Plasma Levels of Estrone, Estrone Sulfate, and Estradici and the Percentage of Unbound Estradici in Postmenopausal Women with and Without Breast Disease1

Home , Estradiol, Estrone, Estrone sulfate

[CANCER RESEARCH 43, 3940-3943, August 1983]

Plasma Levels of Estrone, Estrone Sulfate, and Estradici and the Percentage of Unbound Estradici in Postmenopausal Women with and without Breast Disease1

M. J. Reed,2 R. W. Cheng, C. T. Noel, H. A. F. Dudley, and V. H. T. James

Department of Chemical Pathology [M. J. R., R. W. C., C. T. N., V. H. T. J.] and Academic Surgical Unit [H. A. F. D.Â¡,St. Mary's Hospital Medical School, London W2 1PG, England

ABSTRACT A preliminary account of some of the results obtained in this study has been presented (11). To investigate the possibility of increased tissue exposure to estrogen in breast cancer patients, plasma levels of estrogens and the percentage of unbound estradiol were measured in SUBJECTS postmenopausal women with benign or malignant breast disease Patients for the present study were recruited from postmenopausal and compared with levels in normal postmenopausal women. women [66 Â±10 (S.D.) years old] attending a breast clinic. Blood (20 ml) The percentage of unbound estradiol in breast cancer patients was taken between 2 p.m. and 4:30 p.m. Steroid analyses were carried [1.85 Â±0.35% (S.D.)] was significantly higher (p < 0.001) than out before the subject's clinical status was known. It was subsequently in normal postmenopausal women [1.52 Â±0.33%] and was still established that some of the women had breast cancer, some had benign significantly higher when patients were matched with control breast disease, and others were women who had previously had breast subjects for weight (p < 0.001) or ideal body weight (p < 0.001). cancer (4 months to 18 years previously) who were undergoing follow- The binding capacity of sex hormone binding globulin was similar up examination. No distinction has been made in the results between women with breast cancer and those who had previously had breast in both groups of women. No significant differences in the plasma cancer. levels of estrone, estradiol, or estrone sulfate were detected Blood samples were obtained from normal postmenopausal women between breast cancer and normal subjects. (57 Â±11 years old) between 9 a.m. and 6 p.m. These women were in It is concluded that, given similar concentrations of estradiol good health and without any endocrinological disorder. None of the in plasma of normal and breast cancer subjects, the significant subjects had received any hormone replacement therapy in the 3 months increase found in the unbound estradiol fraction may result in a preceding the study. very small increment in tissue exposure to estrogens in breast Blood obtained from patients and control subjects was centrifuged, cancer subjects. However, even such a small increase in tissue and the plasma was removed and stored at -20Â° until assayed. exposure to estradiol may be significant, given the length of time required for breast tumor development. MATERIALS AND METHODS

INTRODUCTION Plasma levels of estrone, estradiol, and estrone sulfate were measured by methods described previously (4, 15). The percentage of unbound The circumstantial evidence suggesting that estrogens may estradiol was measured in undiluted plasma using a Dianorm dialysis be involved in the development of tumors in hormone-dependent machine (23). Analysis of a plasma pool gave values of 8.3% (n = 10) and 8.7% (n = 15) for the intra- and interassay coefficients of variation tissues such as the breast has resulted in a search for evidence for the measurement of the unbound fraction by this method. The fraction of estrogen excess in women with breast cancer (10). Several of estradiol not bound to SHBG3 was measured using a precipitation studies have been made of urinary estrogen excretion by women technique (16), and intra- and interassay coefficients of variation were with and without breast cancer (24), but few investigations have 4.2% (n = 21) and 5.5% (n = 24), respectively. The binding capacity of been carried out to measure plasma levels of estrogens. In the SHBG was measured by the method of Rosner (20) as modified by present study, plasma levels of estrone and estradiol have been Anderson er al. (2). A detailed description of the techniques used to measured in postmenopausal women with benign or malignant measure the fractions of unbound and non-SHBG-bound estradiol has been published previously (11). A subject's ideal body weight was breast disease and have been compared with levels in normal calculated by comparison of a subject's weight with tables for the postmenopausal women. Plasma levels of estrone sulfate have average weight of women of the same age (6). also been measured in subjects with breast cancer, inasmuch Statistics. Data were analyzed using Student's t test and linear as this hormone is a potential source of unconjugated estrone regression, using the method of least squares. and estradiol. Because measurement of plasma estrogen con centrations may not indicate the level of biologically available estrogen (21), the percentage of unbound estradiol was meas RESULTS ured. In addition, for some patients, the fraction of estradiol Plasma Estrogen Concentration. Plasma levels of estrone, bound to albumin was also determined. estradiol, and estrone sulfate are shown in Table 1. Plasma levels of estrone and estradiol in postmenopausal women with 1This investigation was supported by grants from the Cancer Research Cam benign or malignant breat disease and plasma levels of estrone paign (CP2) and Wellcome Trust (7964/1.5). 2 To whom requests for reprints should be addressed. Received November 17, 1982; accepted May 6, 1983. 3The abbreviation used is: SHBG, sex hormone binding globulin.

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Table1 = -0.36; p < 0.01) and also between SHBG binding capacity Plasma concentrations of estrone, estrone sulfate, and estradici in and subjects' percentage of ideal body weight (r = -0.33; p < postmenopausal breast disease patients and normal postmenopausal women 0.05). sulfate(pg/mi)302 (pg/ml)28.2Â±11.2"(35)i> (pg/ml)14.0 35 Normal Â±197(46) Â±6.0(32) Benign 21 .8 Â± 5.4 ( 5) 11.4 Â±5.2(10) CancerEstrone 31 .4 Â±13.4 (26)Estrone 307 Â±177(17)Estradici13.5 Â±6.1(43) ' Mean Â±S.D. ' Numbers in parentheses, number of subjects.

50 60 70 80 90 100 Body weight (Kg) Chart 2. Correlation between body weight and plasma levels of estradiol (E2)in normal postmenopausal women (â€¢)and in postmenopausal women with benign (A) or malignant (â€¢)breast disease.

40 50 60 70 80 90 100 700 Body weight (Kg) Chart 1. Correlation between body weight and plasma levels of estrone (E,) in 600 normal postmenopausal women (â€¢)and in postmenopausal women with benign (A) or malignant (â€¢)breast disease. 500

sulfate in patients with breast cancer did not differ from the 400 levels in normal postmenopausal women. Significant correlations CD were found between plasma levels of estrone (Chart 1) and a subjects' body weight and also between plasma levels of estra 300 dici and body weight (Chart 2). No such correlation was found between plasma levels of estrone sulfate and body weight (Chart 200 3) for patients with breast disease (r = 0.07, not significant). 100 r=oi3 Percentage of Unbound Estradici, Percentage of Non- N.S. SHBG-bound Estradici and SHBG Binding Capacity. The frac

tion of unbound estradici in plasma from normal women and 40 50 60 70 80 90 100 patients with breast disease is shown in Chart 4 and Table 2. Body weight (Kg) The fraction of unbound estradici was significantly higher (p < Chart 3. Lack of significant correlation between body weight and plasma levels 0.001 ) in the breast cancer group than in normal women and of estrone sulfate (Â£,S)in postmenopausal women with breast cancer. also in women with benign breast disease (p < 0.02). Because the binding capacity of SHBG is reduced in obese women, the unbound estradiol fraction was compared in a group (16) (33) (60) of weight-matched controls and breast cancer patients and also rP<0-02n rP<0-001, in a group matched for ideal body weight (Chart 5), and a - significant difference (p < 0.001) was still found. ui 2 The fraction of estradiol not bound to SHBG (I.e., unbound plus mainly albumin bound) was also measured in the women with breast disease (Table 2), but there was no significant difference between women with benign or malignant disease. There was no significant difference in the binding capacity of SHBG (expressed as ng 5 a-dihydrotestosterone per 100 ml plasma) in patients with breast cancer [2.1 Â±1.2 (S.D.)], in those

with benign breast disease [2.5 Â±1.8], or in normal postmeno Benign Normal Breast Cancer pausal women [2.0 Â±1.4] (Table 2). For patients with breast Chart 4. Percentage of unbound estradiol (EÂ¡)in plasma from normal postmen disease, significant negative correlations were found between opausal women and those with benign or malignant breast disease. Numbers in the fraction of unbound estradiol and SHBG binding capacity (r parentheses, number of subjects.

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Table 2 Tabte3 Percentage ol unbound estradici, percentage oÃnon-SHBG-bound estradiol, and Concentrations of unbound, non-SHBG-bound, SHBG-bound, and albumin-bound SHBG binding capacity in postmenopausal breast disease patients and normal estradiol in postmenopausal women with benign or malignant breast disease postmenopausal women Albumin- binding ca Non-SHBG- SHBG-bound bound pacity (Â¿ig5a-dihy- Unbound estradiol bound estradiol estradiol estradiol of unbound of non-SHBG- drotestosterone/ (pg/100 ml) (pg/100 ml) (pg/100 ml) (pg/100 ml) estradiol1.52 boundestradiol31 100ml)2.0 19.5Â±10.0s(10)" Benign 411Â±331(8) 838Â±227(8)434Â±318(8) Â±0.33" (33)" Normal Â±1.4 (32) Cancer 24.1Â±12.4(42) 540Â±363(24)1056Â±408(24)530Â±346(24) 1.79Â±0.22C(16) Benign .2 Â±12.7 (10) 2.5 Â±1.8 (15) a Mean Â±S.D. 1.85 Â±0.35" (60)% Cancer% 31 .8 Â±11.3 (27)SMBG 2.1 Â±1.2(58) b Numbers in parentheses, number of subjects. ' Mean Â±S.D. 6 Numbers in parentheses, number of subjects. c p < 0.02 compared with normal postmenopausal women. d p < 0.001 compared with normal postmenopausal women.

100(- (25) (25) 2-5 p<0001 NJL 2-0 S 75 1-5 f SO 1-0 j

25 -0-5 m

0 0 50 60 70 80 90 100 (2_1) (21) Body weight (Kg) N.S p<0001 100 2-5 Charte. Lack of correlation between body weight and free estradiol (Â£2)in normal postmenopausal women (â€¢)and in postmenopausal women with benign to (A) or malignant (â€¢)breast disease. N.S., not significant. I 75 i 1-5 !=Â» the unbound fraction in cancer patients suggests that breast 1-0 tissue may receive increased estrogen exposure although the difference between the 2 groups is very small. Others (8, 12) I 25 0-5 have also failed to find a significant difference between plasma 0 levels of estradiol in postmenopausal breast cancer patients and Normal Cancer Normal Cancer normal controls. In 2 recent reports (7,14), significantly elevated Chart 5. Percentage of unbound estradiol (E2) in breast cancer subjects and in control women matched for weight and ideal body weight. Numbers in parentheses, plasma levels of estradiol were found in postmenopausal women number of subjects. W.S., not significant. with breast cancer. In these 2 studies, the mean plasma estradiol levels of approximately 25 pg/ml in normal postmenopausal The concentration of unbound estradiol was calculated from women and 50 pQ/m\ in postmenopausal breast cancer subjects the fraction of unbound estradiol and plasma level of estradiol were much higher than those found in the present study. There for patients with breast disease. These values are shown in is no obvious reason for the difference between these results Table 3 together with the calculated concentrations of non- and our own, other than possible differences in methodology. SHBG-bound, SHBG-bound, and albumin-bound estradiol. Al However, recent studies (3, 13, 18) have reported mean values though no significant difference was found between the concen for estradiol concentrations in plasma from normal postmeno trations of these fractions in women with benign or malignant pausal women of 13.9, 13.4, and 7.2 pg/ml, respectively, which disease, it is apparent that the concentration of estradiol bound are similar to those reported in our study. to albumin (mean, 482 pg/100 ml) is 22 times greater than the The reason for the increase in the fraction of unbound estradiol concentration of unbound estradiol (mean, 21.8 pg/100 ml). No seen in breast cancer patients is not known. Although patients signficant correlation was found between the concentration of with breast cancer tended to be older than control subjects, it is unbound estradiol and subjects' body weight (r = 0.23, not unlikely that this could account for the difference inasmuch as significant) (Chart 6). no correlation was found between the fraction of unbound estra diol and age in breast disease and control subjects. In the present DISCUSSION study, however, there was no difference in the binding capacity of SHBG between cancer patients and normal women, in con The results of the present study confirm preliminary findings trast to the results obtained by Moore ef al. (14). Although weight (11, 21) of a statistically significant increase in the fraction of affects the binding capacity of SHBG (5), a significant difference unbound estradiol in postmenopausal cancer patients. Moore ef was still found in the present study when patients were carefully al. (14) also recently reported that the unbound fraction of matched with controls for weight and ideal body weight. It is estradiol was increased in women with breast cancer. In the possible that, as suggested by Siiteri (21), differences in plasma present study, however, the mean plasma concentration of lipids may account for the higher percentage of unbound estradiol estradiol in breast cancer patients was not significantly different seen in breast cancer patients. from that in normal women. However, the significant increase in The recent studies of Pardridge ef al. (17) have suggested

3942 CANCER RESEARCH VOL. 43

that, in addition to the free fraction, steroids bound to albumin human and simian pregnancy. Clin. Endocrinol., 5: 657-669,1976. 3. Badawy, S. Z. A., Elliott, L. J., Elbadawi, A., and Marshall, L. D. Plasma levels may be biologically available. In the present study, although the of oestrone and oestradiol-17.; in postmenopausal women. Br. J. Obstet. concentration of the albumin-bound fraction of estradiol was 22 Gynaecol., 86: 56-63,1979. times greater than the unbound fraction, no difference between 4. Braunsberg, H., Reed, M. J., Short, F., Dias, V. O., and Baxendale, P. M. Changes in plasma concentrations of oestrogens and progesterone in women patients with benign or malignant breast disease was detected. during anaesthesia and gynaecological operations. J. Steroid Biochem., 14: Because it is possible that women with benign breast disease 749-755,1981. are at an increased risk for developing breast cancer (9), further 5. DeMoor, P., and Joosens, J. V. An inverse relation between body weight and the activity of the steroid binding ,1 globulin in human plasma. Steroidologia, studies are required to resolve the importance of the albumin- 7: 129-136,1970. bound estradiol fraction in the development of breast cancer. 6. Documenta Geigy Scientific Tables, p. 711. Basel: J. R. Geigy SA, 1970. 7. Drafta, D., Schindler, A. E., Milcu, S. M., Keller, E., Stroe, E., Horodniceanu, Plasma levels of estrone have been measured previously in E., and Balanescu, I. Plasma hormones in pre- and postmenopausal breast postmenopausal women with breast cancer (1, 7). Drafta ef al. cancer. J. Steroid Biochem., 73: 793-802, 1980. (7), in agreement with the results of the present study, found no 8. England, P. C., Skinner, L. G., Cottrell, K. M., and Sellwood, R. A. Serum oestradiol-17{i in women with benign and malignant breast disease. Br. J. significant difference between cancer and normal subjects, al Cancer, 30:571-576,1974. though the plasma levels of estrone found by Drafta ef a/, are 9. Hutchinson, W. B., Thomas, D. B., Hamlin, W. B., Roth, A. V., and Williams, higher than those in the present study. In contrast, Adami ef al. B. Risk of breast cancer in women with benign breast disease. J. Nati. Cancer Inst., 65: 13-20, 1980. (1) found significantly higher plasma levels of estrone in post 10. James, V. H. T., and Reed, M. J. Steroid hormones and human cancer. In: S. menopausal women with breast cancer. lacobelli, R. J. B. King, H. R. Lindner, and M. E. Lippman (Eds.), Hormones As far as we are aware, only one other investigation has been and Cancer, pp. 471-487. New York: Raven Press, 1980. 11. James, V. H. T., Reed, M. J., and Folkerd, E. J. Studies in oestrogen carried out to measure levels of estrone sulfate in postmenopau metabolism in postmenopausal women with cancer. J. Steroid Biochem.,75: sal women with breast cancer (19). It has been shown that in 235-246,1981. vitro estrone sulfate is a potential source of estrone and estradiol 12. McFayden, J. J., Prescott, R. J., Groom, G. V., Forrest, A. P. M., Golder, M. P., Fahmy, D. R., and Griffiths, K. Circulating hormone concentrations in in MCF7 human breast cancer cell lines (22). In the present study, women with breast cancer. Lancet, 2:1100-1102,1976. however, no difference in the plasma levels of estrone sulfate 13. Meldrum, D. R., Davidson, B. J., Tataryn, I. V., and Judd, H. L. Changes in circulating steroids with aging in postmenopausal women. Obstet. Gynecol., was detected, thus confirming the results obtained by Remy- 57:624-628,1981. Martin ef al. (19). In the small number of subjects studied thus 14. Moore, J. W., Clark, G. M. G., Bulbrook, R. D., Hayward, J. L, Murai, J. T., far, however, no significant correlation was found between Hammond, G. L., and Suteri, P. K. Serum concentrations of total and non- plasma levels of estrone sulfate and patient's weight, as reported protein-bound oestradiol in patients with breast cancer and in normal controls. Int. J. Cancer, 29:17-21, 1982. previously for normal postmenopausal women (15). 15. Noel, C. T., Reed, M. J., Jacobs, H. S., and James, V. H. T. The plasma In conclusion, the results of the present study suggest that concentration of oestrone sulphate in postmenopausal women: lack of diumal variation, effect of ovariectomy, age and weight. J. Steroid Biochem., 74: breast tissue in patients with breast cancer may be exposed to 1101-1105,1981. 16. O'Connor, S., Baker, H. W. G., Dulmanis, A., and Hudson, B. The measurement a small increase in the amount of biologically active estradiol of sex steroid binding globulin by differential ammonium sulphate precipitation. compared with that in women without breast cancer, although J. Steroid Biochem., 4: 331-339,1973. the difference is very small. However, it is possible that even 17. Pardridge, W. M., Mietus, L. J., Frumar, A. M., Davidson, B. J., and Judd, H. L. Effects of human serum on transport of testosterone and oestradiol into rat such a small increase may be significant during the long period brain. Am. J. Physiol., 239: E103-E108,1980. required for the development of breast cancer. Although SHBG- 18. Poortman, J., Thijssen, J. H. H., and DeWaard, F. Plasma oestrone, oestradiol bound or albumin-bound estradiol fractions may also be involved and androstenedione levels in postmenopausal women: relation to body weight and height. Maturitas, 3: 65-71,1981. in determining tissue estrogen exposure, it remains important to 19. Remy-Martin, A., Prost, 0., Nicollier, M., Burnod, J., and Adessi, G. L. Estrone discover the mechanism responsible for the increase in the sulphate concentrations in plasma of normal individuals, postmenopausal unbound fraction of estradiol seen in many breast cancer pa women with breast cancer, and men with cirrhosis. Clin. Chem., 29: 86-89, 1983. tients, because this may provide further insight into factors that 20. Rosner, W. A simplified method for the quantitative determination of testos- govern the development of this disease. terone-estradiol-binding globulin activity in human plasma. J. Clin. Endocrinol. Metab., 34. 983-988, 1972. 21. Siiteri, P. K. Extraglandular oestrogen formation and serum binding of oestra REFERENCES diol: relationship to cancer. J. Endocrinol., 89:119p-129p, 1981. 22. Vignon, F., Terqui, M., Westley, B., Derocq, D., and Rochefort, H. Effects of 1. Adami, H-O.. Johansson, E. D. B., Vegelius, J., and Victor, A. Serum concen plasma estrogen sulphates in mammary cancer cells. 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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1983 American Association for Cancer Research. Plasma Levels of Estrone, Estrone Sulfate, and Estradiol and the Percentage of Unbound Estradiol in Postmenopausal Women with and without Breast Disease

M. J. Reed, R. W. Cheng, C. T. Noel, et al.

Cancer Res 1983;43:3940-3943.

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