0021-972X/90/7103-1387$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1990 by The Endocrine Society
Vol. 71, No. 5 Printed in U.S.A.
Influence of Estrogens on Serum Free Fatty Acid Levels in Women* F. PANSINI, G. BONACCORSI, F. GENOVESI, M. R. FOLEGATTI, B. BAGNI, C. M. BERGAMINI, AND G. MOLLICA Institute of Obstetrics and Gynecology and Institute of Biochemistry (C.M.B.), University of Ferrara, and the Service of Nuclear Medicine, St. Anna Hospital (B.B.), 4410 Ferrara, Italy
ABSTRACT. A relationship between plasma levels of FFA and incidence of hormone-dependent breast cancer has been suggested. This observation has drawn our attention to possible complementary actions of ovarian steroids on circulating FFA levels. Measurements taken in normal women during the menstrual cycle and in ovariectomized women with and without
E
Mg/24 h (Estraderm, TTS-50, Ciba-Geigy, Italy, Orriggio) for 3 of 4 weeks, with 1 intervening treatment-free week. All of them were followed up over 6 months. Patients were encouraged to maintain their standard diets to avoid possible nutritional interferences in the study. In every case informed consent was obtained for this clinical investigation. All blood samples were taken after overnight fasting and
VIDENCE has been collected for a promoting action of FFA in estrogen-dependent breast cancer (1-3), probably through an increase in the availability of estrogens to the tissue or a decrease in their inactivation in situ. Indeed, it has been proved that FFA provoke a direct displacement of estrogens from their serum carrier proteins in order to increase the fraction of free estradiol (4), a tightening of estradiol binding to estrogen receptors in target tissues incubated in the presence of FFA (5), and a decrease in metabolic clearance of the hormone through 17/3-hydroxysteroid-dehydrogenase inhibition (6). To investigate the correlation between estrogens and plasma FFA, we planned the present study with the aim of ascertaining whether ovarian hormones themselves influence FFA serum levels in women during the normal menstrual cycle, upon ovariectomy, and under estrogen substitutive therapy (ERT).
were allowed to clot at room temperature. In the case of patients
receiving ERT, blood samples were taken during the last week of treatment. The concentrations of estradiol and progesterone were determined in frozen stored serum by RIA (7), while FFA levels were measured in unfrozen serum immediately after centrifugation by a colorimetric procedure (8). Analysis of variance testing was used for statistical evaluation of data (9) using the SPSS package; when necessary, the Scheffe procedure was used as a post-hoc test. Results
Materials and Methods Serum FFA levels were determined in 25 normal women during the secretory and proliferative phases (8th and 24th days of the cycle) and in 14 patients undergoing hysterectomy plus bilateral ovariectomy for uterine leiomyomatosis. Among the ovariectomized patients, only 7 received ERT by a transdermal therapeutic system delivering estradiol at a rate of 50 Received April 17, 1990. Address all correspondence and requests for reprints to: Francesco Pansini, M.D., Institute of Obstetrics and Gynecology, University of Ferrara, Via Savonarola 9, 4410 Ferrara, Italy. * This work was supported by 60% research funds from the Ministero deH'Universita e della Ricerca Scientifica e Tecnologica.
estrogen replacement therapy demonstrate that 1) levels of FFA present in serum are lower during the luteal phase than during the follicular phase; 2) levels of FFA are significantly higher after ovariectomy; and 3) these are again reduced by substitutive estrogen therapy. (J Clin Endocrinol Metab 7 1 : 1387-1389, 1990)
The measurement of FFA blood levels during the menstrual cycle (Table 1) demonstrated a significant decrease in serum FFA ( P < 0.01) during the luteal phase [0.098 ± 0.009 mg/L (mean ± SEM)] compared to levels in the follicular phase (0.130 ± 0.010). An influence of ovarian function on serum FFA was confirmed by experiments on 14 ovariectomized patients, some receiving E R T and others not; in the absence of estrogenic replenishment treatment, serum FFA levels consistently increased as early as 1 month after surgery and increased further at 6 months. In contrast, administration of exogenous estrogen prevented the increase in serum FFA after ovariectomy and,
1387
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1388
COMMENTS
TABLE 1. Relationship between the levels of circulating estradiol (E2) and progesterone (P), and serum FFA FFA (mg/L)
E2 (pmol/L)
P (nmol/L)
Normal menstrual cycle (n = 25) Proliferative 0.130 ± 0.010 257 ± 22 0.9 ± 0.1 Secretory 429 ± 51 34.3 ± 3.8 0.098 ± 0.009° Untreated ovariectomy (n = 7) Before 0.088 ± 0.009 352 ± 66 17.5 ± 6.7 ND After 30 days 48 ± 4 0.136 ± 0.024* After 180 days 40 ± 4 ND 0.156 ± 0.021" Ovariectomy + ERT (n = 7) Before 0.080 ± 0.005 1.9 ± 0.9 238 ± 84 ND After 30 days 0.023 ± 0.0046 172 ± 29 ND After 180 days 0.028 ± 0.0036 150 ± 22 Values are the mean ± SEM. Statistical analysis was carried out using analysis of variance. ND, Not detectable [lower than the sensitivity of the assay (0.3 nmol/L)]. a P < 0.01, secretory vs. proliferative. b P < 0.05, after vs. before treatment.
instead, led to reduced values compared with the preoperative control level. Regression analysis between the levels of circulating ovarian hormones and FFA failed to show correlation among the variables in all the groups examined.
JCE & M • 1990 Vol 71 • No 5
on insulin secretion and sensitivity in ovariectomized patients (14a) demonstrate a significant increase in insulin release, and hence, the possible implication of insulin in the effect of estrogens on serum FFA levels. In any case, studies are required to demonstrate which factor plays a predominant role in physiological conditions. At present, we may discard the possibility of apparent FFA variations due to changes in sex hormonebinding globulin (SHBG) and in binding of FFA to SHBG, since variations in FFA exceed those reported for SHBG upon induction by estrogens (15); furthermore, our FFA assay method measures both carrierbound and soluble FFA. Previous studies (1-6) have provided convincing evidence that FFA-related changes in estrogen availability to target tissues might be of clinical relevance in the pathogenesis of breast cancer; we believe that it could now be worthwhile to extend the study of estrogen/ progesterone action on FFA balance in relation to pathogenesis of endometrial cancer.
Acknowledgments The authors are grateful to Mrs. S. Ferrazzini for skillful technical assistance and to Mr. G. Gilli for help in the statistical evaluation of the data.
References Discussion The present data document an influence of the ovary on the blood level of FFA, with rhythmic changes during the menstrual cycle and relatively higher levels during the follicular phase. Removal of the ovaries leads to consistently high blood levels of FFA; this effect is prevented by ERT. The effect of ERT on serum FFA levels in ovariectomized patients is clearly related to estrogenic hormones, which are the only variable parameter in these women with low serum progesterone, in pre- and postoperative conditions. Progesterone appears to have a contrary action, if any, as seen by the augmented ability of estrogens to lower serum FFA during ERT compared to levels in the normal luteal phase. In keeping with this interpretation, we observed diminished serum FFA levels on day 24 of the cycle, a time at which estradiol values are higher than on day 8 (our present data and Ref. 10). Several mechanisms might contribute to the reduction of serum FFA by estrogens. In fact, estrogens in vitro increase the hepatic uptake of FFA in the perfused liver or in isolated hepatocytes (11,12), whereas progesterone decreases it. In addition, estrogens decrease adipocyte lipolysis via cAMP-related mechanisms (13). Furthermore, estrogenrelated variations in insulin secretion have been documented (14). Preliminary data on the influence of ERT
1. Reed MJ, Cheng RW, Noel CT, Dudley HAF, James VHT. Plasma levels of oestrone, oestrone sulphate and oestradiol and the percentage of unbound oestradiol in postmenopausal women with and without breast cancer. Cancer Res. 1983;43:3940-3. 2. Bruning PF, Bonfrer JMG. Free fatty acid concentrations correlated with the available fraction of estradiol in human plasma. Cancer Res. 1986;46:2606-9. 3. Reed MJ, Beranek PA, Cheng RW, James VHT. Free fatty acids: a possible regulator of the available oestradiol fractions in plasma. J Steroid Biochem. 1986;24:657-9. 4. Martin ME, Vranckx R, Benassayag C, Nunez EA. Modification of properties of human sex steroid binding protein by nonesterified fatty acids. J Biol Chem. 1986;261:2954-9. 5. Benassayag C, Vallette G, Hassid J, Raymond JP, Nunez EA. Potentiation of estradiol binding to human proteins by unsatured nonesterified fatty acids. Endocrinology. 1986; 118:1-7. 6. Blomquist CH, Kotts CE, Habanson E. Phospholipase A2 inactivation of microsomal 17/3-hydroxysteroid oxidoreductase: rates of phospholipids hydrolysis and enzyme inactivation effects of hydrolysis products and properties of phospholipase A2-treated enzyme. Steroids. 1980;36:97-103. 7. Pansini F, Piccolo R, Bassi P, et al. Basal and forskolin-stimulated cyclic adenosine monophosphate in intact human platelets during the menstrual cycle. Am J Obstet Gynecol. 1986;154:679-82. 8. Regouw BJM, Cornerlissen PJHC, Helder RAP, Spijkers JBF, Weeber YMM. Specific determination of free fatty acids in plasma. Clin Chim Acta. 1971;31:187-95. 9. Armitage P. Statistical methods in medical research. Oxford: Blackwell; 1971;189-216. 10. Richardson GS. Hormonal physiology of the ovary and adrenal cortex. In: Gold JJ, Josimovich JB, eds. Gynecological endocrinology. New York: Plenum Press; 1987;57-81. 11. Kenagy R, Weinstein I, Heimberg M. The metabolism of free fatty acid by perfused livers from normal female and ovariectomized rats. Endocrinology. 1981;108:1613-21.
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COMMENTS 12. Okner RK, Lisenko N, Manning JA, Monroe SC, Burnett DA. Sex steroid modulation of fatty acid binding protein (FABP) concentration in rat liver. J Clin Invest. 1980;65:1013-23. 13. Pecquery R, Leneveu MC, Giudicelli Y. Estradiol treatment decreases the lipolytic responses of hamster white adipocytes through a reduction in the activity of the adenylate cyclase catalytic subunit. Endocrinology. 1986;118:2210-6.
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14. Costrini NV, Kalkhoff RK. Relative effects of pregnancy, estradiol and progesterone on plasma insulin and pancreatic islet secretion, J Clin Invest. 1971;50:992-9. 14a.Pansini F, et al. Ann NY Acad Sci. In Press. 15. Plymate SR, Moore DE, Cheng CY, Bardin CW, Southworth MB, Levinsky MJ. Sex hormone-binding globulin changes during the menstrual cycle. J Clin Endocrinol Metab. 1985;61:993-6.
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Vol. 71, No. 5 Printed in U.S.A.
Influence of Estrogens on Serum Free Fatty Acid Levels in Women* F. PANSINI, G. BONACCORSI, F. GENOVESI, M. R. FOLEGATTI, B. BAGNI, C. M. BERGAMINI, AND G. MOLLICA Institute of Obstetrics and Gynecology and Institute of Biochemistry (C.M.B.), University of Ferrara, and the Service of Nuclear Medicine, St. Anna Hospital (B.B.), 4410 Ferrara, Italy
ABSTRACT. A relationship between plasma levels of FFA and incidence of hormone-dependent breast cancer has been suggested. This observation has drawn our attention to possible complementary actions of ovarian steroids on circulating FFA levels. Measurements taken in normal women during the menstrual cycle and in ovariectomized women with and without
E
Mg/24 h (Estraderm, TTS-50, Ciba-Geigy, Italy, Orriggio) for 3 of 4 weeks, with 1 intervening treatment-free week. All of them were followed up over 6 months. Patients were encouraged to maintain their standard diets to avoid possible nutritional interferences in the study. In every case informed consent was obtained for this clinical investigation. All blood samples were taken after overnight fasting and
VIDENCE has been collected for a promoting action of FFA in estrogen-dependent breast cancer (1-3), probably through an increase in the availability of estrogens to the tissue or a decrease in their inactivation in situ. Indeed, it has been proved that FFA provoke a direct displacement of estrogens from their serum carrier proteins in order to increase the fraction of free estradiol (4), a tightening of estradiol binding to estrogen receptors in target tissues incubated in the presence of FFA (5), and a decrease in metabolic clearance of the hormone through 17/3-hydroxysteroid-dehydrogenase inhibition (6). To investigate the correlation between estrogens and plasma FFA, we planned the present study with the aim of ascertaining whether ovarian hormones themselves influence FFA serum levels in women during the normal menstrual cycle, upon ovariectomy, and under estrogen substitutive therapy (ERT).
were allowed to clot at room temperature. In the case of patients
receiving ERT, blood samples were taken during the last week of treatment. The concentrations of estradiol and progesterone were determined in frozen stored serum by RIA (7), while FFA levels were measured in unfrozen serum immediately after centrifugation by a colorimetric procedure (8). Analysis of variance testing was used for statistical evaluation of data (9) using the SPSS package; when necessary, the Scheffe procedure was used as a post-hoc test. Results
Materials and Methods Serum FFA levels were determined in 25 normal women during the secretory and proliferative phases (8th and 24th days of the cycle) and in 14 patients undergoing hysterectomy plus bilateral ovariectomy for uterine leiomyomatosis. Among the ovariectomized patients, only 7 received ERT by a transdermal therapeutic system delivering estradiol at a rate of 50 Received April 17, 1990. Address all correspondence and requests for reprints to: Francesco Pansini, M.D., Institute of Obstetrics and Gynecology, University of Ferrara, Via Savonarola 9, 4410 Ferrara, Italy. * This work was supported by 60% research funds from the Ministero deH'Universita e della Ricerca Scientifica e Tecnologica.
estrogen replacement therapy demonstrate that 1) levels of FFA present in serum are lower during the luteal phase than during the follicular phase; 2) levels of FFA are significantly higher after ovariectomy; and 3) these are again reduced by substitutive estrogen therapy. (J Clin Endocrinol Metab 7 1 : 1387-1389, 1990)
The measurement of FFA blood levels during the menstrual cycle (Table 1) demonstrated a significant decrease in serum FFA ( P < 0.01) during the luteal phase [0.098 ± 0.009 mg/L (mean ± SEM)] compared to levels in the follicular phase (0.130 ± 0.010). An influence of ovarian function on serum FFA was confirmed by experiments on 14 ovariectomized patients, some receiving E R T and others not; in the absence of estrogenic replenishment treatment, serum FFA levels consistently increased as early as 1 month after surgery and increased further at 6 months. In contrast, administration of exogenous estrogen prevented the increase in serum FFA after ovariectomy and,
1387
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1388
COMMENTS
TABLE 1. Relationship between the levels of circulating estradiol (E2) and progesterone (P), and serum FFA FFA (mg/L)
E2 (pmol/L)
P (nmol/L)
Normal menstrual cycle (n = 25) Proliferative 0.130 ± 0.010 257 ± 22 0.9 ± 0.1 Secretory 429 ± 51 34.3 ± 3.8 0.098 ± 0.009° Untreated ovariectomy (n = 7) Before 0.088 ± 0.009 352 ± 66 17.5 ± 6.7 ND After 30 days 48 ± 4 0.136 ± 0.024* After 180 days 40 ± 4 ND 0.156 ± 0.021" Ovariectomy + ERT (n = 7) Before 0.080 ± 0.005 1.9 ± 0.9 238 ± 84 ND After 30 days 0.023 ± 0.0046 172 ± 29 ND After 180 days 0.028 ± 0.0036 150 ± 22 Values are the mean ± SEM. Statistical analysis was carried out using analysis of variance. ND, Not detectable [lower than the sensitivity of the assay (0.3 nmol/L)]. a P < 0.01, secretory vs. proliferative. b P < 0.05, after vs. before treatment.
instead, led to reduced values compared with the preoperative control level. Regression analysis between the levels of circulating ovarian hormones and FFA failed to show correlation among the variables in all the groups examined.
JCE & M • 1990 Vol 71 • No 5
on insulin secretion and sensitivity in ovariectomized patients (14a) demonstrate a significant increase in insulin release, and hence, the possible implication of insulin in the effect of estrogens on serum FFA levels. In any case, studies are required to demonstrate which factor plays a predominant role in physiological conditions. At present, we may discard the possibility of apparent FFA variations due to changes in sex hormonebinding globulin (SHBG) and in binding of FFA to SHBG, since variations in FFA exceed those reported for SHBG upon induction by estrogens (15); furthermore, our FFA assay method measures both carrierbound and soluble FFA. Previous studies (1-6) have provided convincing evidence that FFA-related changes in estrogen availability to target tissues might be of clinical relevance in the pathogenesis of breast cancer; we believe that it could now be worthwhile to extend the study of estrogen/ progesterone action on FFA balance in relation to pathogenesis of endometrial cancer.
Acknowledgments The authors are grateful to Mrs. S. Ferrazzini for skillful technical assistance and to Mr. G. Gilli for help in the statistical evaluation of the data.
References Discussion The present data document an influence of the ovary on the blood level of FFA, with rhythmic changes during the menstrual cycle and relatively higher levels during the follicular phase. Removal of the ovaries leads to consistently high blood levels of FFA; this effect is prevented by ERT. The effect of ERT on serum FFA levels in ovariectomized patients is clearly related to estrogenic hormones, which are the only variable parameter in these women with low serum progesterone, in pre- and postoperative conditions. Progesterone appears to have a contrary action, if any, as seen by the augmented ability of estrogens to lower serum FFA during ERT compared to levels in the normal luteal phase. In keeping with this interpretation, we observed diminished serum FFA levels on day 24 of the cycle, a time at which estradiol values are higher than on day 8 (our present data and Ref. 10). Several mechanisms might contribute to the reduction of serum FFA by estrogens. In fact, estrogens in vitro increase the hepatic uptake of FFA in the perfused liver or in isolated hepatocytes (11,12), whereas progesterone decreases it. In addition, estrogens decrease adipocyte lipolysis via cAMP-related mechanisms (13). Furthermore, estrogenrelated variations in insulin secretion have been documented (14). Preliminary data on the influence of ERT
1. Reed MJ, Cheng RW, Noel CT, Dudley HAF, James VHT. Plasma levels of oestrone, oestrone sulphate and oestradiol and the percentage of unbound oestradiol in postmenopausal women with and without breast cancer. Cancer Res. 1983;43:3940-3. 2. Bruning PF, Bonfrer JMG. Free fatty acid concentrations correlated with the available fraction of estradiol in human plasma. Cancer Res. 1986;46:2606-9. 3. Reed MJ, Beranek PA, Cheng RW, James VHT. Free fatty acids: a possible regulator of the available oestradiol fractions in plasma. J Steroid Biochem. 1986;24:657-9. 4. Martin ME, Vranckx R, Benassayag C, Nunez EA. Modification of properties of human sex steroid binding protein by nonesterified fatty acids. J Biol Chem. 1986;261:2954-9. 5. Benassayag C, Vallette G, Hassid J, Raymond JP, Nunez EA. Potentiation of estradiol binding to human proteins by unsatured nonesterified fatty acids. Endocrinology. 1986; 118:1-7. 6. Blomquist CH, Kotts CE, Habanson E. Phospholipase A2 inactivation of microsomal 17/3-hydroxysteroid oxidoreductase: rates of phospholipids hydrolysis and enzyme inactivation effects of hydrolysis products and properties of phospholipase A2-treated enzyme. Steroids. 1980;36:97-103. 7. Pansini F, Piccolo R, Bassi P, et al. Basal and forskolin-stimulated cyclic adenosine monophosphate in intact human platelets during the menstrual cycle. Am J Obstet Gynecol. 1986;154:679-82. 8. Regouw BJM, Cornerlissen PJHC, Helder RAP, Spijkers JBF, Weeber YMM. Specific determination of free fatty acids in plasma. Clin Chim Acta. 1971;31:187-95. 9. Armitage P. Statistical methods in medical research. Oxford: Blackwell; 1971;189-216. 10. Richardson GS. Hormonal physiology of the ovary and adrenal cortex. In: Gold JJ, Josimovich JB, eds. Gynecological endocrinology. New York: Plenum Press; 1987;57-81. 11. Kenagy R, Weinstein I, Heimberg M. The metabolism of free fatty acid by perfused livers from normal female and ovariectomized rats. Endocrinology. 1981;108:1613-21.
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COMMENTS 12. Okner RK, Lisenko N, Manning JA, Monroe SC, Burnett DA. Sex steroid modulation of fatty acid binding protein (FABP) concentration in rat liver. J Clin Invest. 1980;65:1013-23. 13. Pecquery R, Leneveu MC, Giudicelli Y. Estradiol treatment decreases the lipolytic responses of hamster white adipocytes through a reduction in the activity of the adenylate cyclase catalytic subunit. Endocrinology. 1986;118:2210-6.
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14. Costrini NV, Kalkhoff RK. Relative effects of pregnancy, estradiol and progesterone on plasma insulin and pancreatic islet secretion, J Clin Invest. 1971;50:992-9. 14a.Pansini F, et al. Ann NY Acad Sci. In Press. 15. Plymate SR, Moore DE, Cheng CY, Bardin CW, Southworth MB, Levinsky MJ. Sex hormone-binding globulin changes during the menstrual cycle. J Clin Endocrinol Metab. 1985;61:993-6.
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