Development of bioluminescent enzyme immunoassay for S-equol using firefly luciferase and its application to the assessment of equol-producer status

Article abstract of DOI:10.1248/cpb.59.84

In this study, we developed a specific bioluminescent enzyme immunoassay (BLEIA) for S-equol, employing firefly luciferase as a labeling enzyme, as an alternative to HPLC methods. Satisfactory correlation (r=0.992) was shown when this S-equol BLEIA was compared with HPLC. The cross-reactivity with R-equol as its diastereoisomer is <5%, and that with daidzein, which is the substrate of equol, is 0.02%. Frequencies of Japanese equol producers determined using two distinct approaches were compared: a threshold value for urinary S-equol concentration of 232 ng/ml gave frequencies of 32% of men and 19% of women. These values correspond to the results for log₁₀-transformed urinary S-equol to daidzein ratio threshold of -1.75, namely, 34% of men and 19% of women. When the changes in concentration of urinary equol and daidzein were measured after ingestion of isoflavone, the maximum concentration (C _max) of urinary equol appeared after 9.6 h of isoflavone consumption; this C_max was 2 h later than that for daidzein. The S-equol BLEIA documented in this study is expected to be an important tool for the assessment of equol producer status and demonstration of the bioavailability of isoflavone.

Full text of DOI:10.1248/cpb.59.84

84
Regular Article
Chem. Pharm. Bull. 59(1) 84—87 (2011)
Vol. 59, No. 1
Development of Bioluminescent Enzyme Immunoassay for S-Equol Using
Firefly Luciferase and Its Application to the Assessment of Equol-
Producer Status
Takayuki MINEKAWA,*,a,b Akira KAMBEGAWA,c Kumiko SHINDOME,d Hiroshi OHKUMA,d Katsushi ABE,d
b
Hiroaki MAEKAWA,a and Hidetoshi ARAKAWA
a Biochemical Research Laboratory, Eiken Chemical Co., Ltd.; 143 Nogimachi-nogi, Shimotsuga-gun, Tochigi 329–0114,
Japan: b School of Pharmaceutical Science, Showa University; 1–5–8 Hatanodai, Shinagawa-ku, Tokyo 142–8555, Japan:
c Kambegawa Lab.; 3–8–5 Motoizumi, Komae, Tokyo 201–0013, Japan: and d Biochemical Research Laboratory, Eiken
Chemical Co., Ltd.; 1381–3 Shimoishigami, Ohtawara, Tochigi 324–0036, Japan.
Received August 17, 2010; accepted October 6, 2010; published online October 12, 2010
In this study, we developed a specific bioluminescent enzyme immunoassay (BLEIA) for S-equol, employing
firefly luciferase as a labeling enzyme, as an alternative to HPLC methods. Satisfactory correlation (rꢀ0.992)
was shown when this S-equol BLEIA was compared with HPLC. The cross-reactivity with R-equol as its di-
astereoisomer is ꢃ5%, and that with daidzein, which is the substrate of equol, is 0.02%. Frequencies of Japanese
equol producers determined using two distinct approaches were compared: a threshold value for urinary S-equol
concentration of 232 ng/ml gave frequencies of 32% of men and 19% of women. These values correspond to the
results for log10-transformed urinary S-equol to daidzein ratio threshold of ꢁ1.75, namely, 34% of men and 19%
of women. When the changes in concentration of urinary equol and daidzein were measured after ingestion of
isoflavone, the maximum concentration (Cmax) of urinary equol appeared after 9.6 h of isoflavone consumption;
this Cmax was 2 h later than that for daidzein. The S-equol BLEIA documented in this study is expected to be an
important tool for the assessment of equol producer status and demonstration of the bioavailability of isoflavone.
Key words S-equol; bioluminescent enzyme immunoassay; equol producer; firefly luciferase
Equol [7-hydroxy-3-(4ꢀ-hydroxyphenyl)-chroman] is a ternative method, enzyme immunoassay is the most useful.
metabolite produced in vivo from the soy isoflavone daidzein Brouwers et al. reported a time-resolved fluoroimmunoassay
by the action of enterobacteria. It is known to be estrogenic, (TR-FIA) method that uses rabbit polyclonal antibodies.11)
so exposure to equol could have significant biological effects Talbot et al. reported successful development of monoclonal
on humans. Epidemiological studies suggest that it might be antibodies and applied them to a TR-FIA method.12) As a re-
beneficial in the prevention of many diseases, including sult, a large number of samples have become measurable.
breast cancer,1) prostate cancer,2,3) and osteoporosis.4) The
Equol, unlike the soy isoflavones daidzein and genistein,
production of equol in humans varies: only 30—50% of any has a chiral center, and therefore it can occur as two distinct
population group can produce equol after ingestion of soy diastereoisomers, R-equol and S-equol. When equol is chem-
foods, and they are called equol producers.4) It is thought that ically synthesized, it is the (ꢁ) equol that is usually obtained.
certain bacteria in the intestinal microflora are greatly in- S-Equol has a high affinity for estrogen receptor b (Kiꢂ
volved in equol bioconversion. Recently, bacteria that con- 0.73ꢁ0.2 nmol/l), whereas R-equol is relatively inactive
vert daidzein to equol were isolated from human feces (ex. (Kiꢂ15.4ꢁ1.3 nmol/l). The exclusive product of human en-
Lactocossus garvieae).5—7) A clinical trial intended to iden- terobacterial synthesis from soy isoflavones is S-equol.13)
tify soy health benefits is taking place, using S-equol. Ishi- Therefore, a measurement system that can specifically meas-
wata et al. reported that S-equol supplement improved mood- ure S-equol is anticipated in clinical research. However, con-
related symptoms in premenopausal/postmenopausal equol ventional methods have drawbacks with regard to their speci-
non-producers.8) Jackson et al. reported using pure synthetic ficity for S-equol.
S-equol for the clinical indications of vasomotor symptoms
The measurement of urine samples is known to be influ-
(VMS, hot flashes) in women and benign prostatic hyperpla- enced by the matrix effect. The extraction of equol from
sia (BPH, enlarged prostate) in men, as well as osteoporo- urine reduces this influence, but it results in relatively low
sis.9)
throughput. Dilution of sample for measurement reduces the
The major methods used today for the determination of influence of the matrix effect, but it causes a decrease of sen-
equol are high-performance liquid chromatography (HPLC) sitivity. Therefore, in this study, we overcame this decrease of
and liquid chromatography-mass spectrometry (LC-MS).10) sensitivity by developing a bioluminescent enzyme im-
These methods are useful for basic study that measures mul- munoassay (BLEIA) using firefly luciferase as a labeling en-
tiple molecules, such as equol, daidzein and genistein, and zyme, with a bridge heterogeneous combination of antiserum
they show excellent specificity. However, these methods re- and enzyme-labeled antigen. BLEIA, with firefly luciferase
quire sample extraction and have relatively low throughput, used as a labeling enzyme, has a high sensitivity because
but the measurement of a large number of samples is neces- firefly luciferin-luciferase bioluminescence has high quantum
sary for epidemiological study to examine the evidence that yield (41.0ꢁ7.4%).14) The different bridge haptenic deriva-
the clinical efficacy of isoflavones in humans depends on the tives used for the immunogen and the enzyme-labeled anti-
production of an enterobacterial metabolite, equol. As an al- gen (bridge heterogeneous combination) are more sensitive
© 2011 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: Takayuki_Minekawa@eiken.co.jp

January 2011
85
than a homologous combination.15)
Thus, we report the development of an assay system spe-
cific for S-equol using firefly luciferase as a labeling enzyme,
namely, a bridge heterogeneous immunoassay method.
evaporated at reduced pressure, and 0.96 mg/ml of the residue was incubated
in 14.46 mg/ml streptavidin (SA) and 0.05 mmol/l carbonate bicarbonate
buffer (pH 9.6) for 30 min at room temperature. The crude S-equol-CPE-SA
conjugate was then purified on a Sephadex G-25 fine gel filtration column
(GE Healthcare U.K. Ltd., Little Chalfont, England). The fractions contain-
ing SA were collected. A 25 ml aliquot of S-equol-CPE-SA solution
(10.2 mg/ml) was added to 70 ml of 8.24 mg/ml bL248 and 8.26 ml of conju-
gate storage buffer (0.1 mol/l phosphate buffer (PB), 0.4 mol/l sodium chlo-
ride, 2 mmol/l EDTA·2Na, 5 mmol/l AMP, 0.2% bovine serum albumin
(BSA), 0.02% casein and 0.05% sodium azide). The solution was incubated
at 25 °C for 60 min. This conjugate solution (S-equol-CPE-SA-bL) was di-
Experimental
Materials R,S-Equol was obtained from LC Laboratories (Woburn,
MA, U.S.A.). S-Equol was obtained from Toronto Research Chemicals Inc.
(Ontario, Canada). b-Glucuronidase Type III from Ampullaria was obtained
from Nippon Biotest Laboratories Inc. (Tokyo Japan). Charcoal stripped
human serum was purchased from Scantibodies Laboratory, Inc. (Santee, luted to 100 nmol/l with conjugate storage buffer and stored at 4 °C.
CA, U.S.A.).
Synthesis of Immunogens (R,S-Equol-carboxymethylether (CME)-
Daidzein, Genistein and adenosine-5ꢀ-monophosphate (AMP) were pur- BSA) R,S-Equol-carboxymethylether (CME)-BSA was synthesized using
chased from Sigma (St. Louis, MO, U.S.A.). Streptavidin (SA) was procured
from MP Biomedicals, LLC (Solon, Ohio, OH, U.S.A.). Ethylenediamine-
the same method except for a change of SA-bL to BSA and the following re-
action of derivative synthesis by a method similar to that for S-equol-CPE-
N,N,Nꢀ,Nꢀ-tetraacetic acid disodium salt dehydrate (EDTA·2Na) and 2-mor- SA-bL. R,S-Equol-CME was prepared from bromoacetic acid methyl ester
pholinoethanesulfonic acid monohydrate (MES) were obtained from Do-
(Tokyo Chemical Industry Co., Ltd., Tokyo, Japan). In the presence of
jindo Laboratories (Kumamoto, Japan). Goat anti-rabbit immunoglobulin G 225 mg of potassium carbonate, 150 mg of R,S-equol dissolved in 600 ml of
(IgG) was prepared in our laboratory. Magnetic particles (Dynabeads M280, DMSO and 60 ml of bromoacetic acid methyl ester was added, and the reac-
Tosyl-activated) were obtained from Invitrogen Corp. (Carlsbad, CA,
U.S.A.). Biotinylated luciferase (bL248) was supplied by Kikkoman (Chiba,
Japan). Other chemicals were of analytical reagent grade.
tion mixture was stirred for 2.5 h at 25 °C.
Production of Polyclonal Antibodies Immunogen was prepared by di-
luting R,S-equol-CME-BSA conjugate with an equal volume of saline. Five
rabbits were subcutaneously administered with the R,S-equol-CME-BSA
Subjects A total of 68 men and women between the ages of 26 to 53
years (meanꢁS.D.: 41ꢁ7 years) participated in this study. The selected sub- conjugate (500 mg per rabbit) emulsified in complete Freund’s adjuvant at 3-
jects were in good health and had typical Japanese dietary habits. They
fasted for 12 h and the dietary record prior to fasting was not taken.
week intervals. A total of nine immunizations were given to each rabbit.
Sera were collected using a standard procedure. The cross-reactivity, IC20,
Bioavailability of equol was investigated after ingestion of SOYJOY® Raisin was tested with anti-equol antiserum.
Almond (Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan), containing 19 mg
of isoflavone/30 g bar. Urine samples were collected before and after con-
sumption of isoflavone. The curves for urinary daidzein and equol were con-
structed and postprandial maximum concentration (Cmax) was determined.
The study protocol was reviewed and approved by the Human Investigation
Review Committee of Eiken Chemical Co., Ltd.
Preparation of R-Equol and S-Equol The preparations of R-equol and
S-equol using b-cyclodextrin stationary phase liquid chromatography col-
umn under reversed-phase conditions were based on the method of Muthyala
et al.16) Two milligrams of R,S-equol was dissolved in 1 ml of 0.1% formic
acid (aq)/acetonitrile (65 : 35). Then, aliquots were applied to a CY-
CLOBOND I2000 RSP b-cyclodextrin-R,S-hydroxypropyl ether chiral sta-
The cross-reactivity were calculated using the following formula with the
result of a 20% inhibition test (IC20): cross-reactivity (%)ꢂ(ng/ml of com-
pound at IC20)/(ng/ml of cross-reacting S-equol at IC20)ꢄ100.
Preparation of Goat Anti-rabbit IgG-Immobilized Magnetic Particles
The goat anti-rabbit IgG was immobilized on magnetic particles (Dynabeads
M280, Tosyl-activated). The suspension of magnetic particles (4 mlꢄ100
mg/ml) was washed four times with 20 ml of distilled water. Then, the sus-
pension of magnetic particles was washed two times with 20 ml of 0.05%
Tween 20 in 0.1 mol/l PB, pH 7.4. Additionally, the magnetic particles were
washed two times with 20 ml of 0.1 mol/l carbonate buffer (pH 10.0). The
magnetic particles were re-suspended with 20 ml of 0.1 mol/l carbonate
buffer (pH 10.0) containing goat anti-rabbit IgG (0.1 mg/ml), followed by
tionary phase (column 250 mmꢄ4.6 mm, 5 mm particle size, Astec, NJ, mixing at 37 °C for 18 h. After removal of the supernatant, 0.5% Lipidure
U.S.A.) and eluted with 0.1% formic acid (aq)/acetonitrile (65 : 35). Injec- 206 (NOF Corp., Tokyo, Japan) was added and mixed at 37 °C for 60 min.
tion volume was 20 ml, and separations were performed at ambient tempera- The suspension was washed with 20 ml of 0.1 mol/l PB (pH 7.5), after which
ture. The flow rate was 0.75 ml/min and the UV spectra of the peaks were
recorded at 280 nm. Excellent separation (Rsꢂ2.4) of the two enantiometric
forms of racemic equol was obtained under this condition. Peak 1 (retention
it was washed with 20 ml of 0.01% TritonX-100 in 1 mol/l sodium chloride,
followed by mixing at 37 °C for 30 min. Upon completion, the suspension
was washed four times with 20 ml of bead buffer (0.05 mmol/l MES,
time, 19.7 min) and peak 2 (retention time, 21.8 min) represented S-equol 0.15 mol/l sodium chloride, 0.5% Purebright (NOF Corp., Tokyo, Japan), pH
and R-equol, respectively. These were used for evaluation of cross-reactivity.
Synthesis Labeling Antigen (S-Equol-carboxypropylether (CPE)-SA-
6.0). The suspension containing goat anti-rabbit IgG-immobilized magnetic
particles was diluted to 2 mg/ml with bead buffer and stored at 4 °C until
bL) In the presence of 14 mg of potassium carbonate, 10 mg of S-equol use.
was dissolved in 40 ml of N,Nꢀ-dimethyl formamide (DMF), 5.8 ml of 4-
Competitive Bioluminescent Enzyme Immunoassay (BLEIA) for S-
bromo-n-butyric acid (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan)
was added, and the reaction mixture was stirred for 4 h at 25 °C. The pH of
this solution was set below 2.0 using 6 mol/l hydrochloric acid. The mixture
was completely extracted three times with ethyl acetate. Then, after 3 ml of
distilled water (DW) was added, the S-equol-CPE present in the aqueous so-
lution was completely washed. The organic phase was dried over anhydrous
sodium sulfate. Thin-layer chromatography (TLC) of the organic phase de-
Equol This assay method was based on the utility of firefly luciferase as a
labeling enzyme and a competitive immunoassay procedure. Each urine
specimen was diluted 11-fold with diluents (charcoal stripped human serum)
as a dilute sample. Following transfer of 20 ml of the dilute sample to a poly-
styrene test tube (12ꢄ75 mm), 50 ml of deconjugation buffer (3% b-glu-
curonidase in 0.1 mol/l acetate buffer, pH 5.5) was added to the test tube and
mixed for 2 s. The mixture was incubated at 25 °C for 30 min. After the incu-
veloped with 5% methanol/chloroform as the solvent indicated the appear- bation, 50 ml of S-equol-CPE-SA-bL, 20 ml of goat anti-rabbit IgG-immobi-
ance of monocarboxypropyl ether and dicarboxypropyl ether spots in addi-
lized magnetic particles, and 50 ml of anti-equol rabbit antiserum were
tion to the spots at the starting points. The starting material and dicar- added to the mixture and incubated at 25 °C for 15 min, then washed with
boxypropyl ether were removed using this TLC. Then, 500 ml of methanol 0.5 ml of 0.05% Tween 20 in PBS (repeated four times). Luciferin-luciferase
was added; this solution was saponified using 78 ml of 8 mol/l sodium hy-
droxide. The reaction mixture was stirred for 20 min at 50 °C and 3 ml of
DW was added. The pH of this solution was set below 2.0 using 6 mol/l hy-
drochloric acid. The mixtures were completely extracted three times with
ethyl acetate and then washed three times with DW. The organic phase was
reaction and luminescence measurement were performed as reported previ-
ously.17) Most of the equol in human urine exists as a conjugate, such as glu-
curonide, sulfoglucuronide, and sulfate.18) Therefore, the result is shown in
ng/ml, equivalent to deconjugated aglicon.
Competitive Enzyme-Linked Immunosorbent Assay (ELISA) for
dried over anhydrous sodium sulfate. S-Equol-CPE was recrystallized from Daidzein To each well of goat anti-rabbit IgG-immobilized plate, 20 ml of
benzene. S-Equol-CPE (2.8 mg) was added to 28 ml of dimethyl sulfoxide
(DMSO). Then, 2.1 mg of N-hydroxysuccinimide (NHS) and 2.1 mg of 1-
standard (11—3300 ng/ml) in charcoal stripped human serum or urine speci-
men, diluted 11-fold with diluents (charcoal stripped human serum), was
ethyl-3-(3-dimethylaminopropyl)carbodiimide, hydrochloride (EDC) were added. Then, 50 ml of daidzein-labeled horseradish peroxidase (HRP) in de-
added, and the reaction mixture was stirred at 25 °C for 60 min. The S-equol- conjugation buffer (6% b-glucuronidase in 0.1 mol/l acetate buffer, pH 5.5)
CPE-NHS was extracted with ethyl acetate after addition of DW, and the or-
ganic phase was dried over anhydrous sodium sulfate. The solvent was then
was added and mixed well. The mixture was incubated for 30 min at 25 °C.
Additionally, 50 ml of anti-daidzein rabbit antiserum in 0.1 mol/l phosphate

86
Vol. 59, No. 1
buffer, 0.15 mol/l sodium chloride, 0.5% bovine serum albumin, and 0.01%
Tween 20 were added and mixed well. The mixture was then incubated for
1 h at 25 °C. After incubation, the plate was washed with 350 ml of 0.025%
Tween 20 in 0.3 mmol/l phosphate buffer and 100 mmol/l sodium chloride,
pH 7.5 (repeated four times). After washing, 100 ml of ELISA substrate so-
lution (0.05% hydrogen peroxide, 2.2 mg/ml o-phenylenediamine, 50 mmol/l
citrate buffer, pH 4.1) was added to each well and the mixture was allowed
to react for 30 min at 25 °C. The enzymatic reaction was stopped with 100 ml
of 1.5 mol/l sulfuric acid. The color intensities of the wells were determined
using a spectrophotometer at 492 nm.
HPLC Determinations HPLC analysis was done using a modified pro-
tocol of Franke’s methods at SRL, Inc. (Tokyo, Japan).19)
Results and Discussion
Development of BLEIA for S-Equol Upon determina-
tion of the standard curve for S-equol BLEIA in accordance
with the aforementioned protocol, a measurable range of 110
to 8910 ng/ml was evident, displaying good linearity. Intra-
assay coefficients of variation (CV) for 565—3679 ng/ml
equol in human urine were within the range of 3.3—4.4%,
nꢂ10. The inter-assay CV of 592—3543 ng/ml equol in
human urine ranged between 9.4—10.4%, nꢂ10. The recov-
ery test was performed via addition of one volume of antigen
to four volumes of urine. Recovery was 102ꢁ3—129ꢁ4%
(meanꢁS.D., nꢂ3) in all samples. This S-equol was found to
show cross-reactivity to R-equol of 4.83%, and dehydroequol
and dihydrodaidzein, which are metabolic intermediates of
equol, showed values of 0.40% and 0.21%, respectively.
Cross-reactivity of daidzein, which is a substrate of equol,
was only 0.02%. Other similar molecules such as estrogen
showed less than 0.03% cross-reactivity (Table 1). Equol
concentration, which was measured by employing the current
BLEIA method, was compared with that determined with
HPLC. Satisfactory correlation (rꢂ0.992, yꢂ1.07xꢅ303,
nꢂ22) was evident. Therefore, basic validation of the im-
munoassay was achieved; intra- and inter-assay reproduction
and correlation coefficient were excellent.
Fig. 1. Equol Producer Status of Japanese Men (a) and Women (b) Based
on Two Approaches
Urinary equol concentrations plotted for the individual subjects are shown with bars.
Log10-transformed urinary equol to daidzein ratios for individual subjects are shown
with circles. Equol producers were defined by two approaches: those with a log10 uri-
nary equol to daidzein concentration ratio of ꢆꢅ1.7520) and those with a urinary equol
concentration of ꢆ232 ng/ml (960 nmol/l).12) The subjects that gave different results by
the two approaches are shown with closed circles.
Equol Producer Status of Japanese Population We ex- to daidzein concentration ratio. There was good agreement in
amined the equol producer status of the Japanese population the classification of equol producers between the two ap-
using S-equol BLEIA. The result of equol producer status by proaches: that is, 94% (44 of 47) of male and 90% (19 of 21)
S-equol BLEIA was compared to the equol to daidzein ratio. of female subjects were assigned the same classification. In
Urine specimens from 68 Japanese subjects, who had fasted terms of the frequency of equol producers in this study, male
for 12 h, but without the dietary record prior to fasting being (32% or 34%) were higher than female (19%). This result
taken, were analyzed by S-equol BLEIA and daidzein ELISA corresponds to the reports of Morton et al. (58% of men and
(Figs. 1a, b). The over-the-range specimens were re-measured 38% of women with a serum equol concentration ꢆ20
after dilution. This data was grouped by gender and in as- nmol/l)21) and Setchell and Cole (65% of men, 28% of
cending order of equol concentration. The ratio of equol to women).20) However, the results from our study are generally
daidzein was calculated, transformed, and expressed as lower than those observed by Morton et al.21) and Setchell
log10.20)
and Cole,20) even though the samples in both studies were
The approach to define equol producers as those with an collected from Japanese subjects. The difference in results is
urinary equol concentration ꢆ232 ng/ml (960 nmol/l)12) cor- thought to be due to a variation of the subject population.
responds to another approach that defines equol producers as
Postprandial Urine Concentration of Equol and
those with log10 urinary equol to daidzein concentration ratio Daidzein after Intake of Isoflavone To demonstrate the
ꢆꢅ1.75.21) Within the limitations of the small sample size of bioavailability of daidzein to equol, the urinary equol and
this study, the results of classification of male volunteers daidzein concentration changes were measured after inges-
were as follows. On the basis of their urinary equol concen- tion of isoflavone (Fig. 2). Among the volunteers, a man aged
tration, 32% (15 of 47) of the male volunteers were classified 33 years was selected for his urinary equol concentration of
as equol producers. On the basis of the log10 urinary equol to more than 1000 ng/ml. The urinary daidzein concentration
daidzein concentration ratio, the proportion of equol produc- increased rapidly 7.7 h after isoflavone consumption. There-
ers among male volunteers was 34% (16 of 47). On the other after, the urine concentration of daidzein decreased until
hand, the proportion of female volunteers classified as equol 18.4 h. The urine Cmax for daidzein after the consumption of
producers was 19% (4 of 21) in both classification methods, isoflavone was 4704 ng/ml. On the other hand, there was a
namely, urinary equol concentration and log10 urinary equol rapid increase in equol concentration in urine at 9.6 h after

January 2011
87
of equol is 0.02%. The determination of equol producer sta-
tus of Japanese subjects by this S-equol BLEIA corre-
sponded to that using the ratio of equol to daidzein. When
urinary equol and daidzein concentration changes were
measured after ingestion of isoflavone the time difference be-
tween when equol (9.6 h) and when daidzein reached Cmax
(7.7 h) was 2 h.
In this study, we developed S-equol BLEIA that can be
used to measure S-equol specifically. The S-equol BLEIA
documented here is expected to be an important tool to as-
sess equol producer status and demonstrate bioavailability.
References
Fig. 2. Postprandial Equol Concentration in Urine after Intake of a Single
Bar of SOYJOY (19 mg of Isoflavone)
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Cross-reactivity
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S-Equol
R-Equol
100.00%
4.83%
0.40%
0.21%
0.02%
Dehydroequol
Dihydrodaidzein
Daidzein
Daidzin
Genistein
Genistin
Glycitein
Glycitin
E2
BiochaninA
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Apigenin
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0.01%
0.02%
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ꢃ0.01%
ꢃ0.01%
ꢃ0.01%
ꢃ0.01%
0.03%
0.02%
ꢃ0.01%
ꢃ0.01%
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isoflavone consumption. As shown in Fig. 2, the urine con-
centration of equol decreased until 28.4 h. The urine Cmax for
equol after the consumption of isoflavone was 4433 ng/ml.
As such, the time when equol reached Cmax was 2 h later than
that of daidzein. In addition, equol was still detectable for an-
other 10 h after daidzein became undetectable. This delay is
thought to be due to the time during which daidzein is me-
tabolized to equol by enterobacteria.22)
16) Muthyala R. S., Ju Y. H., Sheng S., Williams L. D., Doerge D. R.,
Katzenellenbogen B. S., Helferich W. G., Katzenellenbogen J. A.,
Bioorg. Med. Chem., 12, 1559—1567 (2004).
17) Minekawa T., Ohkuma H., Abe K., Maekawa H., Arakawa H., Lumi-
nescence, 24, 394—399 (2009).
18) Adlercreutz H., van der Wildt J., Kinzel J., Attalla H., Wähälä K.,
Mäkelä T., Hase T., Fotsis T., J. Steroid Biochem. Mol. Biol., 52, 97—
103 (1995).
Conclusion
We developed BLEIA for S-equol and applied it to investi-
gate the equol producer status of Japanese subjects and its
urine concentration after intake of isoflavone. When this S-
equol BLEIA was compared with HPLC, satisfactory corre-
lation (rꢂ0.992) was observed. Additionally, this assay
method is very convenient because it does not require extrac-
tion, and it can be completed in about 1 h. The cross-reactiv-
19)
Franke A. A., Custer L. J., J. Chromatogr. B Biomed. Appl., 662, 47—
60 (1994).
20) Setchell K. D., Cole S. J., J. Nutr., 136, 2188—2193 (2006).
ity with other isoflavones is low, with that for R-equol, which 21) Morton M. S., Arisaka O., Miyake N., Morgan L. D., Evans B. A., J.
Nutr., 132, 3168—3171 (2002).
22) Zubik L., Meydani M., Am. J. Clin. Nutr., 77, 1459—1465 (2003).
is a diastereoisomer that does not exist as a metabolite of
daidzein is ꢃ5%, and that for daidzein, which is a substrate