Urinary excretion of lignans and isoflavonoid in Japanese men and women consuming a traditional Japanese diet14

phytoestrogens

Herman Takeshi

H#{228}m#{228}l#{228}inen,

Adlercreutz, Hasegawa,

ABSTRACT in hormone-dependent

Hideo Honjo, Akane and Hiroji Okada

Epidemiologic cancer

consuming

a traditional

diphenolic

food

arc converted

lignans

equol,

and

and 9 men in a rural

subjects

consumed

and

very

high.

The

cancer

ofJapancse

be due to the high Nutr

KEY WORDS methylangolensin,

much

and

10

Japan.

The

rice and

soy

correlated

men,

respectively,

diet, urine, daidzcin,

soybean,

gas

chromatography,

sex-hormone-binding

compounds

is highest

patients

excretion

correlates

17, 18). Japanese

women

consuming

have

low breast-cancer

larly,

Japanese

although

spec-

lignans

in all studied are diphenolic

and

animal

isoflavonoid and

phytoestrogens,

human

compounds

with

biological

croflora (6-8). Most ofthc mononetin, matairesinol, low

in urine

activities,

oflignans

and

men

women

and

production activity

mortality

with that

(2,

in Hawaii Japanese

(21-24). prostate

the incidence

diet

Simicancer, of pros-

Western men arc similar (25-27). dependent and could potentially

by alterations

report

was

isoflavonoid

consuming

published

of sex-hormone

metabolism

phytoestrogens

in groups

a traditional

as an abstract

diet.

of Japanese

A preliminary

(28).

and methods

but

have

antiestrogenic

The subjects in

in plants

seem

ofsex-hormonc-binding and of the

Am J C/in Nuir

may

in this

sex hormones.

l991;54:l093-1

globulin way

significantly

arc

by intestinal

mi-

such as foroccur only in inves-

shown

many

(1-3);

antiviral

The

higher

100. Printed

(SHBG)

other (11,

in the liver

influence SHBG

in USA.

values

healthy

and

were

participating recruited

in this investigation in a small

rural

village

were apparently south

of Kyoto,

to

compounds

12); and antiproliferative, cytotoxic, and growth-inhibiting effects (3, 13-15). Studies indicate that they most likely stimulate the (2, 14-18)

mortality

their

food

origin

traditional

found

that

offiber-rich

similar

(9, 10). All compounds

estrogenic

producing

and

weights

original plant aglycones, and secoisolariciresinol,

concentrations so far are weakly

biological

in omnivores

caused by lignans and isoflavonoids or by a direct effect of these compounds on their growth. Because ofthe associations between diet and these diseases, we decided to study the urinary excretion

occur-

fluids

molecular

to those of steroid estrogens (1-3). Precursors occur as glycosides (4, 5), and the mammalian produced from plant lignans and isoflavonoids

very

low have

(14).

Participants

Mammalian

tigated

lower

of Japanese

and

of these

protein

breast cancer and excretion of these

the intake

to the original

have

studies

both

and

incidence

men

autopsy

effect

of the

(2, 18, 20). It was shown

women

similar

to the

women with that urinary

with

and

a diet

due synthesis

in vegetarians

breast-cancer

Subjects

feces,

liver

globulin

Introduction

ring

probably

on

be influenced

0-desmass

are

may

isoflavonoids,

genistein,

17-19)

tate cancer in Japanese and These cancers arc sex-hormone

Am J C/in

lignans,

equol,

(2,

compounds

and

in breast

products.

vegetarians

urinary

100.

enterodiol,

trometry,

of Kyoto, with

Esa

diphenolic

and

in

low mortality

of soybean

Japanese,

enterolactonc,

by

agents. Therefore, (enterolactone,

ofisoflavonoids

The women

intake

199l;54:1093-1

diet

excretion

intake.

which

substances

Fotsis,

Studies in both young and old in various dietary groups indicate

An isotope-dilution gas chromethod was used for the assays. was low but that of the isofla-

oflignans

soybean-product

certain

isoflavonoids,

south

low-fat

spectrometric

vonoids

mortality and men

O-desmethylangolensin)

vegetables.

excretion

prostate

and

Theodore

that

hormone-like

village

a typical

The urinary was

found

may be cancer-protective excretion of these compounds

daidzein,

products, fish, matographic-mass

previously

active

women

with

We

components,

intestinal microflora, we studied urinary enterodiol,

studies revealed low in Japanese women

diet.

to biologically

Higashi,

biological seen

in

© 1991 American

Downloaded from https://academic.oup.com/ajcn/article-abstract/54/6/1093/4715076 by University of Glasgow user on 03 April 2018

I From the Department ofClinical Chemistry, University of Helsinki, Meilahti Hospital, Helsinki, Finland, and the Departments of Obstetrics and Gynecology and Preventive Medicine and the Laboratory of Gas Chromatography-Mass Spectrometry, Kyoto Prefectural University of Medicine, Kyoto, Japan. 2 Preliminary report published as an abstract. 3 Supported by Sigrid Jus#{233}lius and Finnish Cancer Foundations and the Medical Research Council of the Academy of Finland. 4 Address reprint requests to H Adlercreutz, Department of Clinical Chemistry, University of Helsinki, Meilahti Hospital, SF-00290 Helsinki, Finland. Received January 4, 1991. Accepted for publication April 17, 1991.

Society

for Clinical

Nutrition

1093

ADLERCREUTZ

1094 Japan.

Two

(blood

pressure

146/96

were

and

found

180/ 100,

to have

hypertension

respectively).

Most

and

dropped

because

his urine

volume

was

not

known.

urine

of the

were farmers cultivating tea and rice. Originally 10 women volunteered for the study, but 1 man

participants

men

of the women

10 was

Their

main their own

work

ET

AL

saved

for the purpose

scribed. Protection

of the

was in agriculture and they consumed mainly products. The ages of the men and women were 50.4 ± 18.0 and 46.8 ± 1 1.5 y, respectively. Height, weight, and body mass index [BMI, in weight (kg)/height (m)2] were, respectively, 160.8

essary

± 7.8

adex (Pharmacia the acetate form

cm,

58.6

± 6.5 cm,

were

± 5.8

kg,

and

22.7

± 2.3

for men

and

± 7.2 kg, and 22.6 ± 3.5 for women. 15% of normal weight.

All subjects

52.9

within

153.1

only

cartridge

(Waters

described hydrolysis

(33,

Urine

of 0.5

ascorbic tion.

acid.

The

for 48 h in plastic

bottle

was

kept

bottles

in a cool

place

The

urine was mixed and measured as soon as possible and transported

during

and

frozen for analysis. Dietary

containing

2 g

to Finland

was

in dry ice

The study was carried out in October 1985. Before the survey a nutritionist explained how to weigh the food components and how to write down the results on a form. Most of the food was weighed. Some food, such as bread and milk, was recorded as a piece of bread or cup of milk and the nutritionist estimated the weight of these food items afterwards. Food intake was recorded for 3 d and the nutritionist followed all subjects every day during the survey period. Calculation of the food data was made by an experienced nutritionist using the Standard Tables of Food Composition in Japan (29); for fiber calculations the Food Composition Tables ofDietary Fibers, Minerals, Cholesterol, Fatty Acids was used (30). The amount of soy sauce in the diet was calculated from the total sodium chloride content ofthe urine. According to earlier studies Japanese obtain 25.8% oftheir sodium chloride from soy sauce (31). Soy sauce contains 15% NaCl.

The

the following sauce

This is the in Japanese

their

consumption

of soy sauce

is estimated

by using

formula: =

(amount

traditional subjects

of NaCl

in urine)

way to estimate because they do

X 0.258/0.15

soy sauce consumption not add any other salt

It is an estimate and not an exact figure were not included in the correlation analyses.

and

food.

values

Analytical

to the

‘y-butyrolactone;

enterodiol

(End),

methyl]-butane-l,4-diol; daidzein equol (Eci), 4’,7-dihydroxyisoflavan;

Dma), The mining tography

and

estrogen

profile

capillary

of very

low concentrations

by ion-exchange

gas chromatography-mass

in the selected ion-monitoring (32-34). Originally, estrogens

mode (GC-MS-SIM, also were determined

of some

of inhibitors

chromatography

X 5 cm).

For

were

-Eq,

X 5 cm);

C18

out as of the enzyme carried

on a DEAE-Seph-

hydrolysis

and

added

purification

of the

last fraction obtained from the following deuterated

to the

cluate:

d-Enl

and

-End,

and d5-O-Dma

and

elution

ofthe

estrogens,

lignans,

and

sion (0.5 X 3 cm instead of 0.5 X 2.5 cm). Elution phenols was carried out as described and this fraction

Eq with

of the

di-

contains

the isoflavan Eq and the two lignans Ent and End. The two fractions containing lignans and isoflavonoid phytoestrogens and their deutcrated internal standards arc converted to their

trimethylsilyl

ether

by GC/MS by using 386/390; Da, 398/402 448;

out

the

(TMS)

derivatives

(32)

following ion pairs (and 383/387); End,

and

quantified

(mass/charge): 410/416; Enl,

Eq, 442/

and 0-Dma, 459/464 with a Hewlett-Packard

instrument

(36). The measurements were carried 5995 B GC/MS (Avondale, PA) with a Pascal work station and with an

equipped

automatic

injector.

Urinary excretion of < 0.0025 zmol/d and between 0.0025 and 0.005 Mmol/d regarded as semiquantitative. The mean

cannot be measured, the method must be values and interassay

imprecision

sample,

for the

control

pooled-urine

measured

59

times in single assays during 1 y, were as follows Enl, 3.65 mol/d (CV 7.4%); End, 0.364 zmol/d (CV 1 1.6%); and Eq, 0.042 imol/ d (CV 9.4%). For Da interassay imprecision concentrations

at a concentration is 1 1 .0% (n = in this

study,

of 0.028 mol/d, 14) and for 0-Dma

the

interassay

the at the

imprecision

The samples were analyzed in two batches and the values the control sample were almost identical both times and same

is

(CV).

as in analyses

Statistical

(Da), 4’,7-dihydroxyisoflavone; O-desmethylangolensin

in urine

were

before

and

after

these

two

for the

batches.

2,3-bis[(3-hydroxyphenyl)-

l2,4ihydroxyphenyl).2-(4-hydroxyphenyl)-propan-1-one. method used was a modification of a method the

removal

MA)

(nec-

a Sep-Pak

Fine Chemicals, Uppsala, Sweden) column in was done in a smaller column (0.5 X 3 cm

standards

8-10%

The trivial and systematic names ofthe compounds measured and discussed are as follows [structures were shown previously (3)]: enterolactone (Eni), trans- 2,3-bis[(3-hydroxyphenyl)methylj-

Milford,

with

4 mL methanol as described. The modification in this step is that 0-Dma and Da are cluted after this with 4 mL 0.2 mol acetic acid/L in methanol. This fraction is then, after evaporation ofthe solvent, ready for derivatization (trimethylsilyl ethers) and GC/MS. Selective fractionation of estrogens with vicinal cishydroxyls was carried out in a borate column with new dimen-

high

method

by cthoximation

evaporation ofthe DEAE-Sephadex column,

and

could

(35, 36). This was followed by hydrolysis and Sep-Pak extraction; application ofthe methanolic extract directly on the QAE-Sephadex A-25 in the acetate form (0.5

data

Soy

internal

d4-Da

collec-

a sample

Associates,

The

the analyses

extraction

before

the above

was collected

functions and

by ion-exchange

instead

of samples

carbonyl

34).

and

only the lignan and isoflavonoid modifications of the method are de-

for the estrogens),

hydrolysatc,

Collection

was too small

not be repeated. Therefore, values arc presented. Only

fractions,

(0-

the lignan

for deterchroma-

spectrometry

the amount

data arc presented as arithmetic means (±SD) and and phytoestrogen results as arithmetic means (±SD)

and geometric

means.

essary

ofskewness

because

statistical

or GC/MS) but because

methods

The food

gram

analyses

for Macintosh

gree of univariate of

Downloaded from https://academic.oup.com/ajcn/article-abstract/54/6/1093/4715076 by University of Glasgow user on 03 April 2018

timated

as Pearson’s

Geometric

were

ofthe carried

(Abacus

associations correlation

means

were

distribution

out by using Concepts,

between

used when ofthe results.

nec-

the Stat View

pro-

Berkeley,

CA).

The

two variables were coefficients (r). The pairs

The de-

esof

DIET TABLE 1 Intake of various food stuffs by the Japanese consuming a traditional Japanese diet5

AND

women

PHYTOESTROGENS

and men

Women (n = 10)

Nutrient

TABLE

3

Dietary

intake

Men (n

1095

IN JAPANESE of soy products

by the two groups

studied

Women (n = 10)

Soy product

Men (n

9)

=

9)

=

g/d g/d Rice Wheat Potato Sugar Fats Pulses and beans Fruit Green and yellow Other vegetables Pickles Algae Fish Meat Eggs Milk Beer S

578.5

222.5 59.5 ± 46.0 62.6 ± 30.2 8.1 ± 7.0 13.1 ± 7.6 56.5 ± 36.0 228.2± 111.9 60.6 ± 33.3 139.3 ± 69.3 32.9 ± 24.9 1.8±2.0 98.7 ± 46.6 37.0 ± 30.1 38.4 ± 16.6 1 12.7 ± 131.0 5.1 ± 16.1

vegetables

±

764.7 ± 240.3 139.0 ± 113.6 55.2 ± 34.6 8.1 ± 7.4 12.7 ± 6.9 40.9 ± 32.0 146.9± 114.0 55.7 ± 35.2 130.9 ± 77.2 23.2 ± 21.2 0.7 ±0.7 1 13.6 ± 56.5 73.6 ± 58.4 57.4 ± 30.6 90.9 ± 90.2 454.6 ± 647.1

SD.

adjusted group means men) were compared

for the two by nonpaired

groups t test.

studied

(women

and

Results The

Table TABLE

intake

ofvarious

types

2 shows the results

ofthe

offood

are shown

calculations

with

in Table regard

1, and

to energy;

2 intake

of various

nutrients,

and some

Women (n = 10)

Nutrient

ratios

in the two

Men (n

=

9)

Energy

(MJ/d) (kcal/d) Animal protein (g/d) Vegetable protein (g/d) Total protein (g/d) Carbohydrates (g/d) Total fat (g/d) Total fiber (g/d) Animal protein (%)t Proteins(%) Carbohydrates (%) Fats(%) Fat (g/kg body wt)

8.29

±

1.64

1973 ± 391

35.3 ± 38.2 ± 73.6 ± 31 1.4 ± 44.4 ± 16.9 ± 47.2 ± 15.2±2.1 64.6 ± 20.3±5.5 0.86 ±

13.9 10. 1 12.2 77.0 14.4 4.9 15.9 6.8

10.79

±

2569

± 829

3.48

47.8 ± 18.9 45. 1 ± 10.6 93.0 ± 28.4 383.3 ± 100.6 51.0 ± 25.9 15.3 ± 6.0 49.8 ± 7.9 14.6± 1.5 68.2 ± 5.1

wt)

0.31

± 0.37

2.1 ± 0.7 8.8 ± 3.0 0.33 ± 0. 10 2.5 ± 0.9

1.5 ± 6.4 ± 0.26 ± 2.4 ±

0.7 3.0 0.09 0.9

SD.

t Percent f Percent

18.7 ± 28.8 8.5 ±6.4 3.7 ± 4.2 0.8 ± 2.3

0.07 0.9 6.5 19.2 39.2

0.2 2.8 7.8 4.7 36.4

± ± ± ± ±

animal and vegetable and fiber; percentage

and carbohydrate more,

protein; animal

and

we calculated

total proteins, protein and

fat as percent the

fat

intake

carbohydrates, percentage

of total per

calories.

kilogram

fats, protein;

Further-

body

weight,

fiber intake 2). The diet

per J (per 1000 kcal), and the fat-fiber ratio (Table was a low-fat (fat 17.2% and 20.3% oftotal calories

for men

women,

and

respectively),

low-animal-protein

moderate amounts of fiber and a low fat-fiber typical for the traditional Japanese diet (37).

Table expected

3 shows the dietary intake to be the most important

urinary

isoflavonoids

Table

4 shows

enterodiol,

and

daidzcin, equol, 0.22, 0.10, 0.03, respectively

TABLE Urinary Japanese

with

which

is

ofsoy products, which were source of precursors for the

(3).

the mean

excretion

values

phytoestrogens. of cntcrolactonc,

a very

high

excretion

mean values and in Boston and and

for the two lignans

The results show a normal excretion

trogens. The individual results showed larly for equol (from 0 to 10.95 mol/d). that the geometric living in Helsinki

diet ratio,

of isoflavonoid

0-desmethyl-angolensin 2.05, 0.28, 0.32,

phytoes-

large variation, For comparison

in young omnivorous for enterolactone, 0.07,

a relfor

particunote

women enterodiol,

were 2.46, 0.20, and 0.03 tmol/d,

(2).

4 excretion women

of lignans and isoflavonoid phytoestrogens and men consuming traditional Japanese Women (n = 10)

Compound

in diet Men (n

=

9)

mol/d

17.2 ±4.9

0.85

Fiber

(mg/J) (g/1000 kcal) Fiber (g/kg body Fat-fiberratio

25.0 ± 22.9 12.5±6.2 2.6 ± 3.6 4.0 ± 12.7 0.37 ± 0.78 2.4 ± 4.5 7.7 ± 17.8 22.9 ± 6.1 54.4 ± 34.3

SD.

S

and three isoflavonoid atively low excretion

Energy intake, study groups5

S

Tofu (soybean curd) Miso(beanpaste) Aburaage (fried thin tofu) Atuage (fried thick tofu) Koridofu (dried soybean curd) Fermented soybeans Boiled beans Soy sauce Soy products (sauce excluded)

of total protein. of energy.

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Enterolactone Enterodiol Total lignans Daidzein Equol O-desmethylangolensin Total isoflavonoids Total diphenols S

SD (geometric

1.4 0.7 2.1 2.6 2.6 0.7 6.9 9.1

1).

± ± ± ± ± ± ± ±

1.4 (0.89) 1.3 (0.41) 2.6 (1.38) 4.0 (2.55) 4.0 (0.56) 0.6 (0.5 1) 6.8 (4.73) 9.3 (6.7)

1. 1 ± 0.4 ± 1.5 ± 2.2 ± 3.0 ± 0.2 ± 3.9 ± 5.4 ±

0.7 0.3 0.9 2.0 4.6 0.3 3.3 4.0

(0.89) (0.22) (1.13) (1.45) (0.54) (0. 1 1) (2.57) (4.1)

1096

ADLERCREUTZ

Table

S presents

a correlation

ponents

and

urinary

excretion

the total toestrogen

material of 19 subjects data were available.

matrix

of various

of lignans

and

for whom

both

in

Discussion In a previous study oforiental immigrant women from southAsia residing in Hawaii (38), the diet was similar to that consumed by the men and women in the rural village in Japan. In the present study the women had a greater energy intake (an additional ‘-2.l MJ/d, or 500 kcal/d), which may be due to a physically more active life. However, the percentage intake of calories as fat and the dietary fiber and fat-fiber ratio were very similar to the corresponding values in the previous study. Except for the energy intake the values are very different from those seen in Western societies where the fiber intake is similar but the fat-fiber ratio is much higher. Women living in the Boston area had a fat-fiber ratio of 7.7 for the premenopausal women and 4.6 for the postmenopausal women compared with 2.5 for the women in the present study (39). With regard to protein intake, expressed as g/d and as percentage of calories, the mean values in the present study were cast

similar

and

slightly

lower,

respectively,

than

those

of the

im-

5 and

6 g/d)

in the

women

in the

earlier

study

with

which

of

show

a mean

dietary

fiber

16 g/d. This latter value the value of 1 3 g/d for nonstarch

analyses

ofthe

Japanese

diet

intake

is also

‘

in another

study

(40).

investigation,

of dietary

fiber

it may be

in a traditional

ori-

men

(20).

We

yellow

and

women

(Tables

4 and

for the postmenopausal found

a weak

1 A) and

breast-cancer

correlation

was

between

intake

vegetables

which

thus

also

may

be a source

same

in Boston

ofgreen

and excretion of Enl and total 5) but no correlation with rice intake. Because consumed large amounts of rice, it seems justified that refined rice contains very low amounts, if precursors. There was a better correlation with soybeans,

the

patients

and

lignans (Table these subjects to conclude any, of lignan the intake of

of Enl

precursors

(Table 5). It is known that soy sauce contains coniferyl alcohol the building block for lignans and lignin (41). The excretion of the lignan End was also found to be associated with the intake of beans

and

pulses

The excretion in these tamed

Japanese

products

and

living

18). The

more

soy

in general

isoflavonoid

men

in women (2,

and

ofthc

Japanese

Da and

women

in Boston women

20-30

(Table

phytoestrogens compared (2, 20) in the

times

with and

present

more

5).

is very high values

in the

oh-

Helsinki

study

excrete

Eq and 0-Dma

than

and lactovegetarian women living in the abovecities. Of the 19 subjects, 47% and 89% excrete

in an additional

study

group

of nine

subjects,

including

three

(see Appendix A), were not significantly different from those in the two main groups (Tables 4 and lA); they were in fact surprisingly identical. The excretion of matairesinol, the children

whole

agreement found by

polysaccharides

amount

micromole amounts of Eq and Da per day, respectively, a phenomenon very rarely seen in subjects consuming a Western diet but seen in subjects consuming a macrobiotic diet (2). The values

(which

in the

in good

in both

as found

did omnivorous mentioned two

However, according to the the dietary fiber intake was 22.8 g/d in 195 1 and decreased year by year to 17.4 g/d in 1985. These figures are in better agreement with our results obtained in 1985,

is comparable

10 times

may represent crude fiber intake). national nutrition survey in Japan,

group

ental

diet

the

and the present

that

area

from southwest Asia (38). Our results are in good agreement with those from an earlier study of 300 female agricultural workers from 18 regions in Japan (37)except for dietary fiber intake, which was much lower migrants

(between

investigations

concluded

with that in many Western societies (38-40). We may also conclude that the diet ofour subjects was typical for a rural area, where the people to a large extent consumc their own products and have a traditional Japanese diet. The urinary excretion of Enl was, with few exceptions, low

and phy-

food

AL

of these

com-

food

isoflavonoids

ET

precursor lignan for enterodiol, cretion was very high. Genistein laboratories because of its very

On the basis

was very low, but genistein cxis the center ofinterest in many interesting antiproliferative and

TABLES Correlation

matrix

Nutrient Green and yellow vegetables Pulses and beans Algae Total fat Percent fat calories Fat-fiber ratio

of various

food co mponents

Enterolactone

Enterodiol

0.525w 0.541

and urinary Total lignans

0.460 0.492

cxc retion

of ligan s and isofla vonoids

Daidzein

Equol

0.679t 0.561

0.737t

materi al (n

=

19)

Total

Total isoflavonoids

diphenols

0.6l7t

0.668t 0.450t

0.693t 0.430f

0.601 t

0.585t 0.757

0.588t 0.801

O-Desmethylangolensin

0.584t 0.469 0.507

0.507

Meat Soy products (not sauce) Boiled soybeans

0.758

0.481 0.892

0.849*

0.583t 0.632t

S

t

in the whole

P