estrogenic isoflavan produced by gut microflora.
- Equol may possess higher biological activity than its precursor, daidzein.
- High urinary equol levels are linked to reduced breast cancer risk.
Purpose of the Study:
- To develop and validate a time-resolved fluoroimmunoassay (TR-FIA) for equol determination in human plasma and urine.
- To assess the accuracy and reliability of the TR-FIA method compared to gas chromatography-mass spectrometry (GC-MS).
Main Methods:
- Synthesis of 4'-O-carboxymethylequol and its coupling to bovine serum albumin (BSA) for antigen production.
- Development of a europium chelate-labeled tracer using the same equol derivative.
- Application of enzymatic hydrolysis (urine) or hydrolysis and ether extraction (plasma) prior to immunoassay.
Main Results:
- The TR-FIA method demonstrated high specificity for equol in plasma.
- Urine equol measurements using TR-FIA showed overestimation (164%) compared to GC-MS, likely due to matrix effects and interference.
- High correlation coefficients were observed between TR-FIA and GC-MS: 0.98 for plasma and 0.91 for urine.
- Intra-assay and inter-assay coefficients of variation (CV%) were within acceptable ranges for both plasma and urine assays.
Conclusions:
- The developed TR-FIA method is a reliable tool for equol quantification in plasma.
- For urine analysis, a correction formula is recommended to account for matrix effects and ensure comparability with GC-MS results.
- The TR-FIA method offers a sensitive and specific approach for equol analysis in biological samples, supporting research on its health implications.