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ORIGINAL ARTICLE
Hormone replacement therapy reduces lipid oxidation 
directly at the arterial wall: A possible link to estrogens’ 
cardioprotective effect through atherosclerosis prevention
Carlos Gómez Escalante1,2, Silvia Quesada Mora2, Laura Navarro Bolaños2
1Department of Gynecology, San Juan de Dios Hospital, Costa Rica, 2Department of Biochemistry, Faculty of Medicine, School of 
Biochemistry, University of Costa Rica, Washington, USA
ABSTRACT
Background: The	first	step	in	atherosclerosis	formation	is	the	ingurgitation	of	an	oxidized	low‑density	lipid	(LDL)	
molecule by a macrophage which then turns into a foam cell within the vascular wall and initiates a cascade 
of	inflammatory	responses.	Could	it	be	that	the	potential	cardioprotective	effect	observed	in	women	receiving	
hormone replacement therapy (HRT) is modulated by estrogen’s capacity to decrease LDL oxidation in the 
vascular wall and thus decrease atherosclerotic foam cells?
Materials and Methods: Thirty-four adult female Wistar rats were divided into three groups. All were double 
oophorectomized. After recovery, Group 1 received Estradiol Valerate subcutaneous (SC) (2.5 mg/kg/week), 
Group	2	Estradiol	Valerate	SC	(2.5	mg/kg/week)	+	Progesterone	SC	(10	mg/kg/48	h),	and	Group	3	Placebo	
SC.	After	10	weeks,	all	rats	were	sacrificed	and	a	vascular	dissection	performed.	Malondialdehyde	(MDA)	was	
measured directly on the vascular extract to determine lipid oxidative levels and HRTs’ effect. Renal and hepatic 
tissue was also studied. Total antioxidant status (TAS) was measured to determine overall oxidative behavior.
Results: Vascular MDA levels for Group 1 = 80.80 (±16.8) µmol/ml/g, Group 2 = 107.69 (±24.9) µmol/ml/g, and 
Group 3 = 140.96 (±32.4) µmol/ml/g. ANOVA (P < 0.05), with a post hoc Bonferroni corrective t-test, showed that 
both	Group	1	and	2	have	statistically	significant	lower	levels	of	MDA	than	Group	3.	Renal	tissue	showed	less	
oxidative damage in the HRT groups, while hepatic tissue showed an inverse behavior with less lipid oxidation 
in the placebo group. TAS decreased with oophorectomy in all groups but decreased less in both groups that 
received HRT compared to placebo (P < 0.05).
Conclusion: HRT	significantly	reduces	lipid	oxidation	directly	in	the	arterial	wall.
Key Words: Atherosclerosis, hormone replacement therapy, oxidative stress
INTRODUCTION postmenopausal women in Europe and the United 
States.[1] Some developing countries such as Costa Rica 
Cardiovascular disease (CVD), and it is complications, are also showing this trend of  increased cardiovascular 
continues to be, by far, the main cause of  death in mortality, and it has become a main topic of  public 
health.[2]
Address for Correspondence: Dr. Carlos Gómez Escalante, 
Oficina	#314,	Distrito	Cuatro,	Guachipelín	de	Escazú,	 
San José, Costa Rica.  This is an open access article distributed under the terms of the 
E-mail: dr.escalante@gmail.com Creative Commons Attribution-NonCommercial-ShareAlike 3.0 
License, which allows others to remix, tweak, and build upon the 
work non-commercially, as long as the author is credited and the 
Access this article online new creations are licensed under the identical terms.
Quick Response Code:
Website: For reprints contact: reprints@medknow.com
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How to cite this article: Escalante CG, Mora SQ, Bolaños LN. Hormone 
DOI: replacement therapy reduces lipid oxidation directly at the arterial wall: A possible link to estrogens’ cardioprotective effect through atherosclerosis 
10.4103/0976-7800.201967 prevention. J Mid-life Health 2017;8:11-6.
© 2017 Journal of Mid-life Health | Published by Wolters Kluwer - Medknow 11
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Escalante, et al.: HRT and lipid oxidation at the arterial wall
Atherosclerotic lesions have been directly linked to being of  HRT.[17] The 11-year cumulative follow-up of  the 
the cause of  vascular occlusion leading to acute myocardial estrogen-only arm with regards to coronary disease of  the 
infarction and ischemic stroke.[3] The physiopathological Women’s Health Initiative showed a heart rate of  0.96.[18] A 
process leading up to these vascular lesions begins with the meta-analysis done by Salpeter et al., which included only 
migration of  monocytes to the intima layer of  the arteries early postmenopausal women, showed a 39% reduction in 
where they differentiate into macrophages. Once these total mortality.[19] We propose that, during the reproductive 
macrophages come in contact with the fatty streak, oxidized period, estrogens physiologically modify the oxidative 
low-density lipid (ox-LDL) molecules are presented to them status of  the arterial wall, inhibiting or at least delaying the 
through CD-34, these macrophages ingurgitate the ox-LDL oxidation of  LDL molecules and therefore the initiation of  
and become foam cells.[4,5] It is these foam cells that initiate the atherosclerotic plaque. Once women enter menopause, 
a chemotactic process that includes the liberation of  radical and thus, lose the anti-oxidative effect offered by estrogens, 
oxygen species, metalloproteinase, myeloperoxidase, and LDL oxidation rate is increased, and the progression of  the 
interleukin 1, which in turn attract more macrophages atherosclerotic plaque is unopposed. We set out to show 
to the site and also cause oxidative damage to the LDL that estrogens have an anti-oxidative effect on vascular 
deposited in the fatty streak, therefore, promoting a loop lipids and that lipid oxidation would be decreased at this 
which favors the formation of  more atherosclerotic plaque. level with the use of  estrogen-based HRT. The inclusion 
It is this plaque that progressively reduces the lumen of  of  other key organs such as kidney and liver may also shed 
the arteries inducing shear stress which causes turbulent new information in this field.
blood flow at the site and favors the rupture of  the plaque, 
giving way to possibly catastrophic outcomes.[6] The key MATERIALS AND METHODS
point in this pathophysiological process is understanding 
that the LDL molecule has to be oxidized in order for the Animals
macrophage to ingurgitate it, if  the LDL is not oxidized the Due to the nature of  the biological tissue required for 
macrophage will not ingurgitate it, and thus, not become analysis, an animal model was selected to carry out our 
a foam cell.[4-6] protocol. Thirty-four adult female Wistar rats were 
obtained from the University of  Costa Rica’s Center for 
Estrogens, per se, can act as free radical scavengers due to Animal Research. All weighed between 240 and 315 g at 
their phenol ring. Many previous authors have published the beginning of  the study. They were kept in a low-stress 
in vitro studies that show how estrogens can reduce environment with a controlled temperature between 
oxidative damage to molecules such as LDL, CuSO4, and 21 and 25°C. 12 h day–12 h night cycles were maintained 
DNA.[7,8] Estrogens can also induce the formation of  during the greater part of  the study, and all rats were 
other protective enzymatic antioxidants such as superoxide kept in individual cages and received unrestricted water 
dismutase (SOD).[9] In addition, in a previous study, our and daily feeding. The protocol was approved by the 
group was able to show that estrogens have the capacity to University’s Bioethical Committee and was in accordance 
directly decrease lipid oxidation in postmenopausal women to international standards for animal research.
who used hormone replacement therapy (HRT).[10] It is 
through this mechanism of  action that allows us to link Bilateral oophorectomy
estrogens to a possible cardioprotective effect. All the rats had a double oophorectomy performed to 
induce a menopausal model. Antiseptic solution using 
Premenopausal women have relatively low incidence of  topic 3% chlorhexidine was applied on all of  the abdomen. 
CVD compared to men, but once women reach menopause Anesthesia was carried out with an intraperitoneal 
and their estrogen levels decrease, it is quickly observed how injection of  a mixture of  ketamine at a dose of  90 mg/kg 
CVD increases and how it soon surpasses the incidence and xylazine at a dose of  6 mg/kg.[20,21] Great care was 
observed in men.[11,12] The “window of  opportunity” tells carried out to avoid any kind of  pain during and after the 
us that if  we initiate HRT early in postmenopausal women, procedure. Local, subcutaneous (SC) lidocaine was applied 
we will observe a predominately cardioprotective effect.[13] in the area where the incision was going to be carried out. 
Observational studies enrolling only early postmenopausal Preoperative metamizol was applied subcutaneously at a 
women showed an average of  30%–50% decrease of  risk dose of  200 mg/kg.[21] A low, midline incision was carried 
for coronary heart disease with HRT.[14-16] Furthermore, out, with careful dissection the ovaries were identified and 
an open-label prospective study such as The Danish removed bilaterally. Closure was carried out with individual 
Osteoporosis Study has confirmed this data and one of  4-0 nylon sutures. Twenty-four hours after oophorectomy, 
its composite endpoints showed a 49% reduction in risk all the rats received one more dose of  metamizol for pain 
for coronary heart disease over a 16 year period in users control.
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Escalante, et al.: HRT and lipid oxidation at the arterial wall
Groups and medication which would modify the oxidative status of  the animals. 
After allowing a week of  recovery, the rats were divided into Weight and behavioral patterns were normal throughout 
three groups. Group 1 (#12 rats) received Estradiol Valerate the study period.
by SC injection (2.5 mg/kg/week), Group 2 (#12 rats) 
Estradiol Valerate SC (2.5 mg/kg/week) + Progesterone TAS measured at baseline showed no difference among 
SC (10 mg/kg/48 h), and Group 3 (#10 rats) Placebo the three groups reflecting similar antioxidant status 
SC. Based on previous studies using ovariectomized rats, among them. After oophorectomy, all three groups 
intermediate doses were used to try to mimic “normal” showed a statistically significant decrease in their TAS 
hormonal levels and thus better represent menopausal compared to baseline. The placebo group suffered 
hormone therapy in women.[22-28] Since sesame seed oil was the greatest decline in their antioxidant status and was 
used to dilute the progesterone in Group 2, it was also used significantly lower than the two groups that received 
as the placebo in Group 3. HRT [Table 1].
Samples and measurements Regarding lipid oxidation, Table 2 shows that Group 3, 
Serum was obtained from whole blood extracted at which received placebo, had much higher levels of  
baseline (preoophorectomy) and then again at the time of  MDA compared to Groups 1 and 2, which received 
sacrifice to evaluate total antioxidant status (TAS). TAS[29] HRT, (P ≤ 0.05), indicating that the groups that 
was measured using 1, 1-diphenyl-2-picrylhydrazyl (DPPH) did receive hormone replacement had lower levels 
which is a stable purple colored free radical. Once an of  lipid oxidation in their vascular extracts. When 
antioxidant is added to DPPH, it is reduced and its’ color comparing Group 1 with Group 2, there seems to be 
changes to yellow. This change in color is proportional to a lower level of  lipid oxidation in the estrogen-only 
the antioxidants’ capacity of  the added substance and can group compared to the estrogen/progesterone group; 
be measured spectrophotometrically at 517 nm. nevertheless, this difference was not statistically 
significant, ANNOVA (P < 0.05), with a post hoc 
After 10 weeks of  medication, the rats were sacrificed by Bonferroni corrective t-test. Renal MDA levels showed 
guillotine. Immediately after, a complete midline incision was less lipid oxidation in both groups that received 
performed to expose the rat’s circulatory system. Resection HRT (P < 0.05). Hepatic tissue showed an inverse 
of  the aortic arch, the renal arteries and the bifurcation of  behavior, expressing a lower level of  lipid oxidation in 
the iliac arteries all the way to the femoral arteries was carried the group which received placebo, being significantly 
out. The liver and kidneys were also removed. lower than the E2V/Pro and E2V groups (P < 0.05).
Vascular tissue was rinsed first with saline and then with Table 1: Serum total antioxidant status at baseline 
buffer solution, after which they were minced with a scalpel before oophorectomy and 8 weeks after with hormone 
and homogenized during 1 min with an Ultra-Turrax. Tubes replacement according to group
were then centrifuged at 4000 rpm for 10 min at 4°C. The TAS
supernante fraction was removed and analyzed. The whole DPPH (reduction percentage)
liver and both kidneys were also minced separately and Baseline 8 weeks postoophorectomy
processed in a similar fashion. E2V/Pro (n=12) 51.34 (±9.8) 33.98 (±5.8)*
Lipid peroxidation was measured in the tissues using E2 (n=12) 57.90 (±6.6) 23.82 (±4.4)*
,
a standardized TBARS Assay Kit (Cayman Chemicals Placebo (n=10) 52.01 (±6.9) 18.20 (±4.0)* **
#10009055). The measurement of  thiobarbituric *P≤0.05 with regard to baseline, **P≤0.05 with regard to other groups. TAS: Total antioxidant status, DPPH: 1,1‑diphenyl‑2‑picrylhydrazyl
acid (TBA) reactive substances is the most widely employed 
assay used to determine lipid peroxidation by measuring Table 2: Determination of lipid oxidation in rat vascular, 
malonaldehyde, which is a naturally occurring by-product of  renal, and hepatic extracts through malondialdehyde levels 
lipid peroxidation.[30,31] The malondialdehyde (MDA)-TBA according to hormone replacement status
adduct formed by the reaction is measured colorimetrically 
Malondialdehyde (µmol/ml/g)
at 532 nm. All tests were done in duplicates.
E2V/Pro (n=12) E2V (n=12) Placebo (n=10)
RESULTS Vascular tissue 107.69 (±24.9) 80.80 (±16.8) 140.96 (±32.4)*
Renal tissue 108.44 (±28.3) 126.73 (±14.1) 156.44 (±27.7)*
All rats completed the study protocol, and there was no Hepatic tissue 16.37 (±1.6) 16.18 (±1.3) 12.32 (±1.2)*
evidence of  complications from the surgical procedure *P≤0.05 with regard to other groups. E2V: Estradiol valerate, Pro: Progesterone
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Escalante, et al.: HRT and lipid oxidation at the arterial wall
DISCUSSION Previous authors have shown that estrogens have both 
a direct and indirect antioxidative effect. They not only 
TAS has been proven to decrease after menopause. A review act directly as an electron donor, but they also induce the 
of  the literature has shown that this may be due to either formation of  some of  the most potent natural antioxidants 
an increase of  oxidative stress or a decrease of  antioxidant enzymes available, such as SOD.[7-9,37,38] If  estrogen is solely 
enzymes in the body.[32,33] It has also been proven that acting as an antioxidant molecule, as suggested by the work 
HRT improves this antioxidant status in postmenopausal of  Simpkins et al. and his group,[39] one would not expect 
women.[34] TAS of  our laboratory animals showed a similar the progestin to make much difference. On the other hand, 
behavior, with a decrease of  TAS after oophorectomy. if  estrogen is acting to increase biochemical antioxidant 
The greatest decrease in TAS was observed in the group defenses via hormone receptors, then the progesterone 
receiving placebo, and an overall better antioxidant status might well attenuate this effect by causing opposing 
was maintained in both the groups that received hormone changes in cell antioxidant metabolism. Wassmann[40] 
replacement. Nevertheless, this is a marker which shows published findings where they found that progesterone 
the overall status of  an organism and is the sum of  all decreases estrogen-induced synthesis of  SOD in vascular 
the individual processes that are occurring, and thus the smooth muscle. Progesterones’ role in modifying estrogens’ 
importance of  analyzing tissues on an individual basis. antioxidative role in vivo is yet to be defined, but this is a very 
important topic since most of  women reach menopause 
Recent clinical evidence has been able to reinforce the with their uterus and will require the addition of  a progestin 
existence of  the timing hypothesis related to estrogens’ to their HRT.
administration in early menopause.[17] This evidence 
shows that HRT may have a cardio-protective effect if  HRT exhibited a protective effect on lipid oxidation 
given early compared to late in postmenopausal women.[13] in renal tissue. These results are in accordance with a 
Although there is a growing amount of  clinical data in hormone induced renal protection previously documented 
this regard, there is little information on the complete by multiple authors and involves modulation of  
physiopathological mechanism on how estrogen-based STAT3-dependant oxidative responses,[41] AGTR2[42] and 
HRT would achieve the proposed cardioprotective effects. estrogen receptor-α[43] expression. Renal physiology is a 
key determinant of  cardiovascular function and may also 
Our group, as well as other authors, have shown that be contributing to an improved vascular oxidative status.
HRT can decrease overall lipid oxidation in women who 
receive this therapy.[10] Since lipids have a vast distribution Regarding the analysis of  the hepatic tissue we observed 
in the body, a systemic decrease of  lipid oxidation may not a significant increase in lipid oxidation in both groups 
be reflecting what is happening precisely at the vascular which received HRT, thus the placebo group had less 
level where the atherosclerotic plaque is being formed. oxidative damage at this level. Estrogen has been known 
The fact that we were able to isolate vascular tissue and to be a potent inductor of  certain liver enzymes, but 
extract the lipids allowed us to eliminate any confounding when it comes to the oxidative mechanisms involved, 
factors which would modify this analysis. In Table 2, we there has been some mixed results. While Can[44] recently 
see how vascular lipid oxidation levels were lower in both concluded that 17 β-estradiol was not very effective in 
groups which received HRT compared to placebo. Under preventing oxidative-induced liver damage, Sobocanec 
our controlled conditions, we can say that this decrease in et al.[45] proposed that 17 β-estradiol may actually have 
lipid oxidation is due to the administration of  HRT. Wistar a protective effect on age-related liver oxidative damage 
rats conserve 99% of  human genes and physiologically through Nrf2-Keap1 pathways. Compared to other organs, 
resemble humans very well.[35] This 10-week time laps in the liver may have the most complex metabolic pathways of  
rats represent a 7-year period in a human lifespan,[35,36] all and may respond in a grand variety of  ways to specific 
thus suggesting that lipid oxidation at a vascular level metabolites. There is very little information regarding 
may effectively be reduced using HRT during the early specific lipid oxidative pathways at the hepatic level, but 
postmenopausal period. This allows us to hypothesize that we at least infer that estrogen might actually increase an 
if  there is less lipid oxidation directly at the vascular site, and oxidative path or decrease an antioxidative mechanism at 
this is a key step in foam cell formation, then there will be this level.
less foam cell formation, leading up to less atherosclerotic 
plaque and thus a possible cardioprotective effect. To best of  our knowledge, this is the first study that 
analyzes estrogens’ antioxidative capacity to decrease 
Regarding the lower, but not statistically significant, levels lipid oxidation directly at the vascular wall as part of  the 
of  lipid oxidation observed in the estrogen-only group, atherosclerotic process. Physiopathology of  CVD is a very 
there is yet no clear explanation for this phenomenon. complex topic but this protective effect shown by estrogens 
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Escalante, et al.: HRT and lipid oxidation at the arterial wall
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16 Journal of Mid-life Health ¦ Volume 8 ¦ Issue 1 ¦ January-March 2017 Journal of Mid-life Health ¦ Volume 8 ¦ Issue 1 ¦ January-March 2017 PB