THE EFFECTS OF MODERATE ETHANOL CONSUMPTION ON RISK FACTORS

FOR CARDIOVASCULAR DISEASE, OXIDATIVE STRESS AND ANTIOXIDANT

CAPACITY IN RATS

Running Head: Effects of moderate ethanol consumption on risk factors for CVD and
other chronic diseases

Authors:
Mikaela Ambrosini
Jennifer Axford
Kacey Bien
Mee Young Hong, PhD
School of Exercise and Nutritional Sciences, San Diego State
University, San Diego, CA 92182

Corresponding Author:
Mee Young Hong, PhD
Department of Exercise and Nutritional Sciences
San Diego State University
5500 Campanile Drive
San Diego, Ca 92182-7251
(619) 594-2392
mhong2@mail.sdsu.edu

Abstract
Background
In recent years, numerous studies assessing the effects of ethanol consumption
on cardiovascular disease risk factors in both humans and animals have been
published. However, results have shown inconsistent results, have been restricted to
certain population groups and have focused mainly on the negative effects of large
doses of ethanol ingestion.
Objective
To investigate the effects of moderate ethanol cqnsumption on risk factors such
as lipid profiles, oxidative stress and ant1ox1dant capacity as they relate to
cardiovascular and other chronic diseases.
Methods
Twenty-four 109 day old male Wistar rats were equally divided into two groups (n
= 12/group) consuming regular chow and either a 20% ethanol solution (treatment
group) administered on alternate days or plain water ad libitum in control. Body
weights and weight gains were monitored, as well as water , EtOH and regular chow
intake.
Organ weights, general health markers, lipid profiles, oxidative stress and
antioxidant capacity were evaluated after euthanization.
Results
Treatment group demonstrated significant improvements on specific lipid
components (p<0.05). Organ weights did not indicate significant differences
between groups, though epididymal fat reduced.significanUy in treatment group.
General health markers included a significant improvement (p=<0.05) and
/oxidative stress improved significantly withinthe treatment group although
antioxidant capacity results we insignificant between groups
(p=<0.05).
Conclusion
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Moderate ethanol consumption appears to significantly improve specific lipid
profiles, health markers and oxidative stress,suggesting that ethanol may be
beneficial in reducing specific risk factors for developing cardiovascular disease
and other lifestyle related conditions.

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Key Words: Ethanol; rats;lipid profile; antioxidant; oxidative stress

Introduction
Cardiovascular diseases such as coronary heart disease (CHO) and
peripheral arterial disease (PAD), among others, are the leading causes of death
worldwide (1). An estimated 17.5 million deaths in 2012 (representing a staggering
thirty-one percent of all deaths globally) were attributed to CVD; and this number
continues to rise. The World Health Organization predicts that by the year 2030,
nearly 23 million people will die annually from CVD (1). Researchers are now racing
against the clock to find a viable treatment. One compound that has shown some
promise in recent years is ethanol (EtOH). While it was once believed that the
cardioprotective effects associated with moderate drinking were due to polyphenols
such as the ones that exist in wine and dark beers, nearly all recent epidemiologic
studies reported a J-shaped curve with moderate EtOH consumption regardless of
alcohol source (2). With this understanding, the more recent research has focused
on the specific cardioprotective mechanisms associated with ethanol ingestion.
These mechanisms appear to have both direct and indirect functions and work
synergistically to not only reverse damage caused by existing or
prior cardiovascular events, but also trigger preventative measures in the body thereby
reducing the risk factors for CHO and related conditions (2). Some of these risk factors
include, but are not limited to, obesity, oxidative stress, high cholesterol, and diabetes
(2).
It was the preventative measures and their mechanisms whereby these
reductions in risk factors take place that were of the interest to these researchers.

Therefore, the aim of this study was to assess the effects of moderate EtOH
consumption on associated risk factors for cardiovascular disease. In
addition,these researchers were interested in measuring changes in oxidative
stress and antioxidant capacities as the result of EtOH consumption in controlled,
moderate amounts.
Researchers felt it was prudent to explore whether moderate EtOH consumption
would positively influence the risk factors, namely excess body weight, oxidative
stress and reduced antioxidant capacity, in test subjects. Our hypothesis was that
rats consuming moderate EtOH would demonstrate improved lipid profiles and a
reduction in epididymal body fat compared to the control. In addition, rats receiving
EtOH treatment would result in increased antioxidant capacity while demonstrating
a decrease in oxidative stress.

Methods
Animals and Diets
Twenty four male, 109-day old Wistar rats were split into two groups of twelve ,
and housed in two separate wire-bottom cages in a research room at San Diego State
University. Both groups were provided with water and Regular Chow (Lab Diet, St
Louis, MO) in oval pellet form, ad libitum. Each cage contained a feeder designed to
hold approximately 2-3 days worth of chow at once. All procedures and use of the
animals in this study were approved by the San Diego State University Animal
Subjects Committee.
The Laboratory Rodent Diet minimizes biological variation throughout longer
studies by using the highest quality ingredients. Laboratory Rodent Diet manufacturers
guarantee the pellets contain at least 23% crude protein, 4.5% crude fat, 6.0% crude
fiber, and less than 8.0% crude fat. Both groups were fed the Laboratory Rodent Diet
for a duration of 3 months, which translates into approximately 6 years of moderate
human consumption.
Group one functioned as the controlgroup, while the second group was provided
with a 20% Ethanol Alcohol solution, which was administered on alternating days.

Biochemical Assays
Each rat's body weight/weight gain was monitored and recorded daily, as well
as daily water and ethanol solution intake. Generalhealth markers assessed included
serum albumin (Boerne, TX) and s

m glucose. Lipid profiles were monitored

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throughout the study, by measuring serum triglycerides, total cholesterol,HDL: Stanbio

(Boerne, TX), and Serum LDL, de te rm

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Blood samples were collected from each specimen and serum separator tube
was then used to separate the serum from other blood components. The samples were
given 30 minutes to clot in the tube, followed by 10 minutes of centrifugation at 1OOOx
g. Each sample was immediately removed from the centrifuge and stored at <-20
degrees Celsius.
C-reactive protein status was monitored using the Rat C-Reactive Protein (CRP)
ELISA (Enzyme-Linked lmmunosorbent Assay) Kit. This kit was used to detect the
quantity of CRP in the rat serum using a sandwich ELISA test.The standards and
sample serums were added to each well on a plate,causing any existing rat CRP to
bind to the antibody. The plates were then incubated and washed before the
horseradish peroxidase conjugated anti-rat CRP was added. This allowed the
formation of an antibody-antigen-antibody "sandwich." The assay procedure was then
performed according to test directions.The plates were then incubated a second time
and washed prior to adding in the substrate solution.A stop solution was then added
and the final measurement was recorded using a spectrophotometer at a wavelength
of 450nm.
An Oxidative stress test (Cayman, Ann Arbor, Ml) was then performed using
Cayman's TBARS Assay Kit. This assessment tested for Malondialdehyde (MDA),
which is a product of lipid peroxidation.The presence of MDA in the serum is a
specific indicator of oxidative stress in tissues and cells. The measure of
Thiobarbituric Acid Reactive Substances (TBARS) was the method used to screen
and monitor lipid

peroxidation. The instructed amount of TBA was added to the serum and held at 90100 degrees Celsius in acidic conditions, in order to react with the MDA in the serum.
The solution was then measured fluorometrically, at excitation wavelength of 530 nm
and an emission wavelength of 550 nm.
The Cayman's Antioxidant Assay was then used to measure the serum's total
antioxidant capacity (both aqueous and lipid soluble antioxidants). Antioxidants in the
sample inhibit the oxidation of ABTS (2,2'-azino-bis) a compound used to detect
molecules binding to one another. The antioxidant level can then be monitored by
reading the absorbance at 750 or 405 nm.

Statistical Analysis
All data was analyzed using

BM, Armonk, NY) to analyze the C-reactive

protein status, oxidative stress test, antioxidant capacity, as well as lipid profiles, serum
albumin, and serum glucose. Students performed T-tests using determined values and
presented data as mean SE. An alpha level of p < 0.05 was used to determine
whether data was statistically significant.

Results
While organ weights for liver, spleen, kidney and epididymal fat were
reduced in the treatment group, only epididymal fat measurements were
significantly different between the two groups (p=0.030), (Table 1). Total
epididymal fat decreased from 12.13g in the control group to 9.51g in the
ethanol group.
Mean values for triglyceride, total cholesterol and LDL cholesterol were all
lower in the ethanol group versus the control group, and mean HDL cholesterol
values were higher in the ethanol group versus the control group, although the
results for triglyceride and HDL cholesterol were not found to be statistically
significant. Both total cholesterol (p=0.036) and LDL cholesterol (P=0.031) were
significantly lower in the ethanol group versus the control group (Table 2). Total
cholesterol decreased from 178.43mg/dl in the control group to 142.51mg/dl in
the ethanol group. LDL cholesterol decreased from 105.37mg/dl in the control
group to 73.24mg/dl in the ethanol group.
There were no evident differences in the mean albumin values between
the two groups. Results showed the albumin levels for both groups were
4.91g/dl (Figure 1).
Glucose levels were significantly lower in the ethanol group (p=0.030)
(Figure 2) which measured at 114.57mg/dl in the control group and 90.33mg/dl in
the treatment group.

Measurements for C-reactive protein showed no significant difference

between the two groups,with the mean value of the control group being
503.91g/ml versus 493.77g/ml in the ethanol group (Figure 3).
Results for TBARS measurements indicated a significant difference
between the control and the treatment group (p=0.028), (Figure 4). Oxidative
stress was reduced in the ethanol group with the mean value being 2.31M for
the ethanol group versus 3.35M in the control.
Total antioxidant capacity values increased (Figure 5) from 0.26mM in
the control vs 0.47mM in the ethanol group,although the results were not
statistically significant.

Discussion:
Elevated levels of oxidative stress, triglyceride values, blood glucose, LDL
and total cholesterol are all factors that have been found to contribute significantly to
the development of cardiovascular disease (3). Oxidative stress is caused by the
imbalance of free radical production and the inability of the body to neutralize them
through antioxidants (4). A free radical is an unstable oxygen-contai ning molecule
with an unpaired electron. This unpaired electron makes it highly reactive and
eager to steal electrons from other cell components in order to reach a stable state
(5). The simultaneous destabilization of other cellular structures results in a long
chain of free radical reactions and is the root cause of increased levels of oxidative
stress. If antioxidants are present in the cells, they are able to donate an electron to
the free

radical thus preventing the destabilisation resulting in increased levels of oxidative

stress (5).
This study has demonstrated that moderate consumption of ethyl alcohol may

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not only help reduce the risk for cardiovascular disease by decreasing oxidative stress
- .. ... --

...

levels, but may also help decrease blood glucose,body fat, increased antioxidant levels
and improve lipid profiles. Research attributes these benefits to a synergistic effect
between various mechanisms.
One recent study on the effects of alcohol and cardiovascular health suggests
that moderate alcohol consumption increases glucose metabolism and insulin sensitivi!Y..
'7

--

for 12 to 24 hours following ingestion (6). This supports our findings that blood glucose
levels were significantly decreased in rats who had consumed a moderate amount of
ethanol (20% on alternate days). Ethanol improves insulin sensitivity by suppressing the
discharge of fatty acids from adi
adiponectin levels (6).

pose

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while

simultaneously increasing

Adiponectin is a hormone that regulates lipid and glucose metabolism secreted

from adipocytes (fat cells). An increased level of adiponectin improves insulin
sensitivity (7). With fewer fatty acids present, substrate competition in the Kreb's
cycle for skeletal muscle is also reduced, promoting additional glucose metabolism
(7). Although the
liver , spleen, and kidney all showed a decrease in weight following moderate alcohol
consumption, only a decrease in epididymal fat was significant. The increased insulin

.-

sensitivity may play a major role in facilitating the reduction in epididymal body fat.

Lipogenesis is a pathway in which acetyl-CoA is converted to fatty acid for

storage (8). Insulin stimulates lipogenesis by activating two key pathways: Pyruvate
Dehydrogenase (PDH) and Acetyl CoA Carboxylase (ACC) (8). PDH pathway is the
conversion of Pyruvate to Acetyl CoA (8). ACC pathway is the conversion of the Acetyl
CoA produced from the PDH pathway to Malonyl CoA. Malonyl CoA is then used to
build larger fatty acid molecules (8). From this, researchers may conclude that the
increased insulin sensitivity and decreased glucose levels may account for the reduction
in lipogenesis and epididymal fat storage following moderate alcohol consumption.

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Results of another study performed in 2009 indicated that regular moderate
alcohol consumption may stimulate anti-inflammatory processes by inducing
cardioprotective proteins such as Nitric Oxide Synthase, Superoxide Dismutase, and
Heat Shock Proteins (4). Nitric Oxide Synthase (NOS) is a heme protein which acts as
an anti-thrombogeni c agent by suppressi ng muscle proliferation , platelet aggregation,
and monocyte adhesion (4). Following ethanol ingestion, NOS expression is
increased, leading to an increased amount of nitric oxide production. This causes
ethanol-induced
vasodilation, ultimately resulting in an elevated level of myocardial blood flow (4). The

.--

-------

same study found that 8 weeks of moderate alcohol consumption increased the
maximal effects of endothelial-dependent vascular relaxation which were consistent with
the increased expression of NOS (4).

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Heat s h ock proteins also aid in protecting cells from oxidative stress. When
oxidative stress damages proteins, they partially unfold. HSP70 is a specific heat shock
protein which protects proteins by temporarily binding to the exposed hydrophobic

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residues preventing them from aggregating. The increased expression of this protein
likely aids in reducing oxidative stress. Superoxide Dismutase (SOD) is an antioxidant
that acts as an anti-inflammatory agent in the body against superoxide, the most
common free radical in the body. Our study showed a rise in antioxidant levels
following moderate ethanol consumption, which may be a result of the increase in SOD.
Despite the concrete data suggesting that moderate intake of ethanol provides
beneficial effects to the cardiovascular system, skeptics raise questions by examining
surrounding factors. For example, individuals who have the luxury to regularly
consume alcoholic beverages may also correlate to a superior sociodemographic
status, leading to healthier lifestyle choices including diet and exercise routines. This
lifestyle is proposed to have some influence in a reduced risk of Cardiovascular
Disease (3).
In summary, this study demonstrated that moderate consumption of ethanol
reduces cardiovascular disease risk factors by decreasing body fat, improving lipid
profiles and decreasing oxidative stress and blood glucose. These effects may be
attributed to the effects of increased activation of cardioprotective proteins, increased
insulin resistance, and elevated Adiponectin levels. Although results for HDL
cholesterol, albumin, liver, spleen and kidney were insignificant, the trends did follow
those we had predicted thus, additional research on these elements may be beneficial.

REFERENCES
1. Cardiovascular Diseases (CVDs). World Health Organization Web site.
http://www.who.int/medi acentre/factsheets/fs317/en/. Updated January
2015. Accessed May 1, 2015.
2. Krenz M, Korthuis R. Moderate ethanol ingestion and cardiovascular protection:
From epidemiologic associations to cellular mechanisms .J Mo/ Cell Cardiol.
2012;52(1):93-104.
3. O'Keefe JH, Bhatti SK, Bajwa A, DiNicolantonio JJ, Lavie CJ. Alcohol and
cardiovascular health:The dose makes the poison or the remedy. Mayo Clin
Proc. 2014;89(3):382-393.
4. Agarwal DP. Cardioprotective effects of light-moderate consumption of alcohol:
A review of putative mechanisms. Alcohol & Alcoholism. 2002;37(5):409-415.
5. Pagel PS, Kersten JR, Warltier DC. Mechanisms of myocardial protection

D produced

by chronic ethanol consumption.Pathophysiology. 2004;10:121-129.

Martin CG, Agapito W, Obeso A, et al. Moderate ethanol ingestion, redox
status, and cardiovascular system in the rat. Alcohol. 2011;45:381-391.
7. Collins MA, Neafsey EJ, Mukamal KJ, et al. Alcohol in moderation,
cardioprotection, and neuroprotection: Epidemiological considerations and
mechanistic studies. Alcohol Clin Exp Res.2009;33(2):206-219.
8. Poli A, Marangoni F, Avogaro A, et al. Moderate alcohol use and health: A
consensus document. Nutr Metab Cardiovasc Dis. 2013;23:487-504.

Table 1. Organ weight

Liver (g)

Contro
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20.95
1.19

Ethano
l
18.93 0.71

Spleen (g)

0.86 0.03
0.94 0.05

Kidney (g)

3.51 0.13
3.91 0.20

Epididymal fat (g)

9.51 0.42

12.13 1.05

*
Data presented as means SE (standard error).

* P < 0.05 compared to Ethanol group.

Table 2. Lipid profile

Control
Triglyceride (mg/dL)
Total cholesterol (mg/dL)

160.95 19.89
178.43 16.45

*
HDL cholesterol (mg/dL)

40.78 4.81

LDL cholesterol(mg/dl)

105.37 11.65

*
Data presented as means SE.

* P < 0.05 compared to Ethanol group

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Ethanol
149.77 11.73

142.51 8.27

54.91 14.92
73.24 11.24

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Figure 1. Effects of 20% EtOH solutio n on protein status.

Bars represent means SE.

Ethanol

Figure 2. Effects of 20% EtOH solution on glucose

levels. Bars represent means SE.
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Figure 3. Effects of 20% EtOH solution on inflammation.

Bars represent means SE.

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Figure 4. Effects of 20% EtOH solution on oxidative stress.

Bars represent means SE.

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Figure 5. Effects of 20% EtOH solution on total antioxidant capacity.
Bars represent means SE.