Apparent bioavailability of isoflavones after intake of liquid and solid soya foods.

Abstract

Isoflavone (IFL) intake may provide numerous health benefits, but IFL bioavailability differences among soya foods remains uncertain. Urinary IFL excretion (UIE) was shown to provide a reliable surrogate for systemic IFL exposure and therefore can be used as a measure of 'apparent bioavailability' (AB). We investigated the AB of IFL in fourteen healthy adults, consuming two liquid and two solid soya foods in a crossover designed study. Volunteers consumed the foods with a self-selected breakfast, which was kept identical for all four soya items (soya nuts, soya milk, soya protein bar and soya protein powder drink in water; average 23.7 mg IFL, 88-96 % glycosides, by HPLC analysis) and collected all urine up to 26 h. Liquid foods showed initially higher UIE values than solid foods, but this difference was considerably reduced or disappeared entirely after 24-26 h. Conclusive AB results were obtained only after 24-26 h; earlier collections were not reliable. At 26 h, adjusted UIE values for daidzein (DE) were 20 micromol in the milk and bar and 17 micromol for the nut and powder; urinary genistein excretion was the highest in the milk group (10 micromol) followed by the nut, bar (both 6 micromol) and powder groups (5 micromol); the UIE for glycitein was the highest for bars (4 micromol), followed by powder and nuts (3 micromol), and milk (2 micromol). DE makes the largest contribution to urinary total IFL. The AB of IFL was found to be variable depending on the analyte and soya food consumed.

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Apparent bioavailability of isoflavones after intake of liquid and solid
soya foods
Adrian A. Franke1*, Leslie A. Ashburn1, Kerry Kakazu1, Shana Suzuki1, Lynne R. Wilkens1
and Brunhild M. Halm1,2,3
1Natural Products and Cancer Biology Program, Cancer Research Center of Hawaii, 1236 Lauhala Street, Honolulu, HI 96813, USA
2Cancer Prevention and Control Program, Cancer Research Center of Hawaii, 1960 East-West Road, Biomedical Sciences C-105,
Honolulu, HI 96822, USA
3Kapi’olani Medical Center for Women and Children, 1319 Punahou Street, Honolulu, HI 96822, USA
(Received 17 December 2008 – Revised 5 March 2009 – Accepted 14 April 2009)
Isoflavone (IFL) intake may provide numerous health benefits, but IFL bioavailability differences among soya foods remains uncertain. Urinary
IFL excretion (UIE) was shown to provide a reliable surrogate for systemic IFL exposure and therefore can be used as a measure of ‘apparent
bioavailability’ (AB). We investigated the AB of IFL in fourteen healthy adults, consuming two liquid and two solid soya foods in a crossover
designed study. Volunteers consumed the foods with a self-selected breakfast, which was kept identical for all four soya items (soya nuts, soya
milk, soya protein bar and soya protein powder drink in water; average 23·7mg IFL, 88–96% glycosides, by HPLC analysis) and collected all
urine up to 26h. Liquid foods showed initially higher UIE values than solid foods, but this difference was considerably reduced or disappeared
entirely after 24–26h. Conclusive AB results were obtained only after 24–26h; earlier collections were not reliable. At 26h, adjusted UIE values
for daidzein (DE) were 20mmol in the milk and bar and 17mmol for the nut and powder; urinary genistein excretion was the highest in the milk
group (10mmol) followed by the nut, bar (both 6mmol) and powder groups (5mmol); the UIE for glycitein was the highest for bars (4mmol),
followed by powder and nuts (3mmol), and milk (2mmol). DE makes the largest contribution to urinary total IFL. The AB of IFL was found
to be variable depending on the analyte and soya food consumed.
Isoflavones: Bioavailability: Urine: Soya: Liquid foods: Solid foods
Soya and its isoflavones (IFL) are reported to prevent cancer,
heart diseases and other chronic disorders(1–6), especially
when the dietary exposure occurs early in life(7–10). Similar
to the situation with flavonoids, several human studies have
established that isoflavonoid glycosides, the main IFL form
present in a soya containing diet, taken orally, appear in the
circulation as glucuronides and sulphates(11–18). The available
evidence suggests that during digestion, isoflavonoid glyco-
sides are hydrolysed mainly by intestinal bacteria, followed
by passive diffusion through the mucosa and reconjugation
to glucuronides and sulphates on the basolateral side of the
enterocyte and/or in the liver(19,20). Gut bacteria play an
additional vital role because they also form the major IFL
metabolites (dihydrodaidzein, dihydrogenistein, equol (EQ),
O-desmethylangolensin) and further degrade IFL(21–23).
We and others have shown recently that blood and urine
isoflavonoid values, including those in children, are highly
correlated, particularly when timing of collection is considered
accurately(19,24–30). Since bioavailability is defined based
on circulating levels, we suggest the term ‘apparent bio-
availability’ (AB) when using urinary excretion data as surro-
gate to describe systemic exposure(29,31).
In a variety of studies on IFL bioavailability after soya
intake, either men or women (often in one menopausal state)
alone(32–38), solid soya alone(36)or highly processed foods
alone(38)was investigated. Despite variabilities, in particular
related to the patterns of the individual IFL daidzein (DE),
genistein (GE) and glycitein (GLYE), the unifying result
of previous investigations was that background diet plays
an important role(36). For example, fibre decreases IFL
uptake(37)and fat decreases EQ production(39). Additionally,
the IFL from liquid soya foods was absorbed more
quickly(19,35)and more extensively(40). Aglycons as present
in fermented soya products were found to result in higher
IFL uptake by some(33,40–42), but not others(19,34,43)including
non-distinguishable differences(44,45).
While considerable knowledge exists in experimental
settings(41,43,46,47), the uptake efficiency and pharmacokinetic
parameters of IFL from various soya foods are little under-
stood in human subjects. By measuring urinary isoflavonoid
excretion (UIE), the intent of this research was to find out
whether the isoflavonoid exposure differs between different
types of soya foods consumed. We aimed to compare liquid
(soya milk and soya protein drink referred to as ‘milk’ and
*Corresponding author: Dr Adrian A. Franke, fax þ1 808 586 2970, email adrian@crch.hawaii.edu
Abbreviations: AB, apparent bioavailability; DE, daidzein; EQ, equol; GE, genistein; GLYE, glycitein; IFL, isoflavone; UIE, urinary IFL excretion.
British Journal of Nutrition (2009), page 1 of 8
q The Authors 2009
doi:10.1017/S000711450937169X
British Journal of Nutrition

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‘powder’) v. solid (soya nuts and soya protein bars referred to
as ‘nuts’ and ‘bars’) and native v. processed soya foods.
An important part of the present study was to keep the
foods consumed together with the respective study soya item
constant in order to avoid confounding effects by the food
environment. In addition, we aimed to determine the time
frame needed within 26h after soya intake to determine IFL
excretion accurately considering the biphasic UIE profile(19).
Methods
Population
The University of Hawaii Committee on Human Studies
approved the study protocol and consent form. The latter
was signed by all participants. In total fourteen participants
took part in the study. The subjects recruited were healthy,
with no known soya allergies, who did not report antibiotic
use within any 4-week period before consuming the study
soya food. Each participant consented to donating their full
volume of urine immediately before and 2, 8, 24, and 26h
after intake of at least four different soya foods (nuts, milk,
health bar and protein drink), which was designed to happen
on four separate days, each of them approximately 1-week
apart.
Data from urines that were collected with more than 10%
difference to the scheduled time point were omitted. For
soya nuts, eleven people correctly completed the 2 and 8-h
collections, ten completed the 24-h collection and thirteen
completed the up to 26-h collections. For soya milk, eleven
participants completed the 2, 8 and 24-h collections, and thir-
teen completed the up to 26-h collection correctly. For the
soya bar, eleven people correctly collected the 2, 8 and 24-h
collections, and all fourteen completed the up to 26-h collec-
tions. Finally, for the soya powder drink, nine were able to
correctly collect the 2, 8 and 24-h collections, with thirteen
completing the up to 26-h collections (Table 1).
Study procedures
After emptying their bladders for the spot urine collection, the
participants had breakfast with the study soya food and sub-
sequently donated all urine during 0–2, 2–8, 8–24 and
24–26-h time periods. According to HPLC analysis(48), the
serving sizes of the respective soya foods, namely 13·6g
lightly salted and roasted soya nuts (referred to in text as
nuts), 12·7g Peanut Pal soya bar (referred to in text as bar)
containing as IFL source soya protein, 12·6g Strawberry
Banana Bliss soya protein powder drink (referred to in text
as powder) mixed withwater
Physician’s Pharmaceuticals, Kernersville, NC, USA) and
95·2g WestSoy organic unsweetened soya milk (referred to
in text as milk; Hain Celestial Group, Boulder, CO, USA)
contained on average 23·7 (SD 0·03mg IFL (Table 2).
The soya food was consumed together with a breakfast of
the participants’ choice (some participants brought their own
breakfast). The study staff kept the breakfast exactly the
same across each of the 4–5 study days. After an
approximately 1-week washout period, the participants
repeated the same procedure as described earlier with a
different soya food during the following weeks.
(allfromRevivalw,
Urine containers included approximately 200mg boric and
100mg ascorbic acid as preservatives(49). Urine samples
were immediately weighed, followed by storage of 2ml
aliquots at 2208C; if collected urine needed to be stored
longer than 2h, it was kept chilled in coolers that contained
ice packs. Participants completed a validated two-page soya
intake and a twenty-six-page diet and health questionnaire(50),
and provided information regarding their height and weight.
On their worksheets, they recorded time and date of urine
collections, food consumption during the study day and the
time at which the study food was consumed. Participants
avoided eating soya foods for a full 48h before and during
the 26h of the study day. Preliminary data suggested that
allowing participants to consume soya food(s) within 24h
before the baseline urine collection interferes with the study
results. We confirmed by questionnaire that the participants
had refrained from taking oral antibiotics within 4 weeks
before and during their participation, that they had avoided
eating any soya products 48h before the start of the study,
that they had fasted overnight, and that they did not have
any gastrointestinal problems before or on the study day.
Urinary isoflavonoid analysis
DE, GE, GLYE, EQ, O-desmethylangolensin, dihydrogenis-
tein and dihydrodaidzein (the sum of these were calculated
as ‘Total IFL’) were analysed by HPLC with electrospray
ionisation (negative mode) tandem massspectrometry
Table 1. Participant characteristics and urine collection details
(Mean values and standard deviations)
Mean
SD
n
Male (%)
Female (%)
BMI (kg/m2)
Age (years)
Asian (%)
Caucasian (%)
Mixed/other (%)
2-h Collection
Nuts
Milk
Bar
Powder
8-h Collection
Nuts
Milk
Bar
Powder
24-h Collection
Nuts
Milk
Bar
Powder
26-h Collection
Nuts
Milk
Bar
Powder
21
79
3
11
14
14
21·6
39·4
1·97
10·74
14
71
14
2
10
2
1·85*
2·03*
1·92*
1·89*
0·23
0·32
0·13
0·12
11†
11†
11†
9†
7·95*
8·26*
7·87*
7·89*
0·32
0·86
0·09
0·19
11†
11†
11†
9†
23·80*
24·00*
23·86*
23·92*
0·14
0·36
0·11
0·11
10†
11†
11†
9†
25·87*
26·03*
25·96*
25·92*
0·14
0·38
0·18
0·15
13†
13†
14†
13†
Sum of percentage may differ from 100 owing to rounding.
*Mean hours since respective soya intake.
†Total number of participants collecting urine correctly during the given time
periods.
A. A. Franke et al. 2
British Journal of Nutrition

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(model TSQ Ultra, Thermo, San Jose, CA, USA) similar to
our earlier reports(51,52)and as have been detailed most
recently(53). Limits of quantitation for all analytes were 1nM
for DE and GE, 2nM for EQ and 5nM for the other analytes.
Between-day coefficients of variation ranged between 4 and
18% for all analytes, while intra-day variation was half or
less than that.
Creatinine levels were determined by a clinical autoanaly-
zer (Roche Cobas MiraPlus; Roche Diagnostics, Indianapolis,
IN, USA) using a test kit based on the Jaffe ´ reaction (Randox
Laboratories, Crumlin, UK). Isoflavonoid excretion in urine is
expressed in nanomoles per hour by adjusting for urine
volume and collection times. Baseline urine was converted
from nanomoles per milligram creatinine (nmol/mg) units to
nmol/h units using hourly creatinine excretion as available
from each participant from the timed urine collections(29,54).
Instrument calibration was performed daily using stock
solutions that were measured for concentrations using absor-
bance readings as described previously(52).
Calculations
Since the IFL composition was slightly different between the
study foods (Table 2), UIE was adjusted to the IFL doses of
nuts; factors applied (to divide measured UIE values by) for
milk, bar and powder to that in nuts were for DE 1·107,
1·188 and 1·268; for GE 0·866, 0·509 and 0·554; for glycitein
1·000, 3·231 and 3·154; and for Tot IFL 0·987, 0·979 and
1·038, respectively. The UIE 2, 8, 24 and 26h after soya
intake was adjusted for the UIE contribution by the baseline
spot urine if the latter had IFL levels .1000nM as described
in detailed recently(29,54). This cut-off can be considered
conservative because at this level the theoretical amount
after 0–2, 6–8, 8–24 and 24–26h is only 96, 217, 212 and
22nmol,respectively, given
1060mg/l (equivalent to 55mg/h leading to an excretion
rate of 52nmol/h in our cohort) and a 8-h half-life of
IFL(55). This excretion is negligible compared with the
amounts found after soya challenge. Five subjects had levels
,1000nM in baseline spot urine, three participants had IFL
in the baseline spot urine at levels of 1000–3000nM and
a creatinineexcretion of
only one participant had consistently at least one IFL at
levels 3000–5000nM in baseline spot urine, but overall results
did not change when this participant was removed from all
calculations. The correctness of urine collections was checked
by comparing the measured to the theoretically expected urin-
ary creatinine excretion according to the accepted (sex, body
weight and age-dependent) values from healthy adults(29,54).
Ethical approval
The present study was conducted according to the guidelines
laid down in the Declaration of Helinski, and all procedures
involving human subjects were approved by the University
of Hawaii Committee on Human Subjects. Written informed
consent was obtained from all subjects.
Statistical analysis
Paired t tests were performed using Excel 2004 for the Macin-
tosh (Microsoft Inc., Redmond, WA, USA). These tests were
performed with log-transformed UIE values to account for the
non-normality of distributions. A P-value of ,0·05 was
considered suggestive and a P-value ,0·05/6 ¼ 0·008 was
considered significant, after Bonferroni correction for the six
pairwise multiple comparisons of products at each time point.
Results
Fourteen subjects were recruited and thirteen completed the
collection correctly for the soya foods nuts, milk and bar,
whereas fourteen completed the collection correctly for
powder during the cumulative up to 26-h time point, respect-
ively. High compliance was observed by individual monitor-
ing of the collections during the day, since participants were
all part of our institution. Correct urine collections were also
confirmed by the measured creatinine values (data not shown).
There were a total of nine repeat collections for the soya
nuts, four for the soya milk, three for the soya bar and two
for the powder drink, but only the average within each
repeat was used in the present calculations. The mean
coefficient of variation of the four types of repeated soya
Table 2. Isoflavone doses of study soya foods consumed*
Study food† Daidzein GlyciteinGenistein Total isoflavonesGlucosides (%)Malonates (%) Acetates (%) Aglycons (%)
Nuts
mg
mmol
Milk
mg
mmol
Bar
mg
mmol
Powder
mg
mmol
11·2
44·1
1·3
4·6
11·2
41·3
23·7
90·1
346564
12·4
48·9
1·3
4·5
9·7
35·9
23·4
89·3
592884
13·3
52·2
4·2
14·8
5·7
21·0
23·2
88·0
548 27 12
14·2
56·0
4·1
14·6
6·2
22·8
24·6
93·5
57 13247
Total isoflavone values may deviate from the sum of individual isoflavones owing to rounding.
Total isoflavones ¼ all isoflavones as the sum of native aglycons plus all glycosides.
*According to HPLC analysis(48).
†Lightly salted roasted soya nuts 13·6g (Revivalw), unsweetened organic soya milk 95·2g (Westsoy Comp.), Peanut Pal bar 12·7g (Revivalw) and Strawberry Banana Bliss
protein powder 12·6g (Revivalw).
Apparent bioavailability of isoflavones3
British Journal of Nutrition

Page 4

products by analyte was DE 19%, GE 29%, GLYE 28% and
total IFL 14%, which can be considered consistent.
The total IFL dose was consistent in all four soya items,
namely 23·7, 23·4, 23·2 and 24·6mg for nuts, milk, bar
and powder, respectively (mean ¼ 23·7mg, CV ¼ 2·6%);
however, the IFL composition was somewhat different
(Table 2). We therefore adjusted all UIE data to doses as pre-
sent in nuts (see ‘Methods’ for details), in order to perform an
accurate comparison of the AB between each food item by
evaluating each IFL individually. After 2, 8 and 24h relative
to 26h, DE was 10–17, 58–69 and 96–100% excreted,
respectively; for GE, these numbers were 10–17, 49–62
and 85–100 and for GLYE, 7–13, 59–72 and 91–100,
respectively (Fig. 1).
In general, the liquid foods, particularly the milk, showed
initially higher UIE values than the solid foods, but this
difference was much reduced or disappeared entirely after
24 and 26h. Significant differences (P,0·01 by paired t
test) in DE excretion between the foods were observed at
early collection periods, especially at the 2-h time point, but
these were partially lost for later collections. At the 26-h
time point, the milk and bar groups showed urinary DE
excretion of 20mmol, while nuts and powder groups showed
17mmol, respectively. Significant differences (P,0·05 by
paired t test) in DE excretions were observed for the groups
bar v. nuts (þ23%), milk v. powder (þ18%) and bar
v. powder (þ18%). For GE, the pattern changed between
the four soya foods much less over time, in fact stayed very
similar in the last two collection periods (24 and 26-h
collections). After 26h, the urinary GE excretion was the
highest in the milk group (10mmol), followed by the nut,
bar (both 6mmol) and powder groups (5mmol). The differ-
ences were significant (P,0·05 by paired t test) between all
groups except between nuts and bars, i.e. milk v. nuts
þ52%, nuts v. powder þ32%, milk v. bar þ53%, milk
v. powder þ101% and bar v. power þ31%. The UIE for
GLYE was the highest for the bars (4mmol) 26h after soya
intake, followed by powder and nuts (3mmol), and milk
(2mmol). The up to 2-h collection showed significance
(P,0·05 by paired t test) for milk v. nuts (þ52%), bar
v. nuts (þ38%) and powder v. nuts (þ52%). For the up to
8, 24 and 26-h collections, only the pair bar v. milk showed
a significant difference (þ65–67%; P,0·05 by paired t test).
Total IFL 26h after soya intake showed the highest UIE for
the bars (57mmol), followed by those of milk (51mmol),
powder (48mmol) and nuts (39mmol), respectively. This was
significant (P,0·05 by paired t test) for the UIE pairs bar v.
nuts (þ45%), milk v. nuts (þ30%), powder v. nuts
(þ23%) and bar v. powder (þ18%; Appendix 1). The same
pattern was observed for the up to 8 and 24-h periods, but not
for the up to 2-h collection.
The urinary appearance patterns of the metabolites dihydro-
daidzein, dihydrogenistein, EQ and O-desmethylangolensin
are shown in detail in Appendix 1, but the relative short
time of specimen collection after soya intake possibly led to
an incomplete recovery. The urinary recovery relative to
dose 26h after soya intake (Table 3) showed a similar pattern
as described earlier for the individual IFL. Recovery of DE
0
5
10
*
*
*
†
†
†
†‡
†‡
*†
*†
*†
‡
*
*
*
*
†
‡
*
*
*‡‡
*
*
†
†
†
†
*†‡
†
‡ ‡
15
20
25
30(a)(b)
(c)(d)
Time since intake
Urinary excretion (µmol)
0
5
10
15
20
25
30
Time since intake
Urinary excretion (µmol)
0
5
10
15
20
25
30
Up to 2h Up to 8h Up to 24h Up to 26h
Up to 2h Up to 8hUp to 24h Up to 26h
Up to 2hUp to 8hUp to 24h Up to 26h
Up to 2hUp to 8hUp to 24hUp to 26h
Time since intake
Urinary excretion (µmol)
0
10
20
30
40
50
60
Time since intake
Urinary excretion (µmol)
*
*
*
*
*
†
†
†
†
†‡
†‡
†‡
‡ ‡
‡
‡
‡
*
*
*
*†
*†
*†
*†
*†
*†
†
†
†
†
† †
Fig. 1. Urinary excretion of isoflavonoids ((a) daidzein, (b) genistein, (c) glycitein and (d) total isoflavones) over four collection periods after adjustment to dose
present in nuts ( , nut;
, milk; , bar; , powder). *Values for individual and total isoflavones within a collection period with different symbols are signifi-
cantly different (P,0·05); error bars indicate standard error; total isoflavones ¼ daidzein þ genistein þ glycitein þ dihydrodaidzein þ dihydrogenistein þ equol þ
O-desmethylangolensin.
A. A. Franke et al. 4
British Journal of Nutrition

Page 5

and its metabolites were higher in milk (74%) and bars (73%)
than in nuts (61%) and powder (61%). The urinary GE recov-
ery was the highest in the milk group (28%), followed by the
nut (20%), bar (19%) and powder groups (14%). Recovery of
GLYE was the highest for the powder (59%), followed by
bars (56%), milk (54%) and nuts (49%).
Discussion
It was previously demonstrated that UIE profiles reflect
circulating IFL levels accurately(19,28,32)(and also soya
intake(25)) prompting us to use UIE as a reliable surrogate to
determine IFL bioavailability. We emphasise that bioavailabil-
ity is strictly based on measuring levels in the circulation and
we therefore refer to AB when using urinary values. In the
present context, it is important to note that the plasma:urine
ratio is different between the various IFL (for example
different by a factor of 3 comparing DE with GE), but it
will remain unchanged within each IFL(19). Therefore, accu-
rate AB conclusions can be drawn for each individual IFL
when comparing various soya foods or various individuals
exposed to or adjusted to the same dose.
In the present study, we examined the UIE of fourteen
participants after they had a breakfast, which was kept consist-
ent within each subject during the consumption of all four
different soya foods. The soya items were dosed to assure a
consistent total IFL exposure. We allowed for approximately
1-week of washout between the consumption of each soya
food to avoid interferences. Although a previous study
reported no significant difference of UIE when diverse soya
foods were consumed(36), we found that the UIE profiles
differed significantly between the four soya foods we tested
regarding total IFL as well as individual IFL when adjusted
to dose. DE was approximately 18% more bioavailable
when consumed from milk or bars relative to nuts or
powder. By contrast, GE was the most bioavailable from
milk, while nuts and bars were 35% lower, and powder was
50% lower. Again different was the sequence for glycitein
among the soya foods tested with bars leading, followed by
powder (22% lower), nuts (36% lower) and milk (40%
lower). This differential IFL bioavailability pattern depending
on the type of soya food consumed is in agreement with other
studies(32,38).Whencomparing
slight deviations from earlier differences occurred for GE
and glycitein owing to the different doses applied in each
soya food. By and large though, they followed the described
oururinaryrecoveries,
trend even when considering that the urinary recovery data
included the metabolites dihydrodaidzein, EQ and O-des-
methylangolensin with the DE data and dihydrogenistein
with the GE data.
In agreement with other studies(32), we found that DE
appeared in much larger amounts in urine than GE, GLYE,
or the metabolites (Appendix 1). Therefore, DE is the largest
contributor to the total IFL value in urine (in the present study,
approximately 40%) and predominates the latter value.
Consequently, we discourage using the total IFL value as a
reliable UIE data point, because it is biased towards DE and
suggest considering each IFL individually. This approach
will also take into consideration that each IFL has very
distinct pharmacologic properties, for example binding to
and transactivation of the oestrogen receptors(56).
The present results indicate that specimen collections over
at least 24h are needed to determine conclusive bioavailability
data. Shorter periods are insufficient probably owing to the
biphasic IFL appearance pattern in plasma and urine(19,46).
Since the IFL profiles in each of the foods were different,
we adjusted the UIE values to the dose present in nuts; this
made AB comparisons between the foods accurate. This was
also performed for the total UIE values, although little adjust-
ment was needed since serving sizes were designed to keep
the total IFL dose consistent. Some studies have shown that
IFL from liquid soya foods such as soya milk are absorbed
and excreted more quickly than from solid forms of soya
foods(32,40). The present results agree with this finding only
for soya milk, but not for the soya protein powder drink. It
appears that not all liquid forms of soya are equivalent. This
seems to also apply for solid foods because the bars and
nuts we tested showed vast differences in AB. The underlying
mechanisms for the observed differences in urinary IFL
appearance patterns are uncertain, but the matrix of the type
of soya food might have had a distinct influence. For example,
the considerable fat content of the nuts may have caused a
slower uptake(37), while fibre and the solid nature of this
food might have decreased general bioaccesibility. Additional
factors that could have affected the IFL absorption after soya
intake are the intestinal bacteria. IFL absorption will be
decreased when these bacteria that hydrolyse the native IFL
glycosides to the bioavailable aglycons are impaired. On the
other hand, IFL absorption will be increased when bacteria
that degrade IFL aglycons during digestion are impaired.
This fine line of dynamic changes of IFL bioavailability
caused by the gut flora has recently been suggested as an
Table 3. Recovery relative to dose in urine collected up to 26h after soya food consumption
(Mean values with their standard errors of urinary recovery)
NutsMilk BarPowder
Mean
SE
Mean
SE
Mean
SE
Mean
SE
Significance*
Daidzein þ dihydrodaidzein þ equol
þO-desmethyl-angolensin (%)
Genistein þ dihydrogenistein (%)
Glycitein (%)
Total isoflavones (%)
624744 733613NM, NB, MP, BP
20
49
43
3
5
3
28
54
55
5
6
4
19
56
63
3
4
5
14
59
54
2
4
3
NP, MB, MP, BP
NB
NM, NB, NP, BP
NM, nuts v. milk; NB, nuts v. bar; NP, nuts v. powder, MB, milk v. bar; MP, milk v. powder; BP, bar v. powder.
*Significant difference for food pairs at P,0·05 by Student’s paired t test of logged values if letters are shown.
Apparent bioavailability of isoflavones5
British Journal of Nutrition

Page 6

explanation for the variable AB of IFL after oral antibiotic
therapy(29)and may have also played a role in the findings
of the present study. Additional causes for variabilities are
intra-individual differences in IFL uptake depending for
example on hormonal, immunologic or other factors that
play a role during the complex steps of digestion. We tried
to address this issue by including repeated challenges with
the same soya food in a few of our subjects. More extensive
elaborations in this respect would have been desirable, but
could not be done owing to budget restrictions and would
need to be considered in future studies in order to optimise
the robustness of data.
In the present study, the UIE profiles changed significantly
between the time periods of urine collection investigated.
We therefore realise that urine collections of up to 24–26h
are necessary in order to obtain accurate values for AB of
IFL from soya foods. We observed generally no changes
UIE patterns between 24 and 26-h collection periods and
therefore assume that longer times of collection will not add
significantly more information for the unmetabolised IFL.
The reason for less significance between UIE patterns found
at 24 v. 26h are likely due to the lower number of participants
in the 24-h time period. An up to 8-h urine collection may give
an approximate ‘snapshot’ of AB, which, however, could
change over later times and should therefore not be used as
basis for final conclusions. Some of the strengths of the
present study include that subjects were highly compliant,
health professionals, the washout period of 1-week between
consumption of the different soya foods, utilisation of urinary
creatinine to confirm accurate urine collections and several
collections of urine during 26h in order to determine the
appropriate time frame needed to obtain conclusive results
about UIE profiles. Similar to others, we kept the background
diet consistent(35,41), an important factor since the food matrix
has been shown to have an effect of IFL absorption(57).
This eliminated possible confounding factors that would
have been present if participants were allowed to eat different
meals with each soya food. Additional strengths of the present
study were the examination of the participants’ diet history
that assured consistent and healthy dietary behaviours without
carnivorous or vegetarian extremes and lack of medication use
for at least 1 month before and during the study, particularly
antibiotics.
Although our sample size was relatively small and made up
primarily of women, we were able to demonstrate significant
differences in AB of IFL. The highly compliant nature of
the present study participants, mostly health professionals,
possibly contributed to our success. Further studies that
include a larger sample size, more equal sex distribution and
repeated challenges with the same soya food type are
needed to confirm the presented effect of soya food type
(liquid v. solid; processed v. native) on IFL bioavailability.
Acknowledgements
We thank the participants for their time and cooperation and
Physician’s Pharmaceuticals, Inc. for partial support of the
present study including the donation of soya foods. L. A. A.
was responsible for study coordination, urine collection and
assistance with data compilation and manuscript preparation,
K. K. for the urine analysis by LCMS, S. S. for the completion
of the manuscript, L. R. W. for statistical evaluations, B. M.
H. for the study design (she also acted as a consultant in
study performance and medical issues) and A. A. F. for the
overall supervision of all aspects of the study, securing of
research funds and manuscript preparation. We also acknowl-
edge the NIH for instrumentation support S10-RR020890
(there are no conflicts of interest).
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Apparent bioavailability of isoflavones7
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Appendix 1. Urinary isoflavone excretion adjusted for exposure to doses in soya nuts
(Mean values with their standard errors)
Nuts (mmol) Milk (mmol)Bar (mmol)Powder (mmol)
Mean
SE
Mean
SE
Mean
SE
Mean
SE
Significance†
Daidzein
baseline (mmol/h)‡
up to 2 h
up to 8 h
up to 24 h
up to 26 h
Genistein
baseline (mmol/h)‡
up to 2h
up to 8h
up to 24h
up to 26h
Glycitein
baseline (mmol/h)‡
up to 2h
up to 8h
up to 24h
up to 26h
Dihydrodaidzein
baseline (mmol/h)‡
up to 2h
up to 8 h
up to 24h
up to 26 h
Dihydrogenistein
baseline (mmol/h)‡
up to 2 h
up to 8h
up to 24h
up to 26h
O-desmethyl-angolensin
baseline (mmol/h)‡
up to 2h
up to 8h
up to 24h
up to 26h
Equol
baseline (mmol/h)‡
up to 2h
up to 8h
up to 24h
up to 26h
Total isoflavones
baseline (mmol/h)‡
up to 2h
up to 8h
up to 24h
up to 26h
0·07
1·63
9·65
16·11
16·60
0·02
0·26
1·22
1·51
1·37
0·05
3·59
12·99
19·62
20·37
0·02
0·37
1·20
2·06
2·23
0·03
2·52
13·34
20·49
20·42
0·01
0·30
1·00
1·84
1·54
0·02
2·35
11·98
18·02
17·28
0·01
0·26
1·24
1·66
1·40
NM*, NB, NP*, MB, MP
NM, NB*
NB
NB, MP, BP*
0·03
0·69
3·20
5·55
6·50
,0·01
0·13
0·46
1·11
0·90
0·03
1·75
5·36
8·41
9·88
0·01
0·36
0·75
1·36
1·86
0·02
0·72
3·58
5·94
6·45
0·01
0·08
0·38
1·02
1·02
0·01
0·65
3·03
5·15
4·91
,0·01
0·08
0·36
0·92
0·69
NM*, MB*, MP*
NM*, MB, MP*
NM, NP, MB, MP*
NM, NP, MB, MP*, BP
0·01
0·21
1·68
2·55
2·58
,0·01
0·05
0·42
0·46
0·38
0·01
0·32
1·46
2·26
2·44
,0·01
0·04
0·25
0·30
0·26
0·003
0·29
2·41
3·72
4·07
,0·01
0·04
0·78
0·90
0·88
0·004
0·32
2·28
3·16
3·15
,0·01
0·09
0·65
0·62
0·43
NM, NB, NP
MB
MB
MB
0·03
0·02
1·68
4·70
5·13
0·01
0·01
0·65
1·15
1·03
0·01
0·03
1·38
4·00
5·11
,0·01
0·01
0·42
0·81
1·05
0·01
0·02
1·32
4·39
5·66
,0·01
0·01
0·37
1·07
1·09
0·01
0·01
1·30
5·11
5·32
,0·01
,0·01
0·53
1·49
1·20
BP*
NM
0·01
0·03
0·41
1·50
1·77
,0·01
0·01
0·11
0·70
0·69
0·01
0·04
0·70
1·67
1·59
,0·01
0·01
0·45
0·87
0·73
0·01
0·04
0·52
1·12
1·56
,0·01
0·01
0·28
0·50
0·50
0·004
0·02
0·46
0·89
0·90
,0·01
0·01
0·27
0·31
0·26
BP*
0·02
0·02
0·81
3·98
3·74
,0·01
0·01
0·23
0·84
0·71
0·05
0·08
1·25
5·92
5·75
0·04
0·06
0·51
1·45
1·31
0·01
0·02
1·08
3·79
3·78
,0·01
0·01
0·43
0·91
0·76
0·03
0·02
0·50
2·13
2·58
0·02
0·01
0·22
0·53
0·43
NM, NP, MP*, BP
NM, MP, BP*
0·01
0·01
0·11
1·23
1·77
,0·01
0·01
0·06
0·89
0·92
0·003
0·01
0·49
1·52
1·65
,0·01
,0·01
0·42
1·00
0·96
0·005
0·01
0·64
2·20
2·02
,0·01
,0·01
0·58
1·35
1·07
0·003
,0·01
0·66
1·89
1·71
,0·01
,0·01
0·58
1·05
0·78
NP, BP
0·16
2·77
18·07
36·32
38·98
0·04
0·44
2·66
2·70
2·38
0·15
6·30
25·62
47·25
50·55
0·06
0·67
2·12
3·45
3·81
0·07
4·50
30·13
53·66
56·61
0·02
0·45
3·45
4·84
4·24
0·08
4·32
26·69
46·42
47·84
0·03
0·49
3·53
3·18
3·10
NM*, NB*, NP*, MB, MP
NM*, NB*, NP, BP
NM, NB*, NP, BP
NM, NB*, NP, BP
NM, nuts v. milk; NB, nuts v. bar; NP, nuts v. powder; MB, milk v. bar; MP, milk v. powder; BP, bar v. powder.
*P,0·008 (Bonferroni correction for multiple comparisons ¼ 0·05/6).
†Significant difference for food pairs at P,0·05 by Student’s paired t test of logged values if letters are shown.
‡Calculated from creatinine data (mg/h) owing to the lack of values for duration and volume of collected urine at baseline (spot urine).
A. A. Franke et al.8
British Journal of Nutrition