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No Antioxidant Effect of Combined HRT on LDL
Oxidizability and Oxidative Stress Biomarkers in
Treated Post-Menopausal Women
Isabelle Bureau PhDa, François Laporte PhDa, Max Favier MDb, Henri Faure PhDa, Meira
Fields PhD, FACNc, Alain E. Favier PhD, FACNa & Anne-Marie Roussel PhD, FACNa
a LBSO, University J. Fourier, Domaine de la Merci, La Tronche (I.B.,F.L., H.F., A.E.F., A.-
M.R.), FRANCE
b Department of Gynecology, Grenoble Hospital, Grenoble (M.F.), FRANCE
c Human Nutrition Research Center, USDA, Beltsville, Maryland (M.F.)
Published online: 26 Jun 2013.
To cite this article: Isabelle Bureau PhD, François Laporte PhD, Max Favier MD, Henri Faure PhD, Meira Fields PhD,
FACN, Alain E. Favier PhD, FACN & Anne-Marie Roussel PhD, FACN (2002) No Antioxidant Effect of Combined HRT on LDL
Oxidizability and Oxidative Stress Biomarkers in Treated Post-Menopausal Women, Journal of the American College of
Nutrition, 21:4, 333-338, DOI: 10.1080/07315724.2002.10719231
To link to this article: http://dx.doi.org/10.1080/07315724.2002.10719231
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Original Research
No Antioxidant Effect of Combined HRT on LDL
Oxidizability and Oxidative Stress Biomarkers in
Treated Post-Menopausal Women
Isabelle Bureau, PhD, Franc¸ois Laporte, PhD, Max Favier, MD, Henri Faure, PhD, Meira Fields, PhD, FACN,
Alain E. Favier, PhD, FACN, and Anne-Marie Roussel, PhD, FACN
LBSO, University J. Fourier, Domaine de la Merci, La Tronche (I.B., F.L., H.F., A.E.F., A.-M.R.), Department of Gynecology,
Grenoble Hospital, Grenoble (M.F.) FRANCE, Human Nutrition Research Center, USDA, Beltsville, Maryland (M.F.)
Key words: Combined HRT, LDL oxidizability, oxidative stress biomarkers
Objective: To compare oxidative stress and LDL oxidizability in postmenopausal women with and without
HRT.
Methods: In a cross sectional study, two groups of women, with or without combined per os HRT (1.5–2
mg estrogen associated with 10 mg dydrogesteron), were age and duration of menopause matched. Women were
recruited after medical examination at LBSO (Oxidative Stress Laboratory), Joseph Fourier University,
Grenoble, and Department of Gynecology, Grenoble University Hospital, France. Main outcome measures
included determination of lipid profile and oxidative stress biomarkers (TBARS, LDL oxidizability, auto-
antibodies against oxidized-LDL). Measurement of circulating levels of vitamin C, E, ␤-carotene, lycopene and
total antioxidant plasma capacity.
Results: HRT led to decreased plasma total and LDL cholesterol (p Ͻ 0.05), but did not affect oxidizability
and oxidation of LDL. Circulating levels of antioxidant vitamins (␤-carotene, vitamin C, vitamin E/triglycerides)
and total antioxidant capacity of plasma and lipid peroxidation, assessed by plasma TBARs, were not different
from controls in postmenopausal women receiving HRT.
Conclusion: This study suggests that even if combined HRT modifies the blood lipid profile, it does not
appear to influence oxidative status.
has been suggested that increased oxidative stress, related to an
INTRODUCTION
unbalanced pro-oxidant/antioxidant equilibrium, is a potential
Women after menopause experience an increased incidence
of cardiovascular disease [1]. In contrast, women receiving
hormonal replacement therapy (HRT) seem to be protected [2].
The mechanism of HRT-related cardiovascular protection is yet
unclear and seems mediated by several factors [3,4]. It may be
related to favorable changes in several important cardiovascu-
lar risk factors, such as circulating blood low density lipopro-
teins (LDL), high density lipoproteins (HDL) and lipopro-
tein(a) [5], coagulation factors, blood pressure and insulin
tolerance [6] [7]. Oxidative stress has been also implicated as a
risk factor in the pathogenesis of cardiovascular disease [8]. It
inducer of cardiovascular risk [9]. Oxidative modifications of
low-density lipoproteins (LDL) are thought to play a major role
in the process of atherosclerosis [10,11]. The atheroprotective
effect of estrogen might also be partly due to its antioxidant
action [12], resulting in a decrease of LDL oxidation [13]. In
postmenopausal women, hormonal replacement therapy might
either counteract the effect of a possible increased oxidative
stress or improve antioxidant status.
During aging, a decrease of antioxidant enzyme [14] as well
as a lower antioxidant nutriture has been reported [15,16]. In
addition, middle age is often associated with environmental
Abbreviations: LDL ϭ Low Density Lipoprotein, HDL ϭ High Density Lipoprotein, HPLC ϭ High Performance Liquid Chromatogratography, TAS ϭ Total Antioxidant
Plasma Capacity, HRT ϭ Hormonal Replacement Therapy.
This research has been supported by the Region Rhoˆne-Alpes and a grant of the French Ministry for Research.
Address reprint requests to: Pr. A.M. Roussel, LBSO/UFR de Pharmacie, 38700 La Tronche, France E-mail: Anne-Marie.Roussel@ujf-grenoble.fr
Journal of the American College of Nutrition, Vol. 21, No. 4, 333–338 (2002)
Published by the American College of Nutrition
333

Pro/Anti-Oxidant Balance after Menopause
modifications resulting in decreased intakes of micronutrient
[17], which are directly related to antioxidant protective mech-
anisms. Despite the key role of antioxidant micronutrients in
preventing accelerated aging, data related to relationship be-
tween oxidative stress and antioxidant status in menopausal
women are scarce.
This study was designed to compare oxidative stress and
LDL oxidizability in postmenopausal women receiving or not
hormonal replacement therapy. Oxidative process was evalu-
ated in plasma by lipid peroxidation (TBARs), autoantibodies
against oxidized LDL, LDL oxidizability, antioxidant vitamin
status (vitamin C, vitamin E, ␤-carotene, lycopene) and total
antioxidant plasma capacity to monitor the occurrence of oxi-
dative stress taking place in vivo. Since antioxidant status is
partly dependent on antioxidant micronutrient intakes, we also
assess the dietary intakes of the subjects.
and triglycerides (Tg) were measured by enzymatic colorimetric
methods with a Cobas analyzer according to the manufacturer
instructions. The concentration of serum LDL-c was calculated
according to the Dahlen equation [18], in the form as LDL-c ϭ
Chol Ϫ (HDL-c ϩ 0.20 Tg ϩ 0.3 ϫ Lp(a)) (g/L).
Apolipoproteins A1 and B and lipoprotein a (Lpa) were
measured by turbidimetric immunoassays.
HDL-c and LDL-c subfractions were separated by electro-
phoresis in a non-denaturing polyacrylamide gradient gel [19].
LDL size was estimated as the apparent diameter of the major
peak. HDL2 and HDL3 were quantified by scanning the gel
with a CD60 densitometer [20].
Estradiol (E2) serum level was measured by radioimmuno-
assay method.
Plasma Lipoperoxidation and LDL Oxidizability
Lipid peroxidation was measured by thiobarbituric reactive
substance (TBARS) concentration in plasma [21].
METHODS
LDL particles were isolated by density gradient ultracen-
trifugation using a Beckman TL-100 ultracentrifuge with a
fixed angle rotor (100.2). The susceptibility of LDL to oxida-
tion was estimated by incubating isolated LDL at 37°C in PBS
and the reaction was initiated by adding 3 ␮M CuCl2 [22]. The
rate of oxidation and the lag time were determined for each
subjects. The lag time was defined as the intercept between the
baseline at t0 and the slope of the propagation phase.
Auto-antibodies of the IgG class to malondialdehyde
(MDA)-modified LDL were determined by ELISA method
according to Maggi with the following modifications: MDA-
modified LDL were prepared according to Purunen and used as
antigen [23,24]. The assay was calibrated with serial dilutions
of a pool of plasma with high antibody levels. The antibody
titer was calculated after subtraction of the absorbency of a
reagent blank and expressed as the dilution of the standard
corresponding to the absorbency of the sample, taking into
account the dilution (arbitrary unit: AU). Coefficient of varia-
tion is 15.2% (mean ϭ 550UA and n ϭ 30).
Experimental Design
In a cross-sectional study, two groups of women aged
50–60 years old were constituted and compared for their
oxidant/antioxidant status. One group was taking orally com-
bined hormonal replacement therapy (HRT) for at least six
months (group I, n ϭ 18), the other (group II, n ϭ 25) not. Used
orally combined HRT was 1.5–2 mg estradiol associated with
10 mg of dydrogesteron. We checked that women in group I
had not been on HRT prior to the study. Key exclusion criteria
included natural menopause before age 40 years or Ͻ2 years or
Ͼ15 years at time of enrollment, hysterectomy or any surgical
interventions within six months, body mass index Ͼ35 kg/m2,
supplementation with micronutrients within three months and
during the study, current medication that is known to influence
lipoprotein metabolism, abnormal routine biochemical tests
and a medical history (e.g., stroke, endometrial or breast can-
cer, diabetes, liver or renal diseases). We checked that un-
treated and treated women did not receive antioxidant supple-
mentation within three months and during the study.
Antioxidant Plasma Vitamins and Antioxidant
Plasma Capacity
The Ethical Committee of Grenoble University Hospital
approved the study, and each volunteer was given a written
informed consent.
Plasma concentrations of ␤-carotene, lycopene and vitamin
E were quantified using the High Performance Liquid Chro-
matography (HPLC) [25]. Coefficient of variation for vitamin
E, lycopene, ␤ carotene are, respectively: 5.1%, 13.6%, 7.1%.
Total vitamin C: 400 ␮L of plasma was added to 3600 ␮L
aqueous solution of metaphosphoric acid (5% w/v) and stored
at Ϫ80°C until analysis. The determination was done fluoro-
metrically by HPLC after derivatization [26]. Coefficient of
variation for vitamin C is 4.16%.
Blood Sampling and Analysis
Sampling: all blood samples were collected after a 12-hour
overnight fast. Tubes were placed on ice and centrifuged at
4000 ϫ g for 10 minutes at 4°C. Plasma and erythrocytes were
immediately isolated, aliquoted and stored at Ϫ80°C.
Plasma Lipid Profile and Serum Estradiol
Total antioxidant plasma capacity (TAS) was measured
using a Randox kit (Randox 1115 rue H. Boucher, BP 82,
Montpellier Frejorgues, 34131 Mauguio cedex, France).
Plasma levels of total cholesterol, low density lipoprotein
cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL-c)
334
VOL. 21, NO. 4

Pro/Anti-Oxidant Balance after Menopause
Table 1. Baseline Characteristics of the Subjects
Nutritional Assessment
Seven-day dietary records were used to assess dietary in-
takes. Estimation of macronutrients (proteins, carbohydrates,
lipids), alcohol consumption, fiber, vitamins C, E, A, carote-
noids and folates was determined using the REGAL WINDOW
software (CNEVA, Ciqual, Paris, France).
Postmenopausal
Women HRT
Postmenopausal
Women non-
HRT (n ϭ 26)
(n ϭ 18)
Age (years)
54.1 Ϯ 2.2
55.4 Ϯ 3
Menopause duration
(years)
4.5 Ϯ 2.3
23 Ϯ 3
4.8 Ϯ 3.3
24 Ϯ 4
BMI (kg/m2)
Smokersa
1
3
1
3
Statistics
Physically active personb
Alcohol (absolute
ethanol, g/week)
Estradiol (nmol/L)
Data are expressed as means Ϯ S.D. The distribution of all
variables was first checked for normality using the Kolmog-
orov-Smirnov test. Depending upon the distribution of the
population, data were analyzed either using parametric methods
differences where statistical significance was tested using the
Student t test or using non-parametric methods differences
where statistical significance was tested using the Mann-Whit-
ney U test.
56 Ϯ 87
574 Ϯ 399
38 Ϯ 48
72 Ϯ 48*
Values are mean Ϯ SD.
* p Ͻ 0.005.
a Number of smokers in each group, (between 10 and 20 cigarettes/day).
b Number of physically active persons, (three hours or more [maximum six hours]
of regular physical activity/week).
HRT ϭ Hormonal Replacement Therapy.
Statistical evaluations were performed with the analysis
program Statistica (statistical software, Paris, France). Statisti-
cal differences by either Student t test or Mann-Whitney U test
were significant at p Յ 0.05.
Table 2. Plasma Lipids in Postmenopausal Women
Postmenopausal Postmenopausal
Women (HRT)
Women (non-
(n ϭ 18)
HRT) (n ϭ 26)
Cholesterol total (mmol/L)
LDL-Cholesterol (mmol/L)
Apolipoprotein B (g/L)
Triglycerides (mmol/L)
HDL-cholesterol (mmol/L)
Apolipoprotein A1 (g/L)
HDL2/HDL3 (%)
Lipoprotein (a) (mg/L)
Mean:
Median:
6.1 Ϯ 1.0
3.5 Ϯ 0.8
0.82 Ϯ 0.14
1.0 Ϯ 0.2
2.1 Ϯ 0.5
1.75 Ϯ 0.34
1.16 Ϯ 0.64
6.7 Ϯ 0.9*
4.4 Ϯ 0.7**
0.96 Ϯ 0.16**
0.9 Ϯ 0.3
1.8 Ϯ 0.4
1.56 Ϯ 0.23*
1.02 Ϯ 0.42
RESULTS
Study Population
Basic characteristics of the subjects are reported in Table 1.
BMI, alcohol and tobacco consumption, physical activity habits
were not significantly different in the two groups, whereas
mean age and duration of menopause were similar. Women
receiving hormonal replacement therapy exhibited a plasma
estradiol level significantly higher than untreated women.
246 Ϯ 219
182 (112–393)
465 Ϯ 574
279 (42–633)
Values are mean Ϯ SD.
Statistical differences between groups were significant for p Ͻ 0.05 (* p Ͻ 0.05,
** p Ͻ 0.005).
HRT ϭ Hormonal Replacement Therapy.
Lipid Profile
Plasma lipids are reported in Table 2. In untreated post-
menopausal women, significantly higher in plasma cholesterol
levels were observed. This effect was partly due to a higher
LDL-c. Similarly, lipoprotein (a) tended to be lower in HRT-
treated women, but the standard deviation was too high to
achieve a significant difference. HDL-c was non-significantly
raised in the group receiving hormonal replacement therapy,
but the ratio HDL2/HDL3 remained unchanged. Triglyceride
levels were in the normal range.
Table 3. Lipoperoxidation and LDL Oxidazibility of the Subjects
Postmenopausal Women
(HRT)
Postmenopausal Women
(non-HRT)
TBARs (␮mol/l)
2.6 Ϯ 0.3 (n ϭ 18)
0.44 Ϯ 0.08 (n ϭ 18)
10.8 Ϯ 1.3 (n ϭ 16)
48.8 Ϯ 4.5 (n ϭ 16)
27.2 Ϯ 0.4 (n ϭ 18)
533 Ϯ 360 (n ϭ 18)
2.4 Ϯ 0.2* (n ϭ 25)
0.37 Ϯ 0.05** (n ϭ 25)
10.7 Ϯ 1.3 (n ϭ 26)
51.6 Ϯ 5.3 (n ϭ 26)
27.2 Ϯ 0.4 (n ϭ 25)
677 Ϯ 575 (n ϭ 26)
TBARs/Cholesterol (␮mol/mmol)
Oxidation rate of LDL (␮mol diene/minutes/g apo B)
Lag time (minutes)
LDL size (nm)
Auto-antibodies anti oxLDL (AU)
Values are means Ϯ SD.
Statistical differences between groups were significant for p Ͻ 0.05 (* p Ͻ 0.05, ** p Ͻ 0.005).
HRT ϭ Hormonal Replacement Therapy.
JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION
335

Pro/Anti-Oxidant Balance after Menopause
Table 4. Antioxidant Vitamins and Total Antioxidant
Capacity of Plasma (TAS)
Dietary Allowances (ANC) [28]. Intake of energy, fiber, vita-
min C, E, equivalent ␤-carotene, retinol and folates were not
significantly different between groups.
Postmenopausal
Women (HRT)
(n ϭ 18)
Postmenopausal
Women (non-HRT)
(n ϭ 25)
DISCUSSION
Vitamin C (␮mol/L) 57 Ϯ 13
72 Ϯ 15***
1.28 Ϯ 0.92*
1.13 Ϯ 0.67
␤ carotene (␮mol/L) 0.78 Ϯ 0.32
Lycopene (␮mol/L) 0.90 Ϯ 0.49
Vitamin
The higher prevalence of cardiovascular diseases in post-
menopausal women could be related to an increased oxidative
stress. Estrogen has been reported to have antioxidant capabil-
ities and HRT could protect against cardiovascular diseases
throughout this mechanism. The objective of this study was to
investigate LDL oxidizability, plasma lipoperoxidation and
plasma antioxidant capacity in treated and untreated women.
Our data confirmed that hormonal replacement therapy resulted
in beneficial changes in the conventional lipid risk estimators.
However, LDL oxidation and oxidizability were not affected by
this treatment.
E/Triglycerides
(␮mol/mmol)
TAS (␮mol/L)
33 Ϯ 10
44 Ϯ 16**
1.35 Ϯ 0.06 (n ϭ 16) 1.40 Ϯ 0.04** (n ϭ 21)
Values are means Ϯ SD.
Statistical differences between groups were significant for p Ͻ 0.05 (* p Ͻ 0.05,
** p Ͻ 0.01, *** p Ͻ 0.005).
HRT ϭ Hormonal Replacement Therapy.
Lipoperoxidation and LDL Oxidizability
Lipoperoxidation and LDL oxidizability are reported in
Table 3. Oxidizability of LDL, estimated by the lag time and
the rate of oxidation, was not different between groups. Mean
LDL size was in the middle size range in the two groups. This
type of LDL is associated with a low level of oxidation and,
therefore, a low cardiovascular risk [27]. Plasma levels of
auto-antibodies against oxidized LDL were not modified by
hormonal replacement therapy.
The higher incidence of cardiovascular diseases after meno-
pause is well-documented [29,30]. The lipid modifications are
mainly involved in atherogenic risk [31]. Our results confirm
the beneficial effects of hormonal replacement therapy in re-
gard to blood lipid levels. Others have previously reported
these observations [32]. Risks of cardiovascular diseases are
due to several factors including those related to aging and those
specifically linked to hormonal estrogenic deficiency [33]. It
has been reported that estradiol possesses antioxidant proper-
ties [34,35]. However, in our study, the lipoprotein suscepti-
bility to oxidation, assessed by ex vivo analysis, was not af-
fected by hormonal replacement therapy. Our data corroborate
with those of Mc Manus et al. and Wen et al., who showed that
oral equine estrogen therapy, transdermal estradiol or combined
HRT did not protect LDL against oxidation [36,37]. Similarly,
Nenseter et al. demonstrated that non-smoking, hypercholes-
terolemic postmenopausal women were not protected by HRT
in regard to LDL oxidation [38]. More recently, interest has
focused on other plasmatic biomarkers of lipid peroxidation
such as levels of autoantibodies against oxidized-LDL. In the
present study, autoantibodies against oxidized LDL were not
affected by hormonal replacement therapy. Heikkinen has also
Antioxidant Vitamins and Total Antioxidant
Capacity of Plasma
Antioxidant vitamins and total antioxidant capacity of
plasma of all subjects are reported in Table 4. Plasma concen-
tration of ascorbic acid, ␤-carotene, lycopene and the ratio
vitamin E/Triglycerides were significantly different between
the two groups.
Nutritional Assessment
As reported in Table 5, mean dietary intakes were adequate
in the two groups according to the Recommended French
Table 5. Nutritional Assessment
Postmenopausal Women
Postmenopausal Women
ANC (Ͼ50 years)
(HRT) (n ϭ 14)
(non-HRT) (n ϭ 19)
Caloric Intake (KJ/d)
(Kcal/d)
Fiber (mg/d)
Vitamin C (mg/d)
Vitamin E (mg/d)
Eq. ␤ carotene (␮g/d)
Retinol (␮g/d)
7759 Ϯ 1515
(1854 Ϯ 362)
17.4 Ϯ 4.1
97 Ϯ 36
10.6 Ϯ 3.5
4079 Ϯ 1761
403 Ϯ 328
266 Ϯ 66
7470 Ϯ 1590
(1785 Ϯ 380)
18.7 Ϯ 6
7114
(1700)
20
80
12
2880
320
300
99 Ϯ 44
11.1 Ϯ 6.1
3971 Ϯ 2764
328 Ϯ 148
283 Ϯ 91
Folates (␮g/d)
Values are means Ϯ SD.
HRT ϭ Hormonal Replacement Therapy.
336
VOL. 21, NO. 4

Pro/Anti-Oxidant Balance after Menopause
observed that one-year HRT did not influence oxLDL antibody
titer [39]. Controversially, it has been shown that HRT de-
creased the level of oxLDL antibody in postmenopausal
women with coronary heart disease [40]. We observed large
size LDL particles although it has been described that post-
menopausal women had a preponderance of small dense LDL
particles [41]. Since the large size of LDL is less susceptible to
oxidative modification, it is therefore not expected to be asso-
ciated with a higher cardiovascular risk [27].
Plasma lipoperoxidation, monitored by TBARS and TBARS/
cholesterol was different in untreated and treated women. Inal
et al. have reported that malondialdehyde (MDA) levels de-
creased in postmenopausal women receiving hormonal replace-
ment therapy only when associated with a daily supplementation
of vitamin E [43]. This observation points out the fundamental role
of the antioxidant component in the maintenance of pro-oxidant/
antioxidant equilibrium. Furthermore, it has been shown that in-
take of dietary vitamin E is inversely associated with the risk of
death from cardiovascular disease in postmenopausal women [44].
In non-treated women, the lower lipoperoxidation observed
could be due to a protection against oxidative stress related to
optimal level of plasma antioxidants. All the women exhibited
an adequate antioxidant status.
Our data do not agree with studies reporting a risk of deficit
in antioxidants after menopause [17]. The plasma concentra-
tions for vitamins E, C and carotenoids in the non-HRT group
appears likely to be related to an optimal diet in this group. The
recruited women were at an early stage of menopause and their
antioxidant status was optimal. These volunteers were health
conscious as shown by body mass index (BMI) and had healthy
environmental habits (few smokers, no alcohol drinking, low
triglyceridemia regarding aging).
The role of estrogen as antioxidant in vivo is matter of
debate [36,45]. Controversies still exist regarding the beneficial
protecting effect of HRT. In some trials estrogen replacement
therapy had a beneficial effect in prevention of coronary artery
disease, morbidity and mortality [46,47]. However, pooled data
from clinical trials do not support the notion that HRT prevents
cardiovascular events [48]. Moreover, the only randomized
data available to date do not support any beneficial effect in
postmenopausal women with coronary heart disease [49].
In conclusion, the antioxidant effect of combined hormonal
replacement therapy remains controversial. In untreated post-
menopausal women, optimal antioxidant micronutrient intakes
could be a powerful tool in counteracting the effect of hor-
monal modifications in terms of oxidative stress. Under these
conditions, nutrition could offer an interesting alternative way
in preventing cardiovascular risks.
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Received February 7, 2000; revision accepted December 13,
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