ltrt. J . Cancer: 14, 161-167 (1974)

URINE (ESTROGEN PROFILES OF ASIAN AND NORTH AMERICAN WOMEN

Brian MACMAHON, Philip COLE, James B. BKOWN, Kunio AOKI, Tong Ming LIN,

Departmetit o f Epidemiolog-v, Harvard School of ' Public Health, Boston, Massachusetts, USA; Department of Obstetrics and Gynaecology, University o f Melbourne, Victoria, Australia;

Department o f Epidemiology, Aichi Cancer Center, Nagoya, Japan; Department of Public Health, College of' Medicine, National Taiwan University, Taipei, Taiwan; Department of Health Care

and Epidemiology, University of' British Columbia, Vancouver, Canada; atid M. & H. D. Institute o f Radiology, Queen Elizabeth Hospital, Kowloon, Hong Kong

Robert W. MORGAN and Ngai-Chen Woo

Estrone ( E l ) , mtradiol (E2) and mrr io l ( E 3 ) were measured in the urine of Asian and North American women aged 15-19, 20-24 and 30-39 years, during the follicular and Iuteal phases of the menwual cycle. Each urine Jpecimen was churac- terized by its custriol proportion, E3/ ( E l + E2 t E3). In each age group and cycle phase Asians had lower concentrations of mtrone and mtradiol and a higher oestriol pro- portion than North Americans. The diferencc.5 were greatest in the youngest age group. Diferences between the women from two North American citieJ or between women from three Asian areas were small. The data support the idea that the cpstriol proportioii, especially among young women, i s a correlate of subsequent breast cancer risk.

A woman's breast-cancer risk may be related to the pattern of her estrogen metabolism during young adulthood. Specifically, it has been hypothesized that women who, in the decade or so after puberty, have relatively low ratios of cestriol (E3) to estrone ( E l ) and estradiol (E2) are at high risk of breast-cancer (Cole and MacMahon, 1969). The long interval between puberty and the usual age at clinical appearance of breast cancer make this hypothesis difficult to evaluate directly. One method of indirect assess- ment is to determine whether estrogen profiles of young women vary between populations in ways that would be predicted from knowledge of the populations' breast-cancer rates. A pilot study,

comparing urine estrogen profiles of Asian and North American women, provided support for the hypothesis (MacMahon et al., 1971). The larger study which ensued is described herein.

METHODS

The study was conducted in five areas, two, Boston and Vancouver, having Caucasian popu- lations at high risk of breast-cancer and three, Hong Kong, Taipei and Shirakawa Gifu (Japan), having Oriental populations with breast-cancer rates about one-fifth of those seen in North America. In each area, women in each of three age groups, 15-1 9, 20-24 and 30-39 years, were

Received: March 27, 1974. Present address: Department of Preventive Medicine, Faculty of Medicine, University of Toronto,

Requests for reprints should be addressed to B. M. at 677 Huntington Ave., Boston, Massachusetts,

Canada.

USA 021 15.

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included. Because the hypothesis derived in part from the change in estrogen profile during pregnancy, the two younger groups were restricted to nulliparous, and the oldest to parous, women. Consequently, parity is removed as a confounding variable in the comparison of Asian and North American women in this study, but its possible effects as a modifying factor cannot be assessed.

Women were included o n the basis of accessi- bility and willingness to participate. Exclusions were made for: pregnancy within the prior 12 months; usual menstrual cycle of 26 days or less, o r 33 days or more; history of diabetes, thyroid disorder, cancer, breast lump or cyst, liver disease, uterine curettage other than for miscarriage, ovarian surgery, pelvic surgery for any neoplasm, ever having had systemic endocrine therapy, oral contraception within prior 24 months; current use of any medication weekly or more often.

The pilot study indicated that almost identical inferences could be made from a morning urine specimen and from the conventional 24 h collection. The main study was therefore limited to morning specimens. Each subject collected urine on the morning of the 10th day (follicular specimen) and the 21st day (luteal specimen) of a menstrual cycle. All urine passed after retiring, up to and including the morning void, was collected. Urine was chilled a t about 7” C in the women’s homes during the collection period. Upon completion the collection was taken to a local laboratory and aliquots were prepared, frozen (-20“ C) and sent (in dry ice) to the University of Melbourne for biochemical analysis.

Identical, coded aliquot bottles were used in all areas and the source of the bottles was unknown to the Melbourne staff.

For each specimen the concentrations of E l , E2 and E3 were determined. Results were sent to Boston where they were linked to the interview information on individual women. To assess the replicability of the biochemical procedures, 10% of all specimens, selected a t random, were sent to Melbourne in duplicate but were not so identified. Details of the biochemical procedures, their validity and replicability will be reported elsewhere (Brown and Cole, in preparation). The coefficient of variation for duplicate specimens was between 12 and 25% for the individual estrogens and 10% for the oestriol proportion.

The hypothesis to be tested a priori was that the “oestriol ratio ”, E3/(EI +E2), is low in women with high breast-cancer risk, that is, the American women. Data are presented on this index of the estrogen profile. However, the estriol ratio has a log-normal distribution, and we have found a more useful measure of the same relationship to be the *‘ cestriol proportion ”, E3/(EI i-E2$ E3), the proportion of the three estrogens which is E3. In the present data this index is normally distributed, o r nearly so, for each center and age group

RESULTS

The numbers of women in each age group and area, and their mean oestriol proportions, are shown in Table I . The oestriol proportions of the

TABLE I

N U M B E R OF WOMEN A N D MEAN CESTRIOL PROPORTIONS BY AREA, MENSTRUAL PHASE A N D AGE GROUP ’

Ages 15-19 Ages 20-24 Ages 30-39

Area (Estriol proportion CEktriol proportion CEstriol proportion Number Number Number

Follicular Luteal Follicular Luteal Follicular t u t e a l

Hong Kong 27 0.58 0.63 32 0.56 0.57 25 0.56 0.60 Taipei 33 0.62 0.65 25 0.48 0.51 30 0.51 0.53 Shirakawa 39 0.57 0.62 39 0.55 0.61 40 0.49 0.54 Boston 31 0.42 0.46 26 0.38 0.40 22 0.36 0.43 Vancouver 28 0.38 0.41 28 0.39 0.46 13 0.42 0.48

’ The standard errors for Vancouver, luteal phase, are 0.04, all others are 0.03 or less.

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(ESTROGENS OF ASIAN AND AMERICAN WOMEN

Asian women are consistently higher than those of the Americans. Cumulative percentage distri- butions of 15- to 19-year-old subjects, based on follicular cestriol proportions, are shown in Figure 1 . Distributions for other age groups and cycle phases, not shown, are similar to those in Figure 1 . For all groups the distributions of women according to oestriol proportions are similar in the three Asian areas but differ markedly from those in the two North American areas. Therefore, further analyses are presented using data from the three Asian areas combined and compared with data from the two North American areas combined.

Table 11 gives median concentrations of each estrogen, as well as cestriol ratios and propor- tions, for the Asian and North American women. In each age group and cycle phase, the inter- continent difference in mean cestriol proportion

is statistically highly significant ( p ' in all instances). The magnitude of the inter-continent difference is more apparent when distributions rather than means are examined. Figure 2 shows the distributions of the cestriol proportions for follicular phase specimens. The large differences seen are also evident in the luteal phase compa- risons.

The oestriol proportions are most disparate in the youngest women. For the 15- to 19-year age group, mean follicular phase values are 48% higher and luteal values 43% higher for Asian than for North American women. In the 30- to 39-year age group, mean follicular values are only 34% and luteal only 22% higher in the Asians than in North Americans. This trend with age occurs because the mean oestriol proportion of the Asian women declines with age, while that of the North Americans does not change. As

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FOLLICULAR CESTRIOL PROPORTION

FIGURE I

astrio1 proportion, by centre. Wonien aged 15-19 years, follicular phase. Cumulative percentage distribution of women according to their urine

163

MACMAHON ET A L .

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FOLLICULAR OESTRIOL PROPORTION

FIGURE 2 Percentage distribution of women according to urine oestriol proportion,

by continent and age group. Follicular specimens only. Numbers of women in parentheses.

noted, trends with age and parity cannot always be distinguished in these data. However, the sharp decline in the cestriol proportion of Asian women occurs between the age groups 15-19 years and 20-24 years. Since both groups consist of nutli- parae, age is shown to be a relevant variable.

The low cestriol proportions of American women can result either from a low E3 or from high El and E2 concentrations. In general the latter appears to be the case. In each cycle phase and age group, with one exception (follicular

phase, age 15-19 years), little diflerence is seen between E3 concentrations of the Asian and North American women. On the other hand, without exception, concentrations of El and E2 are higher for North Americans than for Asians.

Timirig of collection

l t is more accurate to estimate ovulation as having occurred 14 days prior to onset of the subsequent menses rather than 14 days subsequent to onset of the prior menses. The date of onset of

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ESTROGENS OF ASIAN A N D AMERICAN WOMEN

TABLE I I

COMPARISON OF ASIAN A N D NORTH AMERICAN WOMEN WITH RESPECT TO MEDIAN CONCENTRATIONS OF INDIVIDUAL (ESTROGENS, MEAN (ESTRIOL PROPORTION A N D MEDIAN CESTRIOL RATIO,

BY AGE GROUP A N D MENSTRUAL PHASE

Age group

15-19 20-24 30-39 (Estrogen Cycle or quotient ' phase

Asia North America Asia North America Asia North America

CEstrone ( E l ) F 4.0 6.9 4.5 8.4 5. I 10.6 (Estradiol (E2) F 2. I 3 .5 2.4 4.2 2.6 5.5 (Estriol (E3) F 9.0 6. I 7.7 7.5 8 . 3 9.6 E3/(EI +E2$ E3) F 0.59 0.40 0.53 0.39 0.5 I 0.38 E3/(EI I E2) F I .46 0.65 1.43 0.59 1 . 1 1 0.56

(Estrone L 5.9 12.8 8.0 14.7 6.8 12.4 (Estradiol L 3.2 6.9 4.1 8.4 3.7 5.9 (Estriol L 17.0 15.9 15.4 17.8 13.8 14.0 E3/(EI -tkE+E3) L 0.63 0.44 0.57 0.43 0.55 0.45 E3/(E1 + E2) L 1.82 0.71 1.41 0.74 I . 37 0.75

No. of women 99 59 96 54 95 35

' Median concentrations of estrogens in pg per litre. For the estriol proportion mean values are given, for the estriol ratio, inedians. The standard error of each aestriol proportion is 0.02 or less: each intercontinent difference has a les? than lo- ' probability of being due to chance.

menses following the urine collections was Urine coticenfrafiori ascertained in all areas except Taipei. This allowed computation of the " true " follicular and luteal collection days by assigning the value one to the last day of the collected cycle and counting back in time to the days of collection. In each age group, Asians had a greater mean cycle length than Americans. This ianged from 0.8 days in the middle age group to I .7 days for the youngest women. In the youngest age group the mean true follicular and true luteal collection days were 21.6 and 10.6 respectively, for Asians, and 19.9 and 8.9 for Americans; the standard error of each mean is about 0.7 days. There is a moderately strong relationship between the true collection day and the concentration of estrogens in the urine. Among Asian women the mean follicular- phase cestrone concentrations decreased by about 0.20 pg per litre as the true follicular collection day increased numerically (preceded ovulation) by one, and the value for cestriol decreased by about 0.4,ug per litre. However, in no age- continent group did the value of the estriol proportion change appreciably with advance in the collection day, For Asians, there was a decline of only 3 x 10 as the true follicular day increased by one and for Americans the decline was 2 x 10 J.

The interval during which the urine specimens were collected varied according to the time at which the subject went to bed and arose in the morning. We have no data on this interval. I t may therefore be asked whether the observed differences between continents in estrogen con- centrations reflect differences in the amounts of estrogen excreted. For each specimen, volume and specific gravity were recorded and the weight of each subject was also known; mean values are shown in Table 111. The estrogen concentration values for Asians, who excreted less solids per kilogram of body weight than Americans, were adjusted to the concentrations predicted from their body weight if they had the same ratio of solids to body weight as Americans. The correc- tion factors were 1.25 or less, depending on age and cycle phase. That is, Asian women excreted about one-fourth less grams of solids in their urine, per kilogram of body weight, than did Americans. Multiplying the values for Asians in Table I 1 by 1.25 (for follicular specimens) or 1.1 1 (for luteal) yields values comparable with those of Americans on a per kilogram body-weight basis. The effects of this correction, probably an over-correction, are small. We conclude that the different concen-

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MACMAHON ET AL.

TABLE 111

PHYSICAL CHARACTERISTICS OF T H E FOLLICULAR PHASE URINE SPECIMENS A N D WEIGHT OF THE SUBJECTS, BY CONTINENT A N D AGE G R O U P

Age group

Variable 15-19 20-24 30-39

Asia p2::ita Ratio Asia Ratio Asia Ratio

Volume (ml) 259 305 0.85 275 320 0.86 353 355 0.99 Specific gravity 1.018 1.023 0.79 ' 1.016 1.021 0.77 1.014 1.019 0.73 * Solids (grams per specimen) 4.3 6.5 0.66 3 .8 5.8 0.66 4.5 6.2 0.73 Body weight (kg) 47.9 56.3 0.85 47.5 56.5 0.84 50.1 57.6 0.87

' All are mean values. Ratio of the specific gravities in excess of 1.000.

trations of the individual estrogens between the racial groups reflect differences in the relative amounts of estrogens excreted.

Other variables

There were differences between the Asian and American women in height, weight and Quetelet's index (weight/height'), a favored index of obesity (Khosla and Lowe, 1967), shown in older women in some populations (Valaoras et al., 1969) to be correlated with breast-cancer risk. In the present data this index was correlated neither with the aestriol proportion nor with the concentration of any estrogen.

The strongest risk factor identified for breast- cancer, apart from race, is the age at which a woman delivers her first child (MacMahon et al., 1970). Among women 30-39 (the only parous women), the Asian subjects had a mean age at first birth of 23.1 and the Americans of 24.5 years. An inverse association between age at first birth and estriol proportion was noted. However, the association is too weak to explain the inter- continent difference seen. Adjustment of the follicular cestriol proportion of the Asians to that expected if they had a n age at first birth of 24.5 years reduces the value only from 0.51 to 0.50. Age-at-first-birth associations cannot explain the inter-continent differences among younger women, all of whom were nulliparous.

DISCUSSION

The women in this study are not representative of all women in the areas studied. This is ensured by the criteria for eligibility, such as non-use of

oral contraceptives, as well as by the use of volunteers. It is difficult, however, to conceive of mechanisms that would link a particular estrogen profile to the likelihood of volunteering. The eligibility criteria were introduced to ensure that, so far as possible, the data were applicable t o '' normal '' women in the various populations and that the study groups were comparable. In addition, some selection criteria were introduced to make the young women studied as similar as possible to the generation now in the breast- cancer age range. In Japan, for example, where breast-cancer rates are increasing, women were selected from a rural population in Shirakawa, where diets have not changed as rapidly as in the cities.

However, there is n o way of selecting a group of young women that will certainly experience the same breast-cancer rates as older women of the same population. This study is not, therefore, a direct test of the hypothesis that a low estriol proportion in early adult life is associated with high breast-cancer risk. Nevertheless, the data are consistent with such a hypothesis. The hypothesis is strengthened by the near identity of the estrogen profiles from the three Asian areas and the similarity of the data from the two American areas-despite the different selection procedures employed in each area. Briggs has also reported a n inverse association between the aestriol ratio and the probable risk of breast- cancer among three ethnic groups in Lusaka, Zambia (Briggs, 1972).

Contrary to an impression gained from the few observations previously available (MacMahon rt al., 1971), it appears that the difference in

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(ESTROGENS OF ASIAN AND AMERICAN WOMEN

estriol proportion between Asian and North American women persists throughout premeno- pausal life, although it is most marked under age 20. The significance of the fact that the estriol proportion remains constant with age in North Americans but falls sharply between 15-19 years and 20-24 years in Asians is unknown.

In addition to studies of other populations to evaluate the consistency of the relationship between urine estrogen profile and breast-cancer risk, there is a need to understand the relationship of estrogen levels in urine to those in blood and tissues. Urine concentrations may not directly

reflect blood and tissue levels (Lipsett, 1971). However, it seems likely that differences in urine concentrations would reflect differences in estrogen production and metabolism.

ACKNOWLEDGEMENTS

This work was supported by grants from the American Cancer Society (P-600), the US National Cancer Institute (POI C A 06373), the National Health and Medical Research Council of Australia and the World Health Organization; and a research contract from the US National Cancer Institute (NCI-71-2179).

CONCENTRATIONS DYESTROGENES DANS L’URINE DE FEMMES ASIATIQUES E T AMERICAINES

L’cPstrone ( E l ) , l’mtradiol ( E 2 ) et I’cpstriol ( E 3 ) ont PtP doses dans [’urine de jemmes asiatiques et amtricaines dgees de 15 19, de 20 a 24 et de 30 a 39 ans, pendant les phases jolliculaire et lute‘ale du cycle menstruel. Chaque specimen d’urine a PtP caractbrisk d’apres la proportion d’crstriol, E3J ( E l +E2$ E3). Dans chaque groupe d’dge et pour chacune des phases du cycle, la concentration d’mtrone et d’mtradiol Ctait plus faible et la proportion d’cl‘striol plus PlevPe chez les Asiatiques que chez les AmPricaines. Les differences e‘taient particuli6rement grandes dans le groupe d’cjge le plus jeune. Entre les femmes de deux villes d’Arnkrique du Nord ou entre les femnies de trois rkgions d’Asie, les differences Ptaient faibles. Les constatations con- firment I’idPe que la proportion d’mtriol est en corre‘lation avec le risque subsiquent de cancer du sein, notamment chez les jeunes ferrtnies.

REFERENCES

BRIGGS, M . , Ethnic differences in urinary estrogens. Lancet, 1, 324 (1972).

BROWN, J . B., and COLE, P., Biochemical aspects of epideniiologic studies of the urine estrogen profile. In preparation.

COLE, P., and MACMAHON, B., (Estrogen fractions during early reproductive life in the aetiology of breast cancer. Lancet, i, 604-606 (1969).

KHOSLA, T., and LOWE, C. R., Indices of obesity derived from body weight and height. Brit. J . prev. soc. Med., 21, 122-128 (1967).

LIPSETT, M. , (Estrogen profiles and breast cancer. Lancet, 2, 1378 (1971).

MACMAHON, €3.. COLE, P., BROWN, J . B., AOKI, K., LIN, T. M., MORGAN, R. W., and Woo, N. C., Estrogen profiles of Asian and North American women. Lancet, 2, 900-902 (1971).

MACMAHON, B., COLE, P., LIN, T. M. , LOWE, C. R.,

VALAORAS, V. G. , and YUASA, S . , Age at first birth and breast cancer risk. Bull. Wid Hlth Org., 43,

VALAORAS, V. G. , MACMAHON, B., TRICHOPOULOS, D., and POLYCHRONOPOULOU, A., Lactation and repro- ductive histories of breast cancer patients in Greater Athens, Greece, 1965-67. Int. J . Cancer, 4, 350-363 (1 969).

MIRRA, A. P., RAVNIHAR, B., ~ A L B E R . E. J . ,

209-221 (1970).

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