Am J Clin Nutr 2002;76:1422–7. Printed in USA. © 2002 American Society for Clinical Nutrition
Effects of prune consumption on the ratio of 2-hydroxyestrone to
Sidika E Kasim-Karakas, Rogelio U Almario, Laura Gregory, Heather Todd, Rodney Wong, and Bill L Lasley
Background: A higher urinary ratio of the biologically inactive
estrogen metabolite, 2-hydroxyestrone (2OHE1), to the biologi-
cally active metabolite, 16?-hydroxyestrone (16?OHE1), may be
associated with a lower risk of breast cancer. High fiber intake is
also associated with decreased breast cancer risk.
Objective: We investigated the effects of prunes, which are natu-
rally rich in both soluble and insoluble fiber, on the concentrations
of 2OHE1 and 16?OHE1 and on the ratio of 2OHE1 to 16?OHE1.
Design: Nineteen healthy premenopausal women consumed
their habitual diets for 3 menstrual cycles and then consumed
100 g prunes/d for the next 3 cycles. Concentrations of urinary
2OHE1 and 16?OHE1 were determined during the follicular and
Results: Prune supplementation increased total and soluble
fiber intakes by 4 and 2 g/d, respectively (P < 0.001). Mean
(± SEM) luteal 2OHE1 excretion decreased from 3.92 ± 0.79
to 2.20 ± 0.40 nmol/mmol creatinine during the third cycle
(P = 0.017). Luteal 16?OHE1 excretion decreased from 1.38 ± 0.24
to 0.87 ± 0.10 and 0.87 ± 0.15 nmol/mmol creatinine during the
first and third cycles, respectively (P = 0.018 for both values).
Follicular 16?OHE1 excretion decreased significantly only dur-
ing the first cycle (from 0.82 ± 0.12 to 0.45 ± 0.09 nmol/mmol
creatinine; P = 0.005). The 2OHE1-16?OHE1 ratio did not
change significantly after prune supplementation.
Conclusions: Prune supplementation significantly decreased the
excretion of 16?OHE1 during the follicular phase of the first men-
strual cycle and during the luteal phases of both the first and third
menstrual cycles. The 2OHE1-16?OHE1 ratio did not change
significantly. The potential significance of the decrease in
16?OHE1 excretion, without a change in the 2OHE1-16?OHE1
ratio, on the prevention of estrogen-dependent cancers remains to
Am J Clin Nutr 2002;76:1422–7.
Estrogen exposure is a well-recognized risk factor for breast
cancer (1–3). Research suggests that certain estrogen metabo-
lites may also confer a risk of breast cancer (4). Metabolism of
estrogens involves conversion of estradiol to estrone, which is
then hydroxylated through 2 competing pathways to either
16?-hydroxyestrone (16?OHE1) or 2-hydroxyestrone (2OHE1)
and 4-hydroxyestrone (4OHE1), which are catechol estrogens
(5–8). 16?OHE1 retains its biological activity and therefore is
considered a risk factor. Between the 2 catechol estrogens, 2OHE1
is the major metabolite. It is biologically inactive and may even
1From the Department of Internal Medicine, Division of Endocrinology,
Clinical Nutrition, and Vascular Medicine (SEK-K, RUA, and LG), the Institute
of Toxicology and Environmental Health (HT and BLL), and the Department
of Statistics (RW), University of California, Davis.
2Supported by grants (to SEK-K) from The California Prune Board,
Pleasanton, CA, and the ALSAM Foundation, Los Angeles.
3Reprints not available. Address correspondence to SE Kasim-Karakas,
Division of Endocrinology, University of California at Davis, 4150 V Street,
PSSB Suite G400, Sacramento, CA 95817. E-mail: email@example.com.
Received August 6, 2001.
Accepted for publication February 27, 2002.
have antiestrogenic activity. Thus, 2OHE1 does not promote estro-
gen-dependent cancers and may possibly protect against them.
Therefore, a high ratio of the inactive metabolite, 2OHE1, to the
active metabolite, 16?OHE1, is considered a favorable breast can-
cer risk profile (9, 10).
Several modes of intervention were used to try to increase the
ratio of 2OHE1 to 16?OHE1. Among these, exercise (11), bras-
sica vegetables (12), n?3 fish oils (10, 13), flax seed (14), and
indole-3 carbinol (15, 16) successfully increased the 2OHE1-
16?E1 ratio. However, there are conflicting reports about the
effects of soy protein and isoflavonols (17–19).
The effects of dietary fiber on the 2OHE1-16?OHE1 ratio have
not been conclusively established. High fiber intake is associated
with low estrogen concentrations in plasma and urine and high
concentrations in stool (20–30). Furthermore, this effect may be
independent of the fat content of the diet (31). Nevertheless,
dietary supplementation of insoluble fiber (cellulose and wheat
bran) did not change the 2OHE1-16?OHE1 ratio (10, 14).
Fruits and vegetables, which are natural sources of dietary fiber,
contain soluble as well as insoluble fiber. Animal experiments
show that soluble but not insoluble fiber has protective effects
against breast cancer (32, 33). In addition, phenolic compounds
and xenoestrogens in fruits and vegetables increase the 2OHE1-
16?OHE1 ratio by inducing the cytochrome P450 enzyme and
increasing the production of 2OHE1 (15, 16).
In this study we investigated the effects of prune intake on urinary
excretion of total estrogen conjugates, pregnanediol-3-glucuronide
(PdG), 2OHE1, and 16?OHE1 and on the urinary 2OHE1-
16?OHE1 ratio in healthy women with normal ovarian func-
tion. We hypothesized that prune intake may alter the metab-
olism of estrogens because prunes are a rich source of both
soluble and insoluble fiber and cinnamates (34, 35) and
decrease intestinal transit time (34). Because concentrations
of estrogen metabolites may be affected by the menstrual
by guest on June 9, 2013
EFFECTS OF PRUNE FIBER ON THE 2OHE1-16?OHE1 RATIO 1423
cycle phase (36), estrogen metabolite concentrations were meas-
ured during both the follicular and luteal phases.
SUBJECTS AND METHODS
Twenty-four healthy, premenopausal women with regular men-
strual cycles were recruited from the community. All of the
women signed an informed consent form that was approved by the
Human Subjects Committee of the University of California, Davis.
Exclusion criteria consisted of a habitual dietary intake of <30%
of energy from fat or >20 g fiber/d; habitual use of fiber supple-
ments (ie, psyllium) or laxatives; pregnancy; irregular menses; hir-
sutism; polycystic ovary syndrome; use of birth control pills dur-
ing the preceding 3 mo; intention to get pregnant during the time
period of the study; systemic illnesses, such as renal, hepatic, and
gastrointestinal illnesses; diabetes mellitus; hyperlipidemias;
hypertension that required medication; smoking; and an alcohol
intake of >2 drinks/wk.
The duration of the study was 6 mo. After consuming their
habitual diets for 3 menstrual cycles (control run-in period), the
participants replaced dietary simple sugars with 100 g (?12)
prunes/d for another 3 menstrual cycles (intervention period).
One-hundred grams of prunes contains 1004 kJ, 63 g carbohy-
drates, 2.6 g protein, <1 g fat, 7.2 g fiber (3.8 g soluble and 3.4 g
insoluble), and 78 mg phenolics (68 mg cinnamates). Prunes were
consumed either directly or by adding them to various food items
(salads, muffins, breads, cereals, and cakes). Intakes of energy,
total carbohydrate, fat, protein, and other macronutrients and
micronutrients were not changed.
Seven-day food records were obtained during the follicular
phase of each menstrual cycle and analyzed by using the updated
version of NUTRITION DATA SYSTEMS 93 (University of Min-
Body weight was measured monthly. At the beginning and the
end of the study, waist and hip circumferences and body compo-
sition were measured; the latter was measured by bioelectrical
impedance analysis (Biostat, Isle of Man, United Kingdom).
Because the results of bioelectrical impedance analysis may be
influenced by the water content of the body, these measurements
were obtained only during the follicular phase of the cycle (37).
Sex steroid hormones
Throughout the study (6 menstrual cycles) the participants col-
lected their first morning urine sample every day. The samples
for determinations of estrogen conjugates, PdG, and creatinine
were collected in prelabeled 10-mL tubes, frozen immediately,
stored in the participants’ freezers, and delivered to the labora-
tory once a month. The urine samples for the 2OHE1 and
16?OHE1 assays were collected in ascorbic acid (an antioxidant)
twice a month between the 5th and 8th days and the 19th and
23rd days of the menstrual cycle (follicular and luteal phases,
respectively). The samples were then kept in the refrigerator and
delivered on ice to the laboratory within 8 h of collection. All the
urine samples collected during the first and last menstrual cycles
of the control and prune supplementation periods (cycles 1, 3, 4,
and 6) were assayed for estrogen conjugates, PdG, 2OHE1, and
Estrogen conjugates and PdG were measured by using com-
petitive, microtiter solid-phase enzyme immunoassay methods
(38). Urinary estrogen conjugate and PdG concentrations corre-
late very closely with plasma estradiol and progesterone con-
centrations, with a 1–2-d delay (38). Daily measurements of uri-
nary estrogen conjugates and PdG provide more detailed
information about sex steroid exposure than do measurements of
plasma estradiol and progesterone once or twice a month (39).
Estrogen conjugate and PdG concentrations were indexed to the
creatinine concentration in the same urine sample. All of a sub-
ject’s urine samples obtained during a single menstrual cycle
were assayed in duplicate on the same plate. Urine samples in
which the creatinine concentration was <1.77 mmol/L were con-
sidered to be too dilute to yield accurate measurements, and these
samples were considered as missing. None of the participants had
more than 2 missing samples during a menstrual cycle. The sen-
sitivity of the estrogen conjugate assay was 2.5 nmol/L, and that
of the PdG assay was 0.48 ?mol/L. The intraassay CVs for the
high and low internal controls were 14.7% and 13.1%, respec-
tively, for estrogen conjugates and 15.6% and 12.9%, respec-
tively, for PdG. Cumulative estrogen and progesterone exposure
was assessed by calculating the area under the curve for estro-
gen conjugates and PdG during each menstrual cycle by using
the trapezoidal rule (40).
2OHE1 and 16?OHE1 metabolites were measured in triplicate
by using the ESTRAMET 2/16 kit (Immunacare, Bethlehem,
PA). The intraassay CV was 4%, and the interassay CV was 10%
Of the 24 subjects who began the study, 5 dropped out because
of either relocation (n = 2) or the inconvenience of daily urine
collections (n = 3). Nineteen subjects with a mean (±SE) age of
40 ± 1 y provided at least 95% of the daily urine samples during
the 6-mo study. Data from these subjects were analyzed by using
the MIXED and CORRELATIONS procedures of SAS for TSO40
release 6.12 (43, 44). The participants served as their own con-
trols. Data from the control months were averaged. Data from the
intervention period were not averaged because the duration of
intervention may affect hormonal response independently.
Repeated-measures analysis of variance with an unstructured
covariance matrix was used. Contrasts between the average of 2
control values and values from either the first or the last month of
the study were used to test the significance of the early or late
effects of the intervention. To adjust for the multiple comparisons
(early and late changes from the baseline), the step-up Bonferroni
procedure was used (45). A two-factor analysis of variance with
time (baseline, early, and late), stage (follicular or luteal), and the
interaction of time with stage was used to determine the signifi-
cance of the interaction effects. The step-up Bonferroni procedure
was used to determine which interaction was significant. To eval-
uate the relations between the change in fiber intake and estrogen
metabolites, partial correlations were computed after accounting
for the change in fat intake.
by guest on June 9, 2013
1424KASIM-KARAKAS ET AL
Dietary macronutrient intakes before and after prune supplementation1
(% of energy)
(% of energy)
1x–± SEM; n = 19. t0, t1, and t2, before and 1 and 3 mo after prune sup-
2–4Significantly different from t0:2P < 0.05,3P < 0.01,4P < 0.001.
7.79 ± 0.437.86 ± 0.41 7.47 ± 0.26
64 ± 6
31 ± 2
62 ± 6
29 ± 2
57 ± 52
28 ± 22
20 ± 2
7 ± 1
24 ± 23
9 ± 0.44
24 ± 23
9 ± 0.44
255 ± 16
55 ± 2
111 ± 8
45 ± 6
25 ± 2
24 ± 2
74 ± 5
274 ± 14
59 ± 23
107 ± 9
36 ± 4
41 ± 24
29 ± 22
70 ± 4
266 ± 12
60 ± 23
101 ± 8
35 ± 42
40 ± 24
28 ± 22
67 ± 42
Comparison of the values obtained at the end of the study (sixth mo)
with those obtained at the beginning of the control period showed
that weight (65.2 ± 2.1 compared with 64.3 ± 2.0 kg), body mass
index (in kg/m2; 23.9 ± 1.5 compared with 23.8 ± 0.8), percentage
of body fat (29.6 ± 1.5% compared with 28.9 ± 1.5%), and waist-
to-hip ratio (0.76 ± 0.02 versus 0.76 ± 0.01) did not change.
These changes are shown in Table 1. Total energy intake did
not change significantly. Despite the instructions to keep
dietary fat constant, the percentage of energy from fat
decreased significantly during the third month of prune sup-
plementation (from 31% to 28%; P = 0.011). Simultaneously,
the percentage of energy from carbohydrates increased from
55% to 60% (P = 0.002). Total fiber intake increased from 20 to
24 g, and soluble fiber intake increased from 7 to 9 g. These
increases were significant (P < 0.004). As could be predicted from
the carbohydrate content of prunes, although sucrose intake
decreased significantly, intakes of glucose and fructose increased
significantly. Protein intake also decreased significantly during
the third month of prune supplementation.
Changes in urinary estrogen and progesterone metabolite
Changes in the urinary excretion of estrogen conjugates, PdG,
2OHE1, and 16?OHE1 are shown in Table 2. Prune ingestion did
not significantly alter the urinary excretion of either estrogen con-
jugates or PdG.
When the excretions of 2OHE1 and 16?OHE1 were com-
pared between the follicular and luteal phases of the menstrual
cycle, 16?OHE1 excretion during the luteal phase was signifi-
cantly higher than that during the follicular phase both at base-
line (1.38 ± 0.24 compared with 0.82 ± 0.12; P = 0.01) and dur-
ing the early prune supplementation period (0.87 ± 0.10 compared
with 0.45 ± 0.09; P = 0.0002). Luteal 2OHE1 excretion was
significantly higher than follicular 2OHE1 excretion only during
the early prune supplementation period (2.52 ± 0.35 compared with
1.67 ± 0.20; P = 0.0035). The urinary 2OHE1-16?OHE1 ratio did
not differ significantly between the luteal and follicular phases.
When the changes in urinary estrogen metabolite concentra-
tions from baseline in the early and late periods of prune supple-
mentation were compared, urinary excretion of 16?OHE1
decreased during the luteal phase in both the first and third months
of supplementation (concentrations at baseline and during months
1 and 3 were 1.38 ± 0.24, 0.87 ± 0.10, and 0.87 ± 0.15 nmol/mmol
creatinine, respectively; P = 0.018 for both periods) and during
the follicular phase in the first month of supplementation (from
0.82 ± 0.12 to 0.45 ± 0.09 nmol/mmol creatinine; P = 0.005).
2OHE1 concentrations decreased during the luteal phase in the third
month of supplementation (from 3.92±0.79 to 2.20± 0.40nmol/mmol
creatinine; P = 0.017). These results indicate that prune supple-
mentation altered estrogen metabolism more consistently during
the luteal phase of the menstrual cycle than during the follicular
phase. Although the actual concentrations of 2OHE1 and
16?OHE1 changed significantly, no significant changes were
observed in the 2OHE1-16?OHE1 ratio.
Although this study was designed to maintain a stable fat intake,
dietary fat decreased significantly during the third month. To assess
the relations between the changes in fiber intake and the changes in
correlations were computed after accounting for the changes in fat
intake. During the first month of the study, the change in fiber intake
tended to correlate inversely with the change in follicular 16?OHE1
concentration (r = ?0.529, P = 0.052) and correlated directly with
the follicular 2OHE1-16?OHE1 ratio (r = 0.587, P = 0.027).
Because conventional estrogens account for <50% of circulat-
ing estrogens (46), it is possible that biologically active estrogen
metabolites also confer a significant risk of estrogen-dependent
cancers. This was originally proposed almost 20 y ago as the
“unconventional estrogen hypothesis” (47). In support of this, sev-
eral studies showed a relation between the incidence of breast can-
cer and a low 2OHE1-16?OHE1 ratio (9, 10, 48).
In the present study we investigated the effects of a natural food
that provides both soluble and insoluble fiber. Earlier studies
showed that the amount of prunes used in the present study also
increases stool volume and decreases intestinal transit time (34).
Because estrogen metabolites are subject to enterohepatic circu-
lation (28–30), we postulated that prune supplementation may
increase excretion of estrogen metabolites in the stool and there-
fore decrease them in the plasma and urine. Consistent with our
hypothesis, urinary 16?OHE1 concentrations during the first
month of prune supplementation decreased significantly during
both the luteal and follicular phases of the menstrual cycle. After
3 mo, the decrease in urinary 16?OHE1 persisted only during the
luteal phase. Urinary 2OHE1 concentrations also decreased signi-
ficantly during the luteal phase in the third month of prune sup-
plementation. The persistence of the decreased 16?OHE1 excre-
tion during the luteal phase but not during the follicular phase may
be related to the variance in their concentrations during a men-
strual cycle. In agreement with a previous report (36), the
16?OHE1 concentrations of the subjects at baseline were higher
during the luteal phase than during the follicular phase. Prune sup-
plementation probably increased the intestinal clearance of
by guest on June 9, 2013
EFFECTS OF PRUNE FIBER ON THE 2OHE1-16?OHE1 RATIO 1425
16?OHE1 throughout the menstrual cycle. This may have caused
a compensatory increase in estrogen metabolism. This increase
may have been adequate to negate the effects of prune supple-
mentation during the follicular phase when estrogen concentra-
tions are low. However, during the luteal phase, when plasma
estrogen concentrations are higher, this compensatory increase
may not have been adequate to restore urinary excretion of the
estrogen metabolites to their baseline values.
Despite the decreases observed in 16?OHE1 and 2OHE1 excre-
tion, there was no significant change in excretion of total estro-
gen conjugates. This may be because 2OHE1 and 16?OHE1
account for only 10–40% of total estrogen metabolites (46).
Despite the changes in the urinary concentrations of 2OHE1
and 16?OHE1, there was no significant change in the 2OHE1-
16?OHE1 ratio. The lack of an increase in the 2OHE1-16?OHE1
ratio was due to the proportional changes in both metabolites. An
increase in the 2OHE1-16?OHE1 ratio requires preferential
hydroxylation of the A ring of estrone instead of the D ring. All
the intervention studies to date showed that dietary fiber does not
affect the relative efficiency of these 2 competing hydroxylation
pathways (10, 14, 20, 21). Studies using insoluble fiber did not
even show a decrease in the urinary excretion of either 2OHE1
or 16?OHE1 (9, 10). In the present study we were able to show
decreases in the excretion of both of these metabolites possibly
because we used a food item that contains soluble fiber and
accelerates intestinal transit. Although our intervention was
designed to increase fiber intake by 7 g/d, we achieved only a
4-g/d increase, which was divided equally between soluble and
insoluble fiber. Our subjects had a relatively high baseline fiber
intake (20 g/d) and apparently reduced their fiber intake from
Although our intervention was designed to keep dietary fat
unchanged, we observed a gradual decrease in fat intake. Most of
the interventions that showed decreased estrogen concentrations
with increased fiber intake simultaneously reduced the fat intake
(22–27). Furthermore, cross-sectional data showed that ratios of
dietary fat to total fiber and to soluble fiber correlate directly with
2OHE1-16?OHE1 ratios (49). However, other research provides
evidence that dietary fiber may have independent effects on estro-
gen metabolism (31). In our study, changes in estrogen metabo-
lite concentrations preceded the decrease in dietary fat intake. In
addition, during the third month of the study when the decrease
in dietary fat became significant, estrogen metabolite concentra-
tions did not decrease any further. Finally, during the first month
of prune supplementation, the increase in fiber intake correlated
with the decrease in follicular 16?OHE1 concentrations and with
the increase in the 2OHE1-16?OHE1 ratio independent of the
changes in fat intake. These observations suggest that the changes
in estrogen metabolite concentrations were not due to decreased
fat intake. One caveat remains, however: we did not determine fat
absorption and thus cannot rule out the possibility that prune
intake may have decreased the intestinal absorption of fat.
Certain foods that contain phenolics, indole glucosinolate, or
lignans (ie, brassica vegetables and flax seed) seem to shift estro-
gen metabolism in favor of 2-hydroxylation and increase the
2OHE1-16?OHE1 ratio. Prunes are a rich source of cinnamates
(35). Clearly, cinnamate intake did not influence these hydroxy-
lation pathways. It should be noted, however, that in some studies
the diet-induced increase in the 2OHE1-16?OHE1 ratio was very
small (12), and whether an increase of this magnitude provides
protection against breast cancer is not clear. In addition, hydrox-
ylation of the A ring also results in the production of another cat-
echol estrogen (4-hydroxyestrone) with carcinogenic potential
(6–8). Thus, the overall consequence of inducing the hydroxyla-
tion of the A ring is not yet known.
Another puzzling finding of the clinical studies on the relation
between breast cancer and the 2OHE1-16?OHE1 ratio is the vari-
ability of this relation. For example, in one study the 2OHE1-
16?OHE1 ratio related to breast cancer risk directly in pre-
menopausal women but inversely in postmenopausal women (50).
Importantly, in the same study, urinary concentrations of both
2OHE1 and 16?OHE1 correlated directly with breast cancer
risk regardless of menopausal status. In another study, the
2OHE1-16?OHE1 ratio was related to breast cancer risk only in
postmenopausal women (48). Yet, in 2 other reports in post-
menopausal women, breast cancer risk was not related to the
2OHE1-16?OHE1 ratio but was directly related to urinary con-
centrations of estrone, estradiol, and estriol (51, 52). Thus, it is
not yet clear whether the concentrations of the individual estrogen
metabolites or the 2OHE1-16?OHE1 ratio is a better predictor of
breast cancer risk. If the former is a better predictor, the dietary
intervention that we used may have potential preventive value.
Urinary excretion of estrogen conjugates (EC), pregnanediol-3-glucuronide (PdG), 2-hydroxyestrone (2OHE1), and 16?-hydroxyestrone (16?OHE1) and
the ratio of 2OHE1 to 16?OHE1 before and after prune supplementation1
2OHE1 (nmol/mmol creatinine)
16?OHE1 (nmol/mmol creatinine)
1x–± SEM; n = 19. AUC, area under the curve for one menstrual cycle; t0, t1, and t2, before and 1 and 3 mo after prune supplementation, respectively.
2Significantly different from the follicular phase, P < 0.05, after a significant interaction of time and menstrual phase was found by using two-factor
ANOVA and adjusting for multiple comparisons with the step-up Bonferroni procedure.
3,4Significantly different from t0:3P ≤ 0.018,4P = 0.005.
577 ± 65
2520 ± 256
601 ± 72
2221 ± 197
543 ± 51
1943 ± 161
2.20 ± 0.27
3.92 ± 0.79
1.67 ± 0.20
2.52 ± 0.352
2.03 ± 0.47
2.20 ± 0.403
0.82 ± 0.12
1.38 ± 0.242
0.45 ± 0.094
0.87 ± 0.102,3
0.76 ± 0.14
0.87 ± 0.153
2.83 ± 0.29
3.21 ± 0.48
7.61 ± 2.00
3.00 ± 0.29
3.44 ± 0.67
3.31 ± 0.63
by guest on June 9, 2013
1426KASIM-KARAKAS ET AL
Because our intervention was most effective during the luteal
phase, we speculate that premenopausal women may benefit more
than would postmenopausal women. However, if the 2OHE1-
16?OHE1 ratio is a better predictor, then our intervention is not
likely to be beneficial.
In conclusion, we showed that a natural food item that contains
both soluble and insoluble fiber and accelerates intestinal transit
reduces the urinary excretion of both 2OHE1 and 16?OHE1, espe-
cially during the luteal phase of the menstrual cycle, in healthy
premenopausal women. We observed this effect even with a rela-
tively high preintervention fiber intake and with relatively low
fiber supplementation. Whether such an intervention would be
more effective in women with lower habitual fiber intakes remains
to be seen. The potential clinical significance of the decreased
concentrations of individual estrogen metabolites without a
change in the 2OHE1-16?OHE1 ratio needs to be determined in
large prospective studies.
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