Diet and the excretion and enterohepatic cycling of estrogens

PREVENTIVE

MEDICINE

16, 525-531 (1987)

Diet and the Excretion

and Enterohepatic

SHERWOODL. GORBACH, M.D. ,2~~~B~~ Tufts

University

School

of Medicine,

136 Harrison

Avenue,

Cycling

of Estrogens’

R. GOLDIN,PH.D. Boston,

Massachusetts

02111

Urinary and fecal excretion and plasma levels of estrogens were measured in pre- and postmenopausal women eating different diets. When premenopausal U.S. women eating a “Western diet,” comprising high fat (40% of calories) and low fiber, were compared with age-matched vegetarians eating a moderate-fat (30%), high-fiber diet, it was found that the vegetarians excreted threefold more estrogen in their feces, had lower urinary excretion, and had 15-20% lower plasma estrogen levels. When U.S. pre- and postmenopausal women eating a Western diet were compared with recent Asian immigrants eating a very low-fat diet (20-25% of calories), similar results were obtained except that plasma estrogen levels were 30% lower among Orientals compared with those among Western omnivore women. Correlation analysis of dietary components and plasma estrogen showed that plasma estrogen was positively associated with fat and was negatively associated with fiber. The results indicate that diet can alter the route of excretion of estrogen by influencing the enterohepatic circulation and that this, in turn, influences plasma estrogen levels. o 1987 Academic

Press. Inc.

INTRODUCTION

In women of reproductive age, the primary source of circulating estrogens is the ovary, although estrone production arises in part from the extraglandular aromatization of androstenedione (4). In postmenopausal women, significant amounts of estrogens are synthesized in extraglandular sites (13). Following synthesis, the estrogens circulate in the blood in the unconjugated form and are transported to the target tissues and the liver. The major biologically active estrogens are the unconjugated (free) estrogens, of which estradiol is the most active (19). Estrone is present in nearly equal amounts as estradiol. Estrone possesses weak activity of its own but can be converted intracellularly to estradiol. Estriol is present in only small quantities in the free form and has weak estrogenic activity (15). In the liver, and also in other tissues, the estrogens are metabolized. The major pathway of metabolism is from estradiol to estrone (6). Estrone is then metabolized further to estriol or to the catechol estrogens which are then conjugated, primarily as glucuronides and sulfates (5). Greater than 50% of the metabolism and conjugation of estrogens occurs in the liver. Some of these conjugates reenter tne moodstream, where they are transported to the kidney and excreted. The glucuronide conjugates are excreted in the urine more rapidly than are the sulr Presented at the Workshop on New Developments on Dietary Fat and Fiber in Carcinogenesis (Optimal Types and Amounts of Fat or Fiber), American Health Foundation, New York, March 25-26,

1386.

* To whom reprint requests should be addressed.

525

0091-7435/87 $3.00 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

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GORBACH

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fates. The estrogen sulfates are excreted only slowly and can be hydrolyzed in tissues and act as a source of biologically active estrogen (18). Approximately 50% of the estrogen conjugates, which enter or are formed in the liver, are excreted in the bile, pass into the intestine, and are hydrolyzed by bacteria (17). They are then either reabsorbed or excreted in the feces as the free steroid. The reabsorbed estrogens enter into the portal system; they are conjugated by the splanchnic tissues and either pass into systemic circulation or pass into bile. The excretion of estrogens into bile with subsequent hydrolysis and reentry into the bloodstream comprises the enterohepatic circulation. The hydrolysis of the estrogen-glucoronides is accomplished by the bacterial enzyme p-glucuronidase, which has been shown to be influenced by diet (9, 11, 14). Dietary shifts take 3-4 weeks to affect bacterial p-glucuronidase activity in the gut. Therefore, the possibility exists that diet affects the availability of unconjugated (free) estrogen for reabsorption from the intestine. The intestinal microflora also perform reductive and oxidative reactions on estrogens and androgens which may be influenced by diet and could alter the spectrum of metabolites reabsorbed from the intestine (12). Experiments from the laboratory of Adlercreutz and colleagues first showed that bacterial activity in the bowel can be important in determining estrogen status in women. Administration of oral ampicillin to pregnant women resulted in a 34% decline in urinary estrogen excretion and a 6-fold increase in the excretion of fecal estrogen (1, 2). The concentration of conjugated forms of estrogen in the feces actually increased 60-fold (1). Other studies have confirmed that diet influences the production, metabolism, and excretion of estrogens. Armstrong et al., studying U.S. vegetarian and omnivore women, found differences in serum prolactin and sex hormone-binding globulin as well as urinary estrogens (3). In another study of plasma estrogen levels in vegetarian and nonvegetarian women, 14 premenopausal Seventh-Day Adventist women were compared with 9 premenopausal omnivores (16); the vegetarian women consumed significantly less fat, especially saturated fat, than the omnivores. Plasma levels of estrone and estradiol were found to be lower in the vegetarians . In our laboratory we have conducted a series of studies to determine whether diet has an effect on estrogen excretion and metabolism. We have concentrated on populations that have different dietary intakes of fat and fiber and have attempted to examine these dietary parameters in terms of the enterohepatic circulation of estrogen and circulating plasma levels of estrogen. METHODS

The description of participants and exclusion criteria, collection of samples, analytical procedures used for measuring estrogen and dietary data, and statistical methods have been reported previously (7, 8). RESULTS

The results from two separate studies are described. The first study involved 10 nulliparous Caucasian volunteers ages 20 to 30 years eating a “Western-style

DIET

AND

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ESTROGEN

diet” (omnivores) and 10 vegetarians. The vegetarian and omnivore participants did not differ significantly in their mean ages, heights, or weights. Their mean intakes of calories and protein also were similar, although the omnivores consumed 43% of their protein from animal sources and the vegetarians consumed only 7% from nonvegetable sources, all of it being fish. Omnivores ate an average of 12 g of dietary fiber per day compared with 28 g per day among vegetarians. The omnivores consumed significantly more total fat and saturated fat per 1,000 calories of food intake (P < 0.05). The percentages of calories represented by fat were 40% among omnivores and 30% among vegetarians. Mean fecal excretions of estrone (Et), estradiol (E2), estriol (E,), and total estrogens were significantly higher among vegetarians than among omnivores, being two- to threefold greater per 24 hr (Table 1). Total 24-hr estrogen excretion in the feces of vegetarian women averaged 5.4 nmo1/24 hr compared with 2.3 nmo1/24 hr in those of omnivores. Urinary E, excretion was significantly lower among vegetarians who had a mean 24-hr excretion of 13.2 nmol compared with 21 nmol among omnivores. The vegetarians had 15% lower plasma E, and 19% lower E,, similar testosterone, and 11% higher androstenedione levels than did the omnivores. None of these differences was statistically significant. The vegetarians excreted more than twice the wet or dry weight of feces when compared with omnivores. The 24-hr mean fecal wet weight for vegetarians was 154 g compared with 58 g for omnivores (P < 0.001). There was a positive correlation between an individual’s fecal weight (wet and dry) and her fecal excretion of estrogen, suggesting that the group differences in fecal estrogen excretion may be explainable on the basis of fecal bulk. After adjusting for the effect of fecal bulk in an analysis of covariance, the group means for vegetarians and omnivores were nearly identical, implying that group differences in fecal estrogen excretion were secondary to the differences in fecal weight. This relationship held for total fecal estrogens (E,, E,, and E,), as well as for the individual estrogens (P < 0.002; Fig. 1). An analysis of covariance revealed highly significant (P < 0.003) common slopes for plasma estrogen concentration vs fecal estrogen excretion among both dietary groups; therefore, the model that describes this relationship can be represented by a single regression line (Fig. 2). There was a negative correlation between plasma estrogen levels and fecal estrogen excretion. This was true for both plasma E, and plasma E, (or their sum), regardless of whether we examined fecal TABLE

I

FECAL EXCRETION OF ESTROGENS IN OMNIVORE AND VEGETARIAN WOMEN Group Omnivores Vegetarians

Estrone 0.83 (0.70-0.99)

1.96 (1.68-2.29)b

Estradiol 0.61 (0.52-0.72) 1.52 (1.30-1.78)b

Estriol 0.72 (0.63-0.83) 1.72 (1.50-1.98)'

Estrone + estradiol + estriol 2.33 (2.01-2.70) 5.40 (4.70-6.21)b

a Nanomoles per 24 hr; geometric means (SE range); four collections per subject. bP = 0.03. c P = 0.01.

GORBACH

528

I

AND GOLDIN

1 100

400

200 FECAL

WET

WEIGHT

600

600

(gms.)

FIG. 1. Log-log plot of the correlation between 24-hr fecal excretion of estrogen (sum of estrone, estradiol, and estriol) and 24-hr output of fecal wet weight. The individual points (0 denotes omnivore and V denotes vegetarian) are the geometric mean values from four different fecal collections taken in 1 year. The correlation is significant (P < 0.002). [From Ref. (7).]

E,, E,, or E3 (or their sum). The correlations shown in Figs. 1 and 2 suggest that differences in plasma and fecal estrogens result from changes in daily fecal output. The sum of both fecal and urinary estrogen excretion (total excretion) was only slightly higher in the vegetarians than in the omnivores, suggesting that the increased fecal loss was nearly balanced by decreased urinary excretion. In fact, there was a negative correlation between the two exit routes. But more interesting is the significant inverse relationship between fecal E, and urinary estriol3-glucuronide (E,-3G; P < 0.01). Since E,-3G is formed in the intestinal mucosal cell at the time of intestinal reabsorption of Es, this finding suggests that increased fecal loss causes diminished enterohepatic circulation of estrogens. Fecal bacterial B-glucuronidase activity was lower in vegetarians than that in omnivores (1.0 vs 1.8 pg/min/mg fecal protein; P < 0.05), which together with the data on E,-3G excretion in the urine and the increased fecal estrogens suggests that the dietary effect on gut flora seen in the vegetarians is related to the reduced enterohepatic circulation of estrogens. The lack of large differences in the fat intakes of the omnivore and vegetarian women (40 vs 30%, respectively) in the study described above prompted another project in which estrogen levels were measured in recent Vietnamese and Laotian immigrants from Southeast Asia to Hawaii (8). This population is known to have a low risk for breast cancer and also to have a low intake of dietary fat and a higher intake of fiber. The women had arrived in Hawaii recently (1- 12 months), and although they were not vegetarians, they ate very little meat, Indeed, their fat intakes averaged 20-25% of total calories. The Oriental women had lower plasma and urinary estrogens and higher fecal estrogens, and they were significantly dif-

DIET AND ESTROGEN T .z I

1.40

.

1.20

.

i! 5 p

529

0 1.00

1.0 FECAL

2.0 ESTROGEN

4.0 EXCRETION

12.0

24.0

(nmo1/24hr.)

FIG. 2. Log-log plot of the correlation between 24-hr fecal excretion of estrogen (sum of estrone, estradiol, and estriol) and plasma concentration of estrogen (sum of estrone and estradiol). The individual points (0 denotes omnivore and V denotes vegetarian) are the geometric mean values from four separate fecal collections and four separate W-ml collections of pooled serum, taken on 3 consecutive days during a l-year period. The correlation is significant (P < 0.003). [From Ref. (7).]

ferent in these measurements from both the Boston omnivores and the vegetarians. For example, the premenopausal Orientals excreted three times the amount of estrogen in their feces compared with that of age-matched Caucasians. Total urinary estrogen excretions were 1.45 and 2.58 times greater for Caucasian pre- and postmenopausal women, respectively, compared with those for agematched Orientals. Plasma estrogen was 32% lower in premenopausal Orientals and 30% lower in postmenopausal Orientals when compared with that in agematched Caucasians. The associations between dietary fat and fiber and plasma estrogens were analyzed for the premenopausal Orientals and Caucasians. Total dietary fat intake was positively correlated with plasma estradiol (P < 0.001) and estrone (P < 0.005). There was a negative correlation between dietary fiber intake and plasma estrone (I’ = 0.008) and plasma estradiol (P < 0.001) among the premenopausal women of both groups. DISCUSSION

At least three explanations for our findings can be invoked. The first is that the vegetarian women consumed higher quantities of dietary fiber, accounting for the higher fecal weights. The greater bulk of digesta and nonabsorbed fiber may somewhat shield the estrogens excreted in the bile from bacterial deconjugation and reabsorption. A similar role for dietary fiber has been suggested for the fecal excretion of bile acids, another group of endogenous substances that undergo an enterohepatic circulation and are structurally related to estrogens.

530

GORBACH

AND GOLDIN

A second explanation for our observations, not mutually exclusive with the shielding concept, is that some characteristic of the vegetarian diet decreased the ability of the intestinal flora to deconjugate biliary estrogen conjugates, a necessary step for their reabsorption. The lower p-glucuronidase activity in the feces of vegetarians supports this hypothesis. Previous studies in humans and animals have established that fecal p-glucuronidase activity can be lowered by eating a grain diet or by administering oral antibiotics, and can be increased by feeding beef or fat (9-11, 14). The third explanation, and that which has heretofore been the most popular in the literature, is that high dietary fat levels directly alter estrogen absorption and increase estrogen deconjugation. Currently, our laboratory is conducting studies on premenopausal and postmenopausal women eating their meals in a metabolic unit. The diets are defined and the various components of the diets can be altered independently. Our current work has demonstrated that lowering fat intake from 40 to 20% of calories, decreasing cholesterol from 400 to 150 mg/day, increasing fiber from 12 to 40 g daily, and changing the polyunsaturated:saturated fatty acid ratio from 0.5 to 1 .O results in significant lowering of the serum estrone sulfate levels. These changes occurred after 2 months on the experimental diet. Estrone sulfate is a long-lived estrogen which represents the dominant estrogen in the blood; it is capable of being hydrolyzed to estrone and converted to estradiol in many tissues. These more recent studies, conducted over a relatively short time period and using defined diets, have established that modulation of diet can indeed influence estrogen metabolism in premenopausal women. We have failed to affirm, however, that dietary fiber is the critical factor; rather, the type and quantity of dietary fat seems to play a more important role. It may be necessary for longer-term dietary studies to be done to establish the relative roles of dietary fat and fiber in estrogen metabolism. REFERENCES 1. Adlercreutz, H., Martin, F., Pulkkinen, M., Dencker, H., Rimer, U., Sjoberg, N. O., and Tikkanen, M. J. Intestinal metabolism of estrogens. J. C/in. Endocrinol. Metab. 43, 497-505 (1976). 2. Adlercreutz, H., Martin, F., Tikkanen, M. J., and Pulkkinen, M. Effect of ampicillin administration on the excretion of twelve estrogens in pregnancy urine. Acta Endocrinol. 80, 551-557 (1975). 3. Armstrong, B. K., Brown, J. B., Clarke, H. T., Crooke, D. K., Hahnel, R., Masarei, J. R., and Ratajezak, T. Diet and reproductive hormones: A study of vegetarian and non-vegetarian postmenopausal women. J. Nat/. Cancer Inst. 67, 761-767 (1981). 4. Baird, D. T., Horton, R., Longcope, C., and Tait, J. F. Steroid dynamics under steady state conditions. Recent Prog. Horm. Res. 25, 611-664 (1969). 5. Eriksson, H., and Gustafsson, J. A. Excretion of steroid hormones in adults. Eur. J. Biochem. 18, 146-150 (1971). 6. Fishman, J., Bradlow, H. L., and Gallagher, T. F. Oxidative metabolism of estradiol. J. Biol. Chem. 235, 3104-3107 (1960). 7. Goldin, B. R., Adlercreutz, H., Gorbach, S. L., Warram, J. H., Dwyer, J. T., Swenson, L., and Woods, M. N. Estrogen excretion patterns and plasma levels in vegetarian and omnivorous women. New Engl. J. Med. 207, 1542-1547 (1982). 8. Goldin, B. R., Adlercreutz, H., Gorbach, S. L., Woods, M. N., Dwyer, J. T., Conlon, T., Bohn,

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9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

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E., and Gershoff, S. N. The relationship between estrogen levels and diets of Caucasian American and Oriental immigrants. Amer. J. Clin. Nutr., 44, 945-953 (1986). Goldin, B. R., and Gorbach, S. L. The relationship between diet and rat fecal bacterial enzymes implicated in colon cancer. J. Natl. Cancer Inst. 57, 371-375 (1976). Goldin, B. R., and Gorbach, S. L. Effect of antibiotics on incidence of rat intestinal tumors induced by 1,2-dimethylhydrazine dihydrochloride. J. Natl. Cancer Inst. 67, 877-880 (1981). Goldin, B. R., Swenson, L., Dwyer, J., Sexton, M., and Gorbach, S. L. Effect of diet and lactobacillus supplements on human fecal bacterial enzymes. J. Nat/. Cancer Inst. 64, 255-262 (1980). Lombardi, P., Goldin, B. R., Boutin, E., and Gorbach, S. L. Metabolism of androgens and estrogens by human fecal microorganisms. J. Steroid Biochem. 9, 795-801 (1978). Longcope, C., Kato, T., and Horton, R. Conversion of blood androgens to estrogens in normal adult men and women. J. Clin. Invest. 48, 2191-2201 (1969). Reddy, B. S., Weisburger, J. H., and Wynder, E. L. Fecal bacterial beta-glucuronidase: Control by diet. Science 183, 416-417 (1974). Rotti, K., Stevens, J., Watson, D., and Longcope, C. Estriol concentrations in plasma of normal, non-pregnant women. Steroids 25, 807-816 (1975). Shultz, T. D., and Leklem, J. E. Nutrient intake and hormonal status of premenopausal vegetarian Seventh-Day Adventists and premenopausal nonvegetarians. Nutr. Cancer 4, 247-259 (1983). Sandberg, A. A., and Slaunwhite, W. R., Jr. Studies on phenolic steroids in human subjects. II. The metabolic fats and hepato-biliary-enteric circulation of i4C-estrone and i4C-estradiol in women. J. Clin. Invest. 36, 1266-1278 (1957). Tseng, L., Stolee, A., and Gurpide, E. Quantitative studies on the uptake and metabolism of estrogens and progesterone by human endometrium. Endocrinology 90, 390-404 (1972). Yen, S. S. C. The human menstrual cycle, in “Reproductive Endocrinology” (S. S. C. Yen and R. B. Jaffe, Eds.), pp. 126-151. Saunders, Philadelphia, 1978.