The Effects of Moderate Ethanol Intake on Triglycerides, Total Cholesterol, HDLCholesterol, LDL-Cholesterol, Glucose, and Albumin, in Serum Samples

of Male Rats

Running Head: Effects of ethanol on lipid profile, glucose, and albumin in rats

Authors:
Jessilyn Cauble
Kathryn Enstad

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
Jan Lumibao
Department of Exercise and Nutritional Sciences
San Diego State University
5500 Campanile Drive
San Diego, Ca 92182-7251
(708)745-1398
janlumibao@gmail.com

Abstract
Background: Cardiovascular disease is one of the leading causes of death in todays society.
Studies show that moderate ethanol intake can reduce the risk of cardiovascular disease, suppress
cholesterol synthesis, and decrease lipid levels.
Objective: The purpose of this study is to look further into the risk factors associated with
cardiovascular disease, such as serum triglyceride levels, total cholesterol, HDL cholesterol,
LDL cholesterol, serum glucose, and serum albumin levels.
Methods: Twenty four Sprague-Dawley male rats were equally divided into two groups,
consuming a 20% ethanol solution on alternate days or a control diet of chow and water ad
libidum. The study was conducted over a 3 month span translating into 6 years of moderate
alcohol intake by humans. Serum triglyceride levels, total cholesterol, HDL-cholesterol, LDLcholesterol, serum glucose, and serum albumin levels were tested.
Results: Results from these tests showed there was no statistically significant difference for
serum triglyceride levels, total cholesterol, HDL-cholesterol, and LDL-cholesterol. However,
there was a statistically significant decrease in serum glucose levels given the ethanol solution
(p<0.042) and a statistically significant increase in serum albumin levels (p<0.018).
Conclusion: Moderate ethanol intake appeared to have a beneficial effect on blood glucose and a
significant difference on albumin. It can be concluded that further research on the topic should be
done and inconsistent findings on the subject should be taken into account for future studies.

Key Words: ethanol, cardiovascular disease, triglycerides, cholesterol

Introduction
Cardiovascular disease (CVD), for the past 80 years until present time, has been the
leading cause of death in the United States(1) accounting for roughly 1 in every 3 deaths (2). Due
to its ongoing prevalence numerous studies have investigated preventative methods, ethanol
consumption being among them. Moderate alcohol intake was found to decrease CVD (3) and if
consumed long-term lowered all-cause mortality risk and increased life expectancy (4). The
protective benefits produced are largely attributed to alcohols positive effects on lipid profile, as
much as half stemming solely on an increase in high density lipoprotein (HDL), and to a lesser
extent insulin sensitivity (5). Perissinotto et al. evaluated specific cardiovascular factors of 1896
Italian men, aged 65 to 84 years, who drank wine as a lifelong habit. The results indicated that
moderate consumption produced positive hematological values for HDL and fasting glucose(6).
The study concluded that the men experienced a net benefit of health just from these two
parameters.
Previous studies have failed to evaluate all mediating mechanisms involved in disease
prevention, develop a consistent definition of moderate consumption, and have inconsistent
results. Graphically, the relation between ethanol consumption and CVD incident has been
depicted as J shaped; a visual depiction that represents benefits being dependent upon the
number of drinks consumed(4). No widely accepted standard has been set leaving researchers to
rely on conspicuous terms of light, moderate, or excess defined in their own terms to represent
intake. Often studies are restricted to certain populations, i.e. post-menopausal women,
women(4), preexisting heart disease, or specified ethnicities. Roy et al. comprised a study on the
effects of ethanol on coronary heart disease in Indian men only(7). This limits the results from
being applicable to the majority and narrows the efficacy. The purpose of this study is to

determine the effect of moderate ethanol consumption on lipid profiles (serum triglycerides, total
cholesterol, low density lipoprotein (LDL), and HDL), blood glucose levels, and albumin in rats
treated with a 20% ethanol solution. The basis for this was to evaluate ethanols biological effect
on a wide span of parameters. The hypothesis was that rats moderately consuming ethanol will
display significantly lower levels of serum triglycerides, total cholesterol, and LDL and increase
HDL than those in the control group. The rats consuming moderate ethanol will also display
significant lower levels of fasting serum glucose.
Methods
Rats and diets. Twenty four male Sprague-Dawley rats were housed for 3 months in individual
wire bottom cages at San Diego State University. The research room pertaining the 28 day old
rats was on a 12-hr light dark cycle and held at a consistent temperature and humidity of 20-24
degrees Celsius and 40-45%, respectively. The training and procedures administered were
approved by the San Diego State University animal subject committee. The rats were divided
into 2 groups of 12 (n=12/group) and administered water ad libitum (control) or a 20% EtOH
solution (EtOH) on alternate days. The EtOH group in total received ethanol exposure for 1.5
months, a time frame equivalent to 6 years of moderate human consumption. Regular chow and
water were also administered at all times to the rats.
Experimental design. During the study a 48 hour food and water intake was measured and an
initial and final body weight was recorded (data not shown). The lipid profiles were assessed
through the measurements of serum triglycerides (TG), total cholesterol (TC), high density
lipoprotein (HDL), and a calculated low density lipoprotein (LDL). The general health markers
of serum glucose and albumin were evaluated.

Serum Triglycerides. Serum Triglycerides were assessed through the use of the Stanbio
Triglyceride LiquiColor Procedure No. 2200 kit (Boerne, TX). The procedure instructed to pipet
3 ul of the Triglyceride Liquicolor Reagent (cat. no. 2201) in the first well, 3 ul of the
Triglyceride Standard, 200mg/dL (cat. no. 2103) in the next well, followed by 3 ul of each
unknown sample in the subsequent wells. Unknown samples 1 through 12 were from control
group and sample 13 through 24 from EtOH. The triglyceride reagent in the amount of 300 ul
was added to all samples and incubated at room temperature for 5 minutes. The absorbance was
read at 500 nm, values were recorded, and the TG concentrations (mg/dL) were calculated. The
blank value was subtracted from the sample value then divided by the difference of the blank and
standard; the resulting number was multiplied by 200. This assay relies on the enzymatic
hydrolysis of triglycerides by lipase to produce fatty acids and glycerol. The released glycerol is
oxidized through a series of enzymatic reactions that result in the production of quinoneimine
with a quantitative colormetric reading deteactable at the given 500 nm absorbance.
Serum Total Cholesterol. Concentrations of total serum cholesterol were evaluated through the
use of the Cholesterol LiquiColor Procedure No. 1010 assay kit by Stanbio (Boerne, TX). The
protocol consisted of pipetting 3 ul of Enzymatic Cholesterol Reagent (ref. no. 1011) into the
first curvet, 3 ul of Cholesterol Standard (ref. no. 1012) into the second curvet, and 3 ul of the 24
serum samples in subsequent curvets. An additional 300 ul of cholesterol reagent was added to
the sample curvets, incubated for 5 minutes, and read at 500 nm. Cholesterol concentration
(mg/dL) was calculated by subtracting the blank value from the unknown then divided by the
difference of the blank and standard; the resulting number was multiplied by 200. The principle
of this assay is to utilize cholesterol esterase and cholesterol oxidase in combination with

enzymatic cholesterol reagent, Cat. No. 1011 to produce a colormetric measure that quantifies
both cholesterol esters and free cholesterol present in the serum.
HDL Cholesterol. HDL cholesterol was assessed through the use of a Stanbio kit (Boerne, TX).
A two part procedure was conducted, separating the HDL fraction and evaluating the HDL
cholesterol concentration. To separate, 200 ul of serum from each of the 24 rat samples was
placed in their respective tubes and 20 ul of HDL Cholesterol Reagent (PTA/MgCl) was added to
each. Contents of the tube were mixed, held at room temperature for 5 minutes, and then placed
in centrifuge for 10 minutes at 1000xg. Clear supernatants utilized to determine serum HDL
cholesterol. To evaluate concentrations, 7.5 ul of HDL reagent pipetted into first well, 7.5 ul
standard in second well, and in the following wells pipet 7.5 ul of the 24 clear supernates
previously obtained. To each of the samples 300 ul cholesterol reagent added and held for 2
minutes. Absorbance of each well was read at 500 nm, values were recorded, and HDL
concentration was calculated. The blank value assessed was subtracted from the sample value
then divided by the difference of the blank and standard; the resulting number was multiplied by
55. This assay relies on selective precipitation of serum using magnesium chloride in conjunction
with phosphotungstic acid. The HDL fraction remaining in the serum is assayed for cholesterol
concentration.
LDL Cholesterol. LDL cholesterol was calculated through application of a formula. HDL was
subtracted from total cholesterol, and triglycerides were divided by 5 then subtracted from the
previous value.
Serum Glucose. Glucose was determined through the use of Stanbio Glucose Liquicolor
(oxidase) Procedure No. 1070 kit (Boerne, TX). The Experimental laboratory procedures
followed were to pipet 3 ul of the reagent in first well, 3 ul of the standard in second well,

followed by 3 ul of each of the 24 rat serums in subsequent wells. To each of the sample serums
300 ul of the reagent was added and read at an absorbance of 500 nm. The principle of this assay
is that the intensity of the redviolet quinone complex resulting from the coupled enzymatic
reactions on glucose parallels the serum concentration. Glucose oxidase oxidizes glucose to
hydrogen peroxide which in turn reacts, in the presence of peroxidase, with phenol and 4aminoantipyrine to form the detectable product.
Serum Albumin. Serum Albumin was evaluated by the Stanbio Liquicolor Procedure No. 0285
kit (Boerne, TX). The protocol instructed to pipet 3 ul of Albumin Reagent (cat. no. 0286) in first
well, 3 ul of Albumin Standard (cat. no. 0287) in second well, followed by 3 ul of each of the 24
rat serums in subsequent wells. To each of the sample serums 300 ul bromocresol green (BCG)
was added and read at an absorbance of 550 nm. Albumin concentration (g/dL) was calculated by
subtracting the blank value from the unknown then divided by the difference of the blank and
standard; the resulting number was multiplied by 6. This assay relies on dye binding technique
using BCG and citrate as buffer to form an evident reading with an absorption at 550 nm.
Statistical Analysis. All data was analyzed using SPSS 22 software (IBM, Armonk, NY).
Independent samples t-tests were implemented to compare the difference in means between the
control and EtOH groups of TG, TC, HDL, LDL, glucose, and albumin. The level of statistical
significance was set at alpha level p < 0.05 and data presented as means SEs.

Results
Analysis of the results proved the differences between the control and EtOH groups for
serum TG, TC, HDL, and LDL were statistically insignificant. Table 1 illustrates the effects of
the specified diets on the serum lipid profile. The rats receiving ethanol treatment demonstrated a

significantly lower serum glucose than those abstaining (p<0.042) and displayed in Figure 1.
Serum concentrations of albumin were significantly higher in EtOH group (p<0.018). The
significant difference between the control and EtOH group are represented in Figure 2.
Discussion
The present study was done to further investigate the effects of ethanol on lipid profiles.
Although other studies have been conducted that relate to this, the primary focus has not been on
evaluating the effects on serum triglyceride levels, total cholesterol, HDL-cholesterol, and LDLcholesterol. There have been inconsistent results produced among different studies, and certain
results are population restricted. After attaining the results presented in this study, it was clear
that there was no significant difference between levels of TG, TC, HDL, and LDL. However,
there was a significant difference between the two groups in levels of serum glucose and serum
albumin. These findings conflict with the original hypothesis that moderate consumption of
ethanol will significantly lower levels of serum TG, total cholesterol, LDL cholesterol, serum
albumin, and increase HDL cholesterol. The study did however confirm the hypothesis that
fasting serum glucose levels would be significantly lower after moderately consuming ethanol.
That being said, further research has been done that support these findings.
Shahryari et al. conducted a study that looked at the effects of opium and ethanol
consumption on lipid profiles and atherosclerosis in hamsters. The group given ethanol
demonstrated a significant increase in total cholesterol, TG, and HDL-cholesterol and a decrease
in LDL-cholesterol (8). The difference in findings could be due in part to the use of differing
species. The study completed by Nammi et al. used rats to evaluate the effects of moderate
ethanol consumption on the liver. This study had similar findings to the present study in that
there was no significant difference in total cholesterol and serum triglycerides, but serum glucose

also showed no significant difference (9). These studies confirm the notion that more research
should be done in order to attain more consistent results of the effects of ethanol on TG, TC,
HDL, and LDL.
In addition, the present study raised interests on the effects of ethanol on serum albumin
levels. Findings showed a statistically significant increase in serum albumin levels after the
moderate consumption of a 20% ethanol solution. In a separate study, results showed a
significant decrease in albumin concentration with ethanol exposure, but after exposure to
resveratrol and vitamin E serum albumin levels increased (10). After further research it was
apparent that not many studies have looked into the effects of ethanol on serum albumin and how
that applies to the general health and wellbeing of patients. Along with serum albumin, serum
glucose also showed significant results in the present study.
To continue, the serum glucose findings showed a significant decrease. In an
observational study, fasting glucose results were log transformed to show that moderate alcohol
use has no effect on fasting glucose (11). These differences in findings could be due to the
different populations used, the different methods used, or the difference in the age of the
population. Although the findings in the presented study are significant, further research should
be done because of the differences among studies.
Overall, the need for further research was made apparent after analyzing this study with
the objective to obtain more concrete results in regards to lipid profile, serum glucose, and
albumin. Many studies have previously confirmed the protective effects of moderate ethanol
consumption on cardiovascular disease made evident by Strepple et al.s research. This particular
study sought to distinguish the effects between long-term wine consumption and moderate
ethanol intake. Findings concluded a strong, inverse association between long-term moderate

alcohol intake and cerebrovascular, total cardiovascular, and all-cause mortality (4). With this
information at hand, more studies should look further into the association between ethanol and
the risk factors associated with cardiovascular disease, i.e. triglyceride levels, total cholesterol,
HDL-cholesterol, and LDL-cholesterol. Other parameters, such as blood pressure and weight,
should be taken into account when looking into a study of this nature. These measures all play a
contributing factor in overall heart health and should be taken into consideration when studying
the effects of ethanol and its protective properties on heart health.
Even though several parameters from this study were statistically insignificant, the
gathered information is still beneficial and supplement the valuable knowledge already laid by
previous studies. The greatest impact of this study is its ability to prove conflicting results
continue to persist and to motivate much more research to be done. Concrete evidence to support
the positive effects of ethanol on serum triglyceride levels, total cholesterol, HDL-cholesterol,
LDL-cholesterol, serum glucose, and albumin could make significant advances in the field of
medicine and nutrition.
Acknowledgements
The authors wish to acknowledge the contributions of the Spring 2015 Advanced Nutrition
Laboratory students at San Diego State University who assisted in conducting and evaluating this
research.

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250

200

150

Blood Glucose (mg/dL)

CO

100

EtOH

50

Figure 1: Blood Glucose concentrations in male rats fed the CO or EtOH diet for 3 months. Values are means +/- SE's, n=12 per group. Labeled means do not differ significantly, p<0.05. CO, control; EtOH, 20% ethanol solution.

4.5
4
3.5
3
2.5

Serum Albumin (g/dL)

CO 2

EtOH

1.5
1
0.5
0

Figure 2: Serum total albumin concentrations in male rats fed the CO or EtOH diet for 3 months. Values are means +/- SE's, n=12 per group. Labeled means do not differ significantly, p<0.05. Co, control; EtOH, 20% ethanol solution.