Time-resolved fluoroimmunoassay for equol in plasma and urine.
We present a method for the determination of the isoflavan equol in plasma and urine. This estrogenic isoflavan, which is formed by the action of the intestinal microflora, may have higher biological activity than its precursor daidzein. High urinary excretion of equol has been suggested to be associated with a reduction in breast cancer risk. The method is based on time-resolved fluoroimmunoassay, using a europium chelate as a label. After synthesis of 4'-O-carboxymethylequol the compound is coupled to bovine serum albumin (BSA), then used as antigen to immunize rabbits. The tracer with the europium chelate is synthesized using the same 4'-O-derivative of equol. After enzymatic hydrolysis (urine) or enzymatic hydrolysis and ether extraction (plasma) the immunoassay is carried out. The antiserum cross-reacted to variable extent with some isoflavonoids. For the plasma method the cross-reactivity does not seem to influence the results, which were highly specific. The overestimation of the values using the urine method (164%) compared to the results obtained by a gas chromatography-mass spectrometry (GC-MS) method is probably due to some influence of the matrix on the signal, and interference of structurally related compounds. It is suggested that plasma assays are used but if urine samples are measured a formula has to be used to correct the values making them comparable to the GC-MS results. The correlation coefficients between the time-resolved fluoroimmunoassay (TR-FIA) methods and GC-MS methods were high; r-values for the plasma and urine method, were 0.98 and 0.91, respectively. The intra-assay coefficient of variation (CV%) for the TR-FIA plasma and urine results at three different concentrations vary between 5.5-6.5 and 3.4-6.9, respectively. The inter-assay CV% varies between 5.4-9.7 and 7.4-7.7, respectively. The working ranges of the plasma and urine assay are 1.27-512 and 1.9-512nmol/l, respectively.
Brouwers
Elke
E
Folkhälsan Research Center, Division of Clinical Chemistry, Institute for Preventive Medicine, Nutrition and Cancer, P.O. Box 63, University of Helsinki, FIN-00014 Helsinki, Finland.
L'homme
Rafaëlla
R
Al-Maharik
Nawaf
N
Lapcík
Oldrich
O
Hampl
Richard
R
Wähälä
Kristiina
K
Mikola
Heikki
H
Adlercreutz
Herman
H
D000818:Animals; D000917:Antibody Formation; D002417:Cattle; D002839:Chromans / Q000097:blood* / Q000737:chemistry / Q000276:immunology / Q000652:urine*; D003429:Cross Reactions; D060754:Equol; D005063:Europium / Q000737:chemistry; D015200:Fluoroimmunoassay / Q000379:methods*; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D007106:Immune Sera; D007529:Isoflavones / Q000097:blood / Q000737:chemistry / Q000276:immunology / Q000652:urine; D011817:Rabbits; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine
C007610:4',7-dihydroxy-3,4-dihydroisoflavone; D002839:Chromans; D007106:Immune Sera; D007529:Isoflavones; D012710:Serum Albumin, Bovine; D005063:Europium; D060754:Equol