Objective: To assess the effects of short-term transdermal E, administration on nitric oxide (NO) plasma levels in postmenopausal women. Design: Randomized, placebo-controlled trial. Setting: Healthy volunteers in an academic research environment.
Patient(s): Twenty-eight healthy postmenopausal women. Intervention(s): Transdermal administration of E, (100 pg/d) or placebo on days 1 and 4 of a l-week treatment regimen. Main Outcome Measure(s): Serum concentrations of E, and plasma concentrations of NO stable oxidation products were assessed on day 1, before placement of the patch, and subsequently on days 2, 3, and 6. Result(s): The mean concentration of NO metabolites on days 2, 3, and 6 was significantly greater in the E, group (40.08 5 15.42 pmol/L, 38.05 _+ 18.82 pmol/L, and 42.03 5 16.81 pmol/L on days 2, 3, and 6, respectively) compared with both baseline levels (23.07 + 5.79 PmolL) and the placebo group (23.51 2 4.06 PmolIL, 21.64 + 4.72 Fmol/L, and 21.81 2 4.46 pmol/L on days 2, 3, and 6, respectively). Conclusion(s): During a l-week treatment regimen with transdermal E,, plasma levels of NO in postmenopausal women were significantly higher than baseline levels on days 2, 3, and 6. This suggests that the effect of estrogens on NO synthesis is rapid and that it is maintained with repeated administration. (Fertil SteriP 1998;69:58-61. 01998 by American Society for Reproductive Medicine.

CICINELLI E.

IGNARRO L.J.

MATTEO M.

SCHONAUER L.M

IGNARRO L. J.

SCHONAUER L. M.

MATTEO, MARIA

Archivio Istituzionale della Ricerca- Università degli Studi di Foggia

FERTILITY AND STERILITY” VOL. 69, NO. I, JANUARY 1998 Copyright Q1998 American Society for Reproductive Medicine Published by Elseviar Science Inc. Printed on acid-free paper in U.S.A. Received April 21, 1997. * Department of Obstetrics and Gynecology, University of Bari. t Reprint requests: Ettore Cicinelli, M.D., Department of Obstetrics and Gyn- ecology, University of Bari, Policlinico, Piazza Giulio Cesare, 70124 Bad, Italy. (FAX: +39-80-5473229; e-mail: SCHONAUER@ CIMEDOC.UNIBA.Il-). $ Department of Pharmacology, University of California, Los Angeles. 00150282/98/$19.00 PII s0015-0282(97)00424-x Effects of short-term transdermal estradiol administration on plasma levels of nitric oxide in postmenopausal women Ettore Cicinelli, M. D.,*t Louis J. Ignarro, M. D.,$ Luca Maria Schdnauer, M.D.,* Maria Giuseppina Matteo, M.D.,* Pietro Galantino, M.D.,* Gabriella Balzano, M.D.* University of Bari, Bari, Italy, and University of California, Los Angeles, Los Angeles, California Objective: To assess the effects of short-term transdermal E, administration on nitric oxide (NO) plasma levels in postmenopausal women. Design: Randomized, placebo-controlled trial. Setting: Healthy volunteers in an academic research environment. Patient(s): Twenty-eight healthy postmenopausal women. Intervention(s): Transdermal administration of E, (100 pg/d) or placebo on days 1 and 4 of a l-week treatment regimen. Main Outcome Measure(s): Serum concentrations of E, and plasma concentrations of NO stable oxidation products were assessed on day 1, before placement of the patch, and subsequently on days 2, 3, and 6. Result(s): The mean concentration of NO metabolites on days 2, 3, and 6 was significantly greater in the E, group (40.08 5 15.42 pmol/L, 38.05 _+ 18.82 pmol/L, and 42.03 5 16.81 pmol/L on days 2, 3, and 6, respectively) compared with both baseline levels (23.07 + 5.79 PmolL) and the placebo group (23.51 2 4.06 PmolIL, 21.64 + 4.72 Fmol/L, and 21.81 2 4.46 pmol/L on days 2, 3, and 6, respectively). Conclusion(s): During a l-week treatment regimen with transdermal E,, plasma levels of NO in postmenopausal women were significantly higher than baseline levels on days 2, 3, and 6. This suggests that the effect of estrogens on NO synthesis is rapid and that it is maintained with repeated administration. (Fertil SteriP 1998;69:58-61. 01998 by American Society for Reproductive Medicine.) Key Words: Estradiol, transdermal administration, nitric oxide, postmenopausal women, hormone replacement therapy, nitrite It currently is estimated that the favorable changes in serum lipids and lipoproteins that are induced by postmenopausal estrogen re- placement therapy (ERT) may account for only 30% of the reduction in the incidence of car- diovascular disease that is observed in the treated population (1). Additional mechanisms, such as the direct effect of estrogens on arter- ies, may play an important role in cardiopro- tection. Estrogens induce vasodilation in both pelvic and extrapelvic arteries (2-4), and they poten- tiate the endothelium-dependent vasodilation in both conductance and resistance arterial dis- tricts (5,6). Several studies support the hypoth- esis that estrogens influence the release of ni- tric oxide (NO) from the vascular endothelium (7-l 1). It is known that NO mediates the vas- cular relaxation produced by various endothe- hum-dependent vasodilators, such as acetyl- choline and bradykinin (12, 13). Rosselli and co-workers (11) observed a significant increase in NO plasma levels in postmenopausal women who were treated for 58 at least 6 months with continuous administration of E, (50 lug/d) plus norethisterone acetate (1 mg/d) on days 1 through 12 of each month. These investigators speculated that the effects of estrogens were rapid and receptor-operated, and that the upregulation of estrogen receptors in the vasculature of postmenopausal women contributed to the increased NO synthesis. Accordingly, we recently have demonstrated that plasma levels of NO in postmenopausal women 24 hours after transdermal E, administration are significantly higher than baseline levels, and that the percentage increase in NO plasma levels correlates with serum levels of E, (9). For a better understanding of mechanisms involved in the estro- gen-induced increase in NO production, we measured NO plasma levels on different days during short-term (l-week) E, treatment in postmenopausal women. The production of NO, which is an extremely labile molecule, was assessed by monitoring plasma levels of ni- trite and nitrate, the two stable oxidation products of NO metabolism. Levels of these stable compounds have been used as markers for NO synthase activity in vivo (8-11, 14). Metabolic tracer studies in humans with the use of L-[gua- nidino-‘5N,]arginine have demonstrated that increased ni- trate production in serum in vivo was derived from NO generated from the terminal guanidino nitrogen atom of labeled L-arginine ( 15). MATERIALS AND METHODS The study, designed as a randomized, placebo-controlled trial, was approved by the institutional review board at our institution. Twenty-eight women in spontaneous postmeno- pause who had not undergone hysterectomy were enrolled in the study. All subjects were counseled about the nature and purpose of the study and signed a detailed consent form. Menopausal status was confirmed by the absence of men- struation for at least 1 year and a serum concentration of FSH of >40 mIU/mL. No women had been in menopause for longer than 10 years, and they all were of normal weight. None had ever received hormone replacement therapy or had had coronary artery disease, hypertension, or diabetes, and none had taken any medication known to affect blood pres- sure within the previous 12 months. Alcohol, caffeine, and smoking were prohibited for 24 hours before and during the study. Women were assigned randomly to receive transdermal E, patches (100 pg/d, Estraderm 0.1; CIBA Pharmaceutical, Basel, Switzerland) or a nonmedicated patch on days 1 and 4 of the l-week treatment period. Randomization was done by opening sealed envelopes containing computer-generated block randomization numbers. Blood samples were collected at 8 A.M. after overnight fasting on day 1, before placement of the patch, and on days 2, 3, and 6. Women in both groups were given the same meals containing aliments with poor nitrite content beginning 24 hours before the first blood sampling and continuing throughout the study period. All blood samples were divided into titrated and nonci- trated tubes and centrifuged immediately in a refrigerated centrifuge at 2,000 X g for 10 minutes; both the plasma and the serum were frozen at -20°C until assayed. Serum E, (normal menopausal values, <20 pg/mL) was assayed with the use of a no-extraction iodine-125 RIA (DI RIA-ESTR; Sorin Biomedica, Saluggia, Italy). The sensitivity of the assay was 10 pg/mL and the variation coefficient was ~6% in the low range of values. Nitric oxide production was assessed by monitoring plasma levels of nitrite and nitrate, the two stable oxida- tion products of NO metabolism, using procedures that were described previously by this laboratory (14). Briefly, chemiluminescence detection was used to quantify both nitrite and nitrate in samples of plasma that were depro- teinized by mixing two volumes of cold ethanol with one volume of plasma, then centrifuging to remove the pre- cipitate. Supernatant fractions (0.1 mL) were analyzed by refluxing in acidic vanadium (III) under argon as de- scribed previously (14). This method detects and is quantitative for nitrite plus nitrate, both of which are reduced to NO, which subse- quently is assayed by chemiluminescence detection. This technique is more sensitive than standard calorimetric meth- ods, and it has been validated and used to monitor NO formation both in vitro and in vivo (14). Clinical characteristics of the women in both groups were compared by unpaired Student’s t-test. The variation in NO metabolites and E, concentrations was evaluated statistically with the use of analysis of variance and Student-Newman- Keuls test. Concentrations in the two groups were compared by unpaired Student’s t-test; 95% confidence intervals also were calculated for the observed mean differences. All val- ues were given as means + SD. P < 0.05 was defined as statistically significant. RESULTS The E, and placebo groups were shown to be homoge- neous for age (54.42 + 2.37 years versus 55.35 + 2.68 years), years since menopause (4.07 ? 2.61 years versus 5.07 +_ 1.94 years), and body mass index (23.25 2 1.33 kg/m* versus 22.99 t_ 1.54 kg/m*). The concentrations of NO stable metabolites and E, on different days are reported in Table 1. Baseline E, serum concentrations were in the menopausal range in both groups. In women who received medicated patches, serum E, concentrations on days 2, 3, and 6 were significantly higher than baseline concentrations (F = 332.16, & = 52, P < 0.000001) and higher than the corresponding concentrations in the control group. FERTILITY & STERILITY@ 59 Mean levels of E,, nitrite, and nitrate on different days of treatment in 28 women who received either transdermal E, or placebo on days 1 and 4 of treatment. Variable Ez (pg/d) W/NO, (PmolW Treatment E, group daY (mean F SD) 1 13.78 -c 3.66 2 103.71 i- 11.87 3 83.28 _’ 7.96 6 79.28 _’ 6.22 1 22.43 I 5.45 2 40.08 2 15.42 3 38.05 ? 18.82 6 42.03 2 16.81 Placebo group (mean 2 SD) 15.00 t 3.39 12.14 2 2.62 13.21 2 3.94 15.43 -c 2.17 22.73 ? 3.49 23.51 lr 4.06 21.64 2 4.72 21.81 k 4.46 Mean difference Unpaired Student’s (95% CI) t-test 1.21 (-1.52-3.94) 0.91 91.57 (84.89-98.25) 28.19 70.07 (65.20-74.94) 29.52 63.86 (60.2467.48) 36.27 0.31 (-3.25-3.87) 0.18 16.58 (7.82-25.34) 3.89 16.41 (5.74-27.08) 3.16 20.22 (10.66-29.78) 4.35 P value 0.371 <0.000001 <0.000001 <0.000001 0.860 <O.OOl <0.005 <0.0002 Mean baseline concentrations of NO metabolites were similar in both the treated and placebo groups. At subsequent evaluations, they increased significantly only in the E, group (F = 4.97, df = 52, P < O.OOS), in which mean levels of NO metabolites on days 2,3, and 6 were significantly higher than baseline levels. On the same days, the levels of NO metab- olites in the E, group also were significantly higher than those in the control group. DISCUSSION Short-term ERT improves flow-mediated endothelium- dependent vasodilation in postmenopausal women (6). The data in this study demonstrate that this vasodilatory effect may be related to a significant increase in plasma levels of NO induced by estrogens, and they provide further evidence that an NO-mediated mechanism may contribute to the car- dioprotective effects of ERT. The effect of estrogens on NO metabolism already has been investigated in vitro and in vivo, in both animals and humans (7, 8, 10). However, few studies have evaluated the effect of ERT on NO metabolism in postmenopausal women. Rosselli and coworkers (11) reported long-term data of post- menopausal women treated with E, combined with norethis- terone acetate. In a previous study, we demonstrated that transdermal administration of E, in postmenopausal women induces a significant increase in NO plasma levels 24 hours after administration (9). Therefore, we undertook this study to evaluate whether the beneficial effect of E, on NO me- tabolism also is maintained with repeated administration. According to our previous findings, plasma levels of NO metabolites 24 hours after E, administration (day 2 of treat- ment) were significantly higher than baseline levels (9); at subsequent evaluations, on days 3 and 6, the levels did not vary significantly from those observed on day 2. This sug- gests two things: [l] that the positive effect of E, on NO metabolism does not decrease with subsequent administra- tion, and [2] that the effect of estrogens is rapid, probably reaching its peak within the first 24 hours of therapy. The results of this study also indicate that the effect of estrogens does not increase with subsequent administration, which is consistent with the finding that changes in steroid nuclear receptors in most circumstances occur within hours (16). Our results agree in part with those of Rosselli and co-workers (1 l), who also reported low baseline values of NO metabolites in postmenopausal women, probably in re- lation to a postmenopausal hypoestrogenic situation, al- though NO plasma levels during therapy were about 35% lower. We can only speculate on whether this difference is due to the higher E, dose used in our study or to the effects of norethisterone acetate. Nitric oxide is a labile molecule with a half-life of a few seconds that degrades to the stable metabolites, nitrite and nitrate (7-l 1, 14). Plasma levels of nitrite are known to be extremely low (approaching zero) in most normal individu- als, but plasma levels of nitrate are reasonably constant at around 25 to 45 kmol/L (17). The baseline concentrations of NO metabolites in both study groups were slightly lower than 25 pmol/L, probably as a result of the postmenopausal E, deficiency. At subse- quent evaluations, the concentrations of NO metabolites increased significantly only in women who were treated with transdermal E,. This agrees with the previous findings of Ramsay and associates (10) in premenopausal women who were pre- treated first with GnRH analogues, then with oral estrogens. Our results also demonstrate that estrogen-induced NO pro- duction occurs in postmenopausal women, who are much older than those studied by Ramsay and associates (10). Because our study was drug- and diet-controlled, only dif- ferences in E, absorption and NO metabolism could account for the wide intersubject variability that we observed on days 2,3, and 6 in the percentage increase in the concentrations of NO metabolites. We can only guess which cells are the main source of the 60 Cicinelli et al. Short-term ERT and plasma levels of NO Vol. 69, No. 1, January 1998 observed increase in circulating concentrations of NO me- tabolites because several cell types other than endothelial cells also are known to produce NO (18). Further, only endothelial cells and neuronal cells have both constitutive and inducible forms of NO synthase, whereas most of the other cell types have only inducible NO synthase (18). Because E, stimulates the basal release of NO from endo- thelium (7), it is possible that the increases in circulating levels of nitrite and nitrate that we observed are solely endothelium-derived. In conclusion, short-term transdermal administration of E, to healthy postmenopausal women induces a rapid and sustained increase in plasma concentrations of NO compared with baseline levels. Plasma levels of NO on day 2 of treatment were similar to those on day 6 of treatment. This finding supports the hypothesis that the effect of estrogens on NO synthesis is rapid, probably receptor-operated, and maintained with repeated administration. This study pro- vides further evidence that an NO-related mechanism may contribute to the cardiovascular protective effect of ERT in postmenopausal women. Acknowledgments: The authors acknowledge the expert technical assistance of Mr. Russell Byms (Department of Pharmacology, University of Califor- nia, Los Angeles, Los Angeles, California) and Ms. Elide Cataldi (Depart- ment of Obstetrics and Gynecology, University of Bari, Bari, Italy). References 1. Barret-Connor E. 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Estrogen improves endothelium-dependent, flow-mediated vasodilation in post-menopausal women. Ann Intern Med 1994;121: 936-41. 7. Hayashi T, Fukuto JM, Ignarro LJ, Chaudhuri G. Basal release of nitric oxide from aortic rings is greater in female rabbits than in male rabbits: implications for atherosclerosis. Proc Nat1 Acad Sci USA 1992;89: 11259-63. 8. Rosselli M, Imthurn B, Macas E, Keller PJ, Dubey RK. Circulating nitrite/nitrate levels increase with follicular development: indirect evi- dence for estradiol mediated NO release. Biochem Biophys Res Com- mun 1994;202:1543-52. 9. Cicinelli E, Ignarro LJ, Lograno M, Matte0 G, Falco N, Schonauer LM. Acute effects of transdermal estradiol administration on plasma levels of nitric oxide in post-menopausal women. Fertil Steril 1997;67:63-6. 10. Ramsay B, Johnson MR, Leone AM, Steer PJ. The effect of exogenous oestrogen on nitric oxide production in women: a placebo cornrolled crossover studv. Br J Obstet Gvnaecol 1995:102:417-9. 11. Rosselli M, Imthum B, Keller PJ, Jackson EK, Dubey RK. Circulating nitric oxide (nitrite/nitrate) levels in post-menopausal women substi- tuted with 17/3-estradiol and norethisterone acetate: a two-year fol- low-up study. Hypertension 1995;25:848-53. 12. Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 1980; 288:373-6. 13. Ignarro LJ, Buga GM, Wood KS, Byms RE, Chaudhuri G. Endotheli- urn-derived relaxing factor produced and released from arteries and veins is nitric oxide. Proc Natl Acad Sci USA 1987;84:9265-9. 14. Kiyozo M, Ihnken K, Buckberg GD, Sherman MP, Young HH, Ignarro LJ. Role of controlled cardiac reoxygenation in reducing nitric oxide production and cardiac oxidant damage in cyanotic infantile hearts. J Clin Invest 1994;93:2658-66. 15. Hibbs JB, Westenfelder C, Taintor R, Vavrin Z, Kablitz C, Baranowski RI.,. et al. Evidence for cvtokine-inducible nitric oxide svnthesis from &ginine in patients r&eiving interleukin-2 therapy. J Clin Invest l wJ?: R9~8hl - l l  ____,__ .-_. . 16. Lin AL, Gonzales R Jr, Carey KD, Shain SA. Estradiol-17 beta affects estrogen receptor distribution and elevates progestogen receptor content in baboon aorta. Arteriosclerosis 1986,6:495-504. 17. Lee K, Greger JL, Consaul JR, Graham KL, Chinn BL. Nitrate, nitrite balance and de novo synthesis of nitrite in humans consuming cured meats. Am J Clin Nutr 1986;44:188-94. 18. Ignarro LJ. Physiological significance of endogenous nitric oxide. Se- min Perinatol 1991;15:20-6. FERTILITY 8~ STERILITY@ 61 

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Effects of short-term transdermal estradiol administration on plasma levels of nitric oxide in postmenopausal women.

Effects of short-term transdermal estradiol administration on plasma levels of nitric oxide in postmenopausal women.

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