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Reproductive Endocrinology |
Department of Obstetrics and Gynecology (C.M., G.F.), and Division of Neonatology (G.B., D.G., I.S.), University of Siena, Siena, Italy
Address all correspondence and requests for reprints to: Cosimo Massafra, Department of Obstetrics and Gynecology, University of Siena, Via P. Mascagni, 46, 53100 Siena, Italy.
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Abstract |
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Introduction |
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As hydrogen donors from their phenol-hydroxyl ring, estrogens, like vitamin E, have been reported to have in vitro antioxidant effects on membrane phospholipid peroxidation (5). It is not yet clear whether a relation exists between the sex steroid hormones and the cellular antioxidant enzyme system. An increase in erythrocyte GSH-Px (6, 7) and CAT (7) activities has been shown in prolonged oral contraceptive users. The present investigation was undertaken to compare erythrocyte SOD, CAT, and GSH-Px activities in healthy eumenorrhoic women (EW) in the midfollicular phase of the menstrual cycle, and in hypothalamic amenorrhoic patients (AP), and to determine the effects of 17ß-estradiol (E2), medroxyprogesterone-acetate (MPA), and combined E2-MPA replacement therapies on these activities. Malondialdehyde (MDA) plasma concentrations were also evaluated as an index of lipid peroxidation and peroxidative tissue injury (8).
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Materials and Methods |
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The study population consisted of 20 healthy eumenorrhoic volunteers (EW) (age 19–28 yr; mean age 23.3 yr) evaluated on day 7 of the menstrual cycle, and 48 patients (age 18–26 yr; mean age 21.8 yr) with secondary hypothalamic amenorrhea (AP), with duration ranging between 12 and 36 months. All were nonsmokers. Body weight was normal and diet was typical Mediterranean. Appropriate ovarian function was checked in EW by evaluating basal body temperature during the cycle immediately preceding that of the study. Diagnosis of the hypothalamic amenorrhoic condition was made according to criteria reported elsewhere (9). The subjects did not have a history of intensive exercise. None of the women had been on any type of medication in the previous 6 months, and no other drugs were prescribed or taken during the study. Informed consent was obtained from all subjects before enrollment in the study.
Study design
Plasma samples were collected, and plasma levels of E2 and MDA and the erythrocyte antioxidant enzyme activities of SOD, CAT, and GSH-Px were evaluated at the same time and under the same conditions in EW on day 7 of the menstrual cycle and in AP (time 0). The AP were randomly divided into four subgroups of 12 subjects. The subgroups were treated as follows: transdermal therapy for 30 days with 8-mg E2 patches (changed twice a week) providing a nominal daily dose of E2 of 100 µg (Estraderm TTS 100, Ciba-Geigy, Origgio, Varese, Italy) (subgroup A); 10 mg of oral MPA for 30 days (Farlutal, Farmitalia, Milano, Italy) (subgroup B); or transdermal E2 therapy plus oral MPA for 30 days at the same doses (subgroup C). The fourth subgroup acted as control. Plasma levels of E2 and MDA and the activities of SOD, CAT, and GSH-Px were measured in subgroups A, B, and C after a month of treatment and in the control subgroup on the same day. Erythrocyte pyruvate-kinase (PK) activity, which is correlated with red blood cell age, was also assayed in all subjects as an index of erythrocyte populations (10).
Methods and statistical analysis
Blood samples (10 ml) were taken from the antecubital vein into heparinized tubes at 0800 h after overnight fasting. The plasma was immediately centrifuged and frozen at -30 C. The erythrocytes were immediately washed three times in normal saline, deprived of buffy coats, and stored at -80 C. The samples from EW and AP groups were stored under similar conditions for similar times until the assay, performed in a single matrix at the end of the study period (1 month). E2 was determined by RIA, using a Biodata kit (Estradiol Maia, code 12264, Biodata, Guidonia Montecelio, Roma, Italy). The intra- and interassay coefficients of variation were 2.2–7.1% and 7.9–9.8%, respectively. SOD activity was assayed by the method of Beauchamp and Fridovich (11). The activities of CAT, GSH-Px, and PK were determined by the method of Beutler (12). MDA plasma concentrations were evaluated by HPLC, according to the method of Carbonneau et al. (13).
The data, expressed as means ± SD were analyzed for statistically significant differences by Student’s t test for unpaired data (EW group vs. AP group at time 0 and EW group vs. AP subgroups on day 30) and Student’s t test for paired data (AP subgroups at time 0 vs. the same subgroups on day 30) using the SPSS/PC + 4 statistical package (SPSS Inc., Chicago, IL). A multiple comparison of means was performed by Scheffé’s multiple comparison procedure (14).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows MDA plasma concentrations and SOD,
CAT, and PK in our population. The variations in E2 plasma
levels and GSH-Px activity in the same groups are reported in
Fig. 1. In subgroups A and C, a statistically
significant positive correlation was observed between E2
plasma levels and erythrocyte GSH-Px enzyme activity on day 30 of
treatment (subgroup A: r = 0.752, P = 0.004;
subgroup C: r = 0.717, P = 0.008). The multiple
comparison procedure showed that mean E2 and GSH-Px data
from EW, AP subgroup A, and AP subgroup C were equal and significantly
greater at a 5% confidence level than the same parameters in AP
subgroup B and AP control group. In EW, no correlation was observed on
day 7 between E2 plasma levels and GSH-Px erythrocyte
activity.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In women on prolonged courses of low-dose oral contraceptives, we showed a significant increase first in GSH-Px (sixth cycle), and later in CAT (ninth cycle) (7). These findings confirm those of Capel et al. (6) and suggest that sex steroids may have an antioxidant activity that is mediated by an increase in these enzymes. In the present study, the difference in GSH-Px activity between EW and AP, and the increase in this activity in AP treated with E2 or combined E2-MPA, but not MPA alone, indicate that estrogens stimulate the activity of this enzyme. This is also demonstrated by the significant positive correlation between GSH-Px activity and plasma levels of E2 in these two subgroups of AP 1 month after the start of treatment. The existence of a positive statistical relationship between GSH-Px erythrocyte activity and E2 plasma levels was demonstrated by a time-series analysis during the normal menstrual cycle (our unpublished observations). These results suggest that a lack of ovarian E2 production in women is associated with reduced protection against oxidative stress. This is in line with results by Asada et al. (18), which showed that serum lipid peroxide levels of normal premenopausal women were lower than those of normal postmenopausal women, and that bilateral ovariectomy significantly increases serum lipid peroxide in premenopausal women. On the other hand, our results are in contrast with those of Kanaley and Ji (19), who demonstrated that the GSH-Px activity was significantly higher in AP athletes than EW athletes at rest and during exercise. This result could simply reflect their training status, because training has been reported to increase this enzyme in humans (20). Kanaley and Ji’s study (19) is in fact of little significance as far as an valuation of the relationship between estrogen levels and GSH-Px activity is concerned, because no comparison is reported between AP athletes and AP sedentary women.
Estrogens, like vitamin E, have a substantial capacity to inhibit lipid peroxidation caused by free radicals in vitro (5, 15). The results of the present study indicate that E2 may also have an antioxidant effect via modulation of intracellular GSH-Px activity. This may be the result of an effect of this hormone on bone marrow erythroblast maturation with stimulation of the synthesis of active new GSH-Px molecules. This is an important finding because oxidative damage has a definite pathogenetic role in aging and various disease processes, including cancer, immune complex-mediated disease, rheumatoid arthritis, various lung disorders, and ischemia-reperfusion injury (1, 2, 3). In the case of cardiovascular disease, it is well known that the drop in estrogens occurring at menopause tends to cause a rise in female morbidity and mortality caused by this pathology, approaching levels found in males (21), whereas estrogen replacement in postmenopausal women has been shown to have antiatherogenic effects (17).
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Acknowledgments |
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Footnotes |
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Received May 13, 1996.
Revised August 19, 1996.
Accepted September 11, 1996.
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This article has been cited by other articles:
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  E. J. Ha and A. M. Smith Plasma Selenium and Plasma and Erythrocyte Glutathione Peroxidase Activity Increase with Estrogen during the Menstrual Cycle J. Am. Coll. Nutr., February 1, 2003; 22(1): 43 - 51. [Abstract] [Full Text] [PDF]
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 J. Pfeilschifter, R. Koditz, M. Pfohl, and H. Schatz Changes in Proinflammatory Cytokine Activity after Menopause Endocr. Rev., February 1, 2002; 23(1): 90 - 119. [Abstract] [Full Text] [PDF]
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