Serum vitamin E

Serum vitamin E, Copper and Zinc Levels in Postmenopausal Women Taking Hormone Replacement Therapy

The objectives of this study were to investigate the serum vitamin E, copper and zinc status in postmenopausal women who took hormone replacement therapy (HRT). Sixty healthy postmenopausal women (30 were under HRT and 30 were not) were studied. Biochemical measurements, anthropometric measurements and interviews of the subjects were done. No significant difference was found between HRT and non-HRT groups in terms of mean age, BMI, and daily dietary intakes of vitamin E, copper and zinc. There were no significant differences between levels of vitamin E and zinc in two studied groups .Mean serum copper levels and the copper/zinc ratio were significantly higher in the HRT group. The results of this study confirm the increase in serum copper levels among postmenopausal women who take hormonal replacement therapy.

There is a large body of evidence indicating that cardiovascular diseases (CVD) due to atherosclerosis are the more important causes of death in postmenopausal women (1). It is believed that one reason for this increased risk of CVD in postmenopausal women is due to a reduction in female hormones (1, 2). In view of these data, hormone replacement therapy (HRT) for postmenopausal women has become popular in recent years (3). According to some evidence, estrogen may be of benefit in reducing the risk of heart disease in postmenopausal women. Although this beneficial effect could be attributed to its effects on lipid metabolism, endothelium, and intrinsic antioxidant activity of estrogen, the overall harm or benefit of HRT is still controversial (1).

In the past three decades, the role of dietary antioxidants in human health and prevention of many diseases has received much attention due to expansion of the free radical theory.The contribution of free radical damage to the development of atherosclerosis is well established (4).Vitamin E (a-tocopherol), known as the “antioxidant vitamin”, is a fat-soluble vitamin that can act as an effective free radical scavenger in the biological membrane, where it contributes to membrane stability (5). According to some studies, there is a geographical correlation between low level of vitamin E and high rates of cardiovascular diseases (CVD) and cancer (6).

There are also controversial results with respect to the influence of HRT on antioxidant levels in women. According to some in vitro studies, estradiol can regenerate oxidized vitamin E radicals (7). On the other hand, it has been shown that in young women taking estrogen containing oral contraceptives, the plasma vitamin E level may be decreased (8). Wu et al have not found any significant difference in vitamin E levels between premenopausal and postmenopausal women (3). These authors have also reported no significant change in plasma vitamin E levels following six or twelve months HRT (3, 8). Association between changes in estrogen levels and the altered vitamin C levels has been also mentioned in some in vitro (9) and in vivo (10) studies.

Copper and zinc are components of endogenous antioxidant enzyme systems such as superoxide dismutase (SOD) which regulates the intercellular concentration of superoxide anion by converting it to hydrogen peroxide.A reduction in Cu/Zn SOD may cause oxidative damage to membranes and other cellular structures (11). The interaction between HRT and these trace elements have been investigated by some researchers, but the results are not sconclusive. Recently, the status of trace minerals in postmenopausal women and the effect of HRT on these elements have been examined by Bureau et al (12) and Meram et al (13). In their studies the beneficial influences of HRT on trace mineral status related to menopause have been reported.

Taking into account the risk of nutritional disturbances, particularly vitamin and trace element deficiencies are striking during menopause and the existence of controversial results concerning the possible effects of HRT on antioxidant vitamins and trace elements, in the present study, the serum levels of vitamin E, copper and zinc in postmenopausal women with or without HRT are investigated.

This cross sectional study was carried out on two groups of postmenopausal women who attended the Obstetrics and Gynecology clinic of Tabriz University of Medical Sciences.

All of subjects had not undergone hysterectomy and were amenorrheic for at least one year and had FSH>25 IU/ml. None had a history of liver or vascular disease. None of subjects were receiving any drug known to affect liver metabolism and none of them were receiving vitamin or mineral supplements before the study. No subject had diabetes, or was a cigarette smoker. All subjects provided informed consent.

The HRT- treated group consisted of thirty healthy postmenopausal women with mean age of 51.69 ± 3.46 years who were treated with continuous HRT [0.625 mg conjugated equine estrogen (CEE) and 2.5 mg medroxyprogesterone (MPA) daily] at least for a three months. Non-HRT group consisted of thirty healthy postmenopausal women with mean age of 52.34 ±3.10 years, who were not taking any form of HRT.

Dietary intake was evaluated on three consecutive days by a face-to- face interview using a 24-hour dietary recall method (14). The body mass index (BMI) was calculated as the weight in kilograms divided by the square of height in meters (kg/m²).

The blood sample was obtained from each subject under fasting condition and protected against light. Serum was separated by centrifugation at 2000 g for 15 minute and stored frozen at –70^°C until assay. Serum vitamin E was determined by high performance liquid chromatography as described by Bieri et al (15). Serum copper and zinc were measured with atomic absorption spectroscopy (16).

Data are given as mean ± standard deviation (S.D), and also the number and percent of subjects based on biochemical status. The comparisons between two groups were made by using unpaired Student’s t-test (table 1) and X² test (table 2);results were considered significant if p<0.05.

As shown in table 1,no significant differences between the study groups were observed with regard to any of the anthropometric data. In both groups the mean of height, weight and BMI were similar. The mean daily dietary intake of vitamin E, copper and zinc were also similar in both groups.

No significant difference was found in mean of serum vitamin E levels between HRT and non-HRT groups. The mean serum copper levels was significantly higher in HRT group compared to non- HRT group (144.24 ± 25.47 mg/dl vs. 122.47 ± 24.89 mg/dl, p<0.02), but no significant difference was found in serum zinc levels between two groups. The mean copper/zinc ratio in HRT group was also significantly higher than non-HRT group (p<0.002).

Table 2 shows the distribution of subjects based on serum vitamin E, copper and zinc status (17). Approximately majority of the subjects had serum levels of these nutrients in normal range, so X² test showed no significant differences between two groups.

Table 1: Basic characteristics and biochemical status of the study population (Mean±SD)

	groups		P. value
	HRT n=30	non-HRT n=30	P. value
Age (y)	51.69±3.46	52.34±3.10	0.13
Mean duration of taking HRT (month)	20.33±17.13	-	-
Weight (kg)	68.47±10.52	68.2±11.99	0.93
Height (cm)	154.88±4.13	154.53±5.07	0.78
BMI (kg/m²)	28.51±3.96	28.56±4.82	0.97
Daily Dietary intake:
Vitamin E (mg)	5.30±2.26	6.79±3.52	0.09
Copper (mg)	1.01±0.74	0.84±0.30	0.76
Zinc (mg)	5.21±2.17	4.34±1.55	0.08
Biochemical data (serum levels):
Vitamin E (µmol/l)	26.51±11.58	22.89±8.63	0.18
Copper (µg/dl)	144.24±25.47	122.47±24.89	0.02
Zinc (µg/dl)	92.11±26.06	97.68±27.44	0.42
Copper / zinc ratio	1.70±0.59	1.30±0.30	0.002

	groups
	HRT		non-HRT
	n	%	n	%
Serum vitamin E levels
<12 µmol/l (low)	3	10.0	2	6.7
12-42 µmol/l (normal)	24	80.0	27	90.0
>42 µmol/l (high)	3	10.0	1	3.3
Serum copper levels
<80 µg/dl (low)	-	-	-	-
80-155 µg/dl (normal)	22	73.3	27	90.0
>155 µg/dl (high)	8	26.7	3	10.0
Serum zinc levels
<70 µg/dl (low)	6	20.0	2	6.7
70-150 µg/dl (normal)	22	73.3	26	86.7
>150 µg/dl (high)	2	6.7	2	6.7

According to the results (table 1) two groups of subjects were comparable in age, weight, height and BMI. The mean intakes of vitamins E, copper and zinc were similar between two groups, so the variables mentioned above would not be accounted as confounding factors in interpreting the biochemical results of the study.

Our findings indicated that postmenopausal women taking HRT did not have significantly different serum vitamin E levels compared to non treated group. Mean of serum vitamin E levels in both groups of women were also in normal range. Similarly, in the studies conducted by Wen et al and Wu et al, a-tocopherol levels in serum and red blood cells were not modified in postmenopausal women by HRT supplementation (3,8). Clemente et al also reported, the HRT did not modify significantly a-tocopherol serum levels in postmenopausal women, while a-tocopherol/LDL ratio significantly increased after HRT therapy (18). However, Meram et al reported beneficial effect of HRT on serum levels of vitamin E (13).

It is well known that HRT is associated with wide inter- individual variation in estradiol levels,(10) so it is possible that the dose of estrogen used in our study may not have been enough to increase blood estrogen level sufficiently in all of subjects for affecting serum vitamin E level significantly. However concentration of vitamin E in other tissues may be altered. Chung et al indicated that estrogen replacement in ovarictomized rats produced a marked increase in serum a-tocopherol, with a selective elevation in a-tocopherol carried by HDL and estrogen replacement also produced significant increase in the a-tocopherol concentrations in the liver and kidneys; indicating that estrogen replacement enhances the overall nutritional or body status of the vitamin E (19). Therefore it is suggested that the measurement of vitamin E in red blood cells and in lipoproteins, will be more sensitive accurate indicators for in vivo vitamin E assessment and may reflect it’s concentrations in tissues more directly.

The findings of significantly higher serum copper levels in HRT group compared to non-HRT group and no significant difference in zinc levels between two groups, are consistent with the results of the study by Bureau and et al, which demonstrated high plasma copper concentration in women treated by HRT compared to untreated ones and also no significant difference in plasma zinc levels (12). In the study by Herzberg et al, HRT did not modify significantly zinc serum levels in postmenopausal women (20). Chilvers et al reported that orally administrated ethinyle estradiol increased total copper concentration in postmenopausal women. Approximately 80% of the increase in copper was due to a rise in ceruloplasmin – bound copper and 20% to an increase in the amount of copper bound per gram of albumin (21). Ganoraja et al also suggested a direct effect of estrogen on hepatic ceruloplasmin production/release .Estrogen therapy has been shown to increase the ceruloplasmin mRNA in the liver of rat models (22).

According to the results of the study by Johnson et al, exogenous estrogens did not have effect on copper absorption (23), and taking in to account that in our study ,the mean copper intake was similar in both groups (table 1), so in agreement with other studies (12,21,22), our findings confirm that HRT may increase serum copper concentration in postmenopausal .

Based on the results (table 1) the copper/zinc ratio was significantly higher in women taking HRT than that of non-HRT group and also the percent of subjects(table 2) with serum copper levels above normal limit and with serum zinc levels below normal limit were remarkably higher in HRT group compared to non-treated group. However, from statistical point of view the differences were not significant. There is evident that copper and zinc have pro-oxidant and antioxidant properties, respectively, so their imbalance may be expected to condition oxidative stress (11).

As a conclusion, postmenopausal women taking HRT did not have significantly different serum vitamin E and zinc levels compared to untreated group. High serum copper concentration in HRT treated postmenopausal women may be a concern because, the higher serum levels of copper is considered atherogenic and in cohort studies high serum copper concentration unequivocally was associated with an increased cardiovascular morbidity and mortality (24,25). Larger studies are needed to address the aspects in more detail.

We thank the Vice-Chancellor for Research Office in the Tabriz University of Medical Sciences, Tabriz, Islamic Republic of Iran, for financial support; and the women who participated in the study. This paper was presented in part at the 9^th European Nutrition Conference, Rome, Italy, October 1-4,2003.

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