BIOL2537 Laboratory in Nutritional Science

Practical 5: Serum Triglyceride and

Cholesterol: Effects of Different Dietary
Components
Name: Hung Wing Tung (2009053464)
Group No.: 3
Date: 14/4/2011

Abstract

The serum triglyceride and

cholesterol levels of four 8
weeks old male rats were
determined after each having a
3-week dietary consumption of
20% butter fat/ 0.5% cholesterol
with either (a) cholestyramine,
(b) 5% fiber, (c) 10% fiber or (d)
20% fiber.
15 L of serum from each rat
was mixed with 1500 L of
Stanbio Triglyceride Liquicolor
kit, #2200 430 for the
calculation of serum triglyceride
level after reading the
absorbance at 500 nm. Same
volume of rat serum was mixed
with 1500 L of Stanbio
Cholesterol assay kit, #1010
225 for the calculation of serum
total cholesterol. The serum of
the rat fed with 20% fiber was
found to contain the least
amount of triglyceride (72.92
mg/dL) and cholesterol (46.37
mg/dL), while that of the rat fed
with 5% fiber was found to
contain the highest amount of
triglyceride (173.45 mg/dL) and
cholesterol (103.01 md/dL). It is
concluded that increasing
dietary fiber consumption could
lower serum triglyceride and
cholesterol levels.

Introduction

Dietary lipids, once enter

human body, are subjected to

digestion and absorption. There

are two main categories of
blood lipid: triglycerides and
cholesterols. Due to their
hydrophobic nature, they are
carried by lipoproteins in blood1.
Although lipids are essential
building blocks of cells and
organelles1, a high level of blood
lipid is the major risk factor for
coronary heart diseases2.
High blood lipid can be caused
by genetic factors, or
problematic diet habits, such as
alcoholism and high intake of
saturated fat3. While excessive
saturated fat intake can raise
blood lipid level, high intake of
dietary fiber is known to reduce
it4. Thus, increasing the intake
of dietary fiber serves to be the
first-line approach for blood lipid
reduction5. Besides dietary
intervention, pharmacological
treatment can also lower the
blood lipid profile6. For example,
cholestyramine, a bile acidbinding resin, is often used to
treat people with
hypercholesterolemia7.
The specific aim of this study is
to examine and compare the
effect of cholestyramine and
dietary fiber on the serum lipid
profile after consumption of a
high fat diet. On the course of
experimental treatment, both
triglyceride and cholesterol
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BIOL2537 Laboratory in Nutritional Science

assays will be used to

determine the amount of
triglyceride and total cholesterol
in the rat serum respectively.
The principle of the triglyceride
assay is as follows8:
Lipase

Triglyceride + H2O

Glycerol

Glycerol kinase

Glycerol + ATP
ADP

G-3-P +

Glycerylphosphate oxidase

G-3-P + O2

DAP + H2O2

H2O2 + 4-Aminoantypyrine + 4Chlorophenol

Peroxidase

Quinoneimine + HCl + 4
H2O
And the following is the
principle of cholesterol assay9:
Cholesterol esterase

Cholesterol esters
Cholesterol + Fatty acids

15.0 mM Magnesium Salt, 0.5

mM 4Aminoantipyrine, 4 mM 4Chlorophenol, 1500 U/L
Glycerylphosphate oxidase,
0.01 % Sodium Azide, 4000 U/L
Lipase, 400 U/L Glycerol kinase,
2000 U/L Peroxidase, 50 mM
Goods Buffer (pH 6.7 0.1).
The latter contains 0.25 mmol/L
4-Aminophenazone, 25.0
mmol/L Phenol, > 5.0 U/mL
Peroxidase, > 0.15 U/mL
Cholesterol esterase, > 0.1
U/mL Cholesterol oxidase,
buffers and stabilizers.
Triglyceride and cholesterol
standards were also prepared
by the School. Triglyceride
standard contains glycerol with
surfactant to yield 200 mg/dL
triglycerides as triolein, with
0.01% cSodium Azide being
added as preservative.
Cholesterol standard contains
buffered aqueous solution of
cholesterol with stabilizers,
surfactants and preservative.

Cholesterol oxidase

Cholesterol + O2
en-3-en-one + H2O2

Cholest-4-

H2O2 + 4-Aminophenazone +
Phenol
Peroxidase

H2O + Q-Quinoeimine dye

Materials and Methods

Collection of Materials
A. Reagents
Stanbio Triglyceride Liquicolor
kit, #2200 430 and Stanbio
Cholesterol assay kit, #1010
225 were purchased by the
School of Biological Sciences,
University of Hong Kong. The
former contains 2.0 mM ATP,

B. Animals and diets

4 male (8 weeks old) Sprague
Dawley rats were obtained from
the Laboratory Animal Unit,
University of Hong Kong. Rats
were housed individually and
kept in a controlled environment
at 22C under a 12-h-light/12-hdark cycle with light on from 7
am to 7 pm. They consumed ad
libitum water and laboratory
food (Lab Diet, The Richmond
Standard, PMI Nutritional
International) for 1 week before
receiving the experimental diets
(refer to Annex 1 for the
composition of the diets). All
prepared diets were stored at
20 C.
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BIOL2537 Laboratory in Nutritional Science

Serum Samples
Preparation

Reagen
t
Standar
d
Serum
Water

5%
fibe
r

10 %
fiber

150
0

150
0

1500

15

15
-

15
-

15
-

15
-

150
0

150
0

15

Duplicate of this assays set was

done at the same time. The
cuvettes were incubated at
room temperature for 10
minutes. Absorbance was read
at 500 nm. Blank solution was
used to set the
spectrophotometer to zero
absorbance.

Cholest
yramin

Abdominal cavity of the rats

was exposed after midline
incision. Blood was drawn from
abdominal vena cava into a
syringe, and then transferred
B. Serum Cholesterol Assay
into glass centrifuge tube. Blood
Blank solution containing
was allowed to stand at room
distilled water (B), cholesterol
temp for 30 minutes for blood
standard (S) and 4 serum
clot formation. After that blood
samples (U) were pipetted into
was centrifuged at 2000 rpm
cuvettes with the following
at 4C for 10 minutes. The
volumes (L) (shown in Table b.)
supernatant (serum) was
and mixed well with the
transferred to clean tubes and
Enzymatic Cholesterol Reagent
stored at 2 8 C for
by pipetting up and down.
preparation of assays.
Table b. The required volumes for different
assays
Assays Preparation after testing serum triglyceride level
A. Serum Triglyceride Assay
U
Blank solution containing
10
5%
distilled water (B), triglyceride
B
S
%
15%
fibe
standard (S) and 4 serum
fibe fiber
r
samples (U) were pipetted into
r
cuvettes with the following
150 150 150 150 150
1500
volumes (L) (shown in Table a.)
0
0
0
0
0
and mixed well with the
15
Triglyceride LiquiColor Reagent
by pipetting up and down.
15
15
15
15
15

15
%
fibe
r
150
0

Cholest
yramin

Rats were randomly assigned to

one of the four 20% butter fat/
0.5% cholesterol diets: (i) 8%
w/w
cholestyramine, (ii) 5% fiber,
(iii) 10% fiber, (iv) 20% fiber and
allow free access for 3 weeks.
On day 21, they were
anaesthetized with
pentobarbital (Abbott
Laboratory) intraperitoneal
(50 mg/kg) after a 12-hour food
deprivation overnight. Blood
was drawn from abdominal vena
cava of the rats to get the
serum.

Table a. The required volumes for

different assays for testing serum
triglyceride level

BIOL2537 Laboratory in Nutritional Science

Duplicate of this assays set was

done at the same time. The
cuvettes were incubated at
room temperature for 10
minutes. Absorbance was read
at 500 nm. Blank solution was
used to set the
spectrophotometer to zero
absorbance.

Results
The absorbance at 500 nm of
both triglyceride and cholesterol
assays was read and
summarized in Table c. and
Table d. respectively.
Table c. Absorbance of different
triglyceride assays

Seru
m
sam
ple

Where AU, AS and AB were the

absorbance values of the serum
sample with unknown
concentration of triglyceride or
cholesterol, standard and blank
solution respectively; 200 was
the concentration of triglyceride
or cholesterol standard (mg/dL).
The calculated serum
triglyceride and cholesterol
levels of the rats fed with
different diets were illustrated in
the following tables.
Table e. Calculated serum
triglyceride levels in different
rat serum samples

0.280

0.285

Avera
ge
0.283

0.127

0.159

0.143

0.210
0.175
0.065

0.280
0.196
0.141

0.245
0.186
Table e. Calculated serum cholesterol
0.103

Absorbance
Standard
Cholestyramin
e
5% fiber
10% fiber
20% fiber

Serum Triglyceride or Serum

Total Cholesterol (mg/dL) =
[ (AU AB)/ (AS AB) ] x 200

Rat Serum
Sample
Cholestyramine
5% fiber
10% fiber
20% fiber

Serum Triglyceride
Level (mg/dL)
101.2389
173.4513
131.3274
72.92035

levels in different rat serum samples

Rat Serum
Serum Cholesterol
Table d. Absorbance of different
Sample
Level (mg/dL)
cholesterol assays

Serum
Sampl
e

Standard
Cholestyram
ine
5% fiber

Cholestyramine
Absorbance
5% fiber
0.312
0.253
10% fiber
0.130 20%
0.102
fiber
0.148

0.143

0.129

0.095

82.12389
103.0088
79.29204
46.37168

Bar charts below showed the

comparison of serum
20% fiber
0.068
0.063 triglyceride and cholesterol
levels among the rats fed with
After obtaining the absorbance
different diets.
readings, serum triglyceride and
serum cholesterol levels were
calculated using the following
equation:
10% fiber

BIOL2537 Laboratory in Nutritional Science

Fig a. Comparison of serum triglyceride levels in rats fed with different diets
200
150
Serum Triglyceride Level (mg/dL)100
50
0
Cholestyramine
Types of diets received

Fig b. Comparison of serum total cholesterol levels in rats fed with different diets
200
150
Serum Total Cholesterol level (mg/dL)100
50
0
Cholestyramine
Types of diet received

Discussion
Effect of Different
Concentrations of Fiber on
Serum Triglyceride Levels
Compared to
Chloestyramine
Referring to the bar chart
illustrated in Fig a, the serum
triglyceride levels were lower in
rats fed with increasing
percentages of fiber. Rat which
consumed 20 % fiber had the
lowest serum triglyceride level
(72.92 mg/dL), followed by that
fed with 10 % fiber (131.32
mg/dL) and lastly 5 % fiber
(173.45 mg/dL). A 20 % fiber

diet also showed a greater

reduction on serum triglyceride
level compared to a diet with
cholestyramine (101.24 mg/dL),
while the effects of 10 % and 5
% fiber diets on serum
triglyceride reduction were less
potent than that of the
cholestyramine diet.

Effect of Different
Concentrations of Fiber on
Serum Total Cholesterol
Levels Compared to
Chloestyramine
Referring to the bar chart
illustrated in Fig b, the serum
total cholesterol levels were
lower in rats fed with increasing
percentages of fiber. Rat which
consumed 20 % fiber had the
lowest serum total cholesterol
level (46.37 mg/dL), followed by
that fed with 10 % fiber (79.29
mg/dL) and lastly 5 % fiber
(103.01 mg/dL). Both 20 % and
10 % fiber diets showed a
greater reduction on serum total
cholesterol levels compared to a
diet with cholestyramine (82.12
mg/dL), while the effect of a 5 %
fiber diet on serum total
cholesterol reduction was less
potent than that of
cholestyramine diet.

Serum Lipids Lowering

Effect of Fiber and
Cholestryamine
Dietary fiber includes for a wide
range of plant substances
resistant to digestive enzymes
in human gastrointestinal
tract10. It can be classified into 2
major groups depending on the
solubility in water. Structural
fibers like lignin and cellulose
are insoluble fibers, while the
gel-forming fibers, such as oats,
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BIOL2537 Laboratory in Nutritional Science

psyllium, pectin and guar gum

are soluble fibers4. Several
reviews have suggested that it
is the soluble fibers carrying the
serum lipid lowering effect11,12.
Insoluble fibers, unless they
displace the food supplying
saturated fat and cholesterol,
had no effect on serum lipid
profile13.
There are many suggested
mechanisms by which fiber
lowers blood lipid level. First,
evidence has shown that some
soluble fibers can bind with bile
acids or cholesterol during the
formation of micelles14. This
lowers the cholesterol content in
liver cells and up-regulates the
LDL (low density lipoprotein)
receptors, clearing more LDL
cholesterol for bile acid
synthesis. Another suggested
mechanism involves an
inhibition of hepatic fatty acid
synthesis by the production of
short-chain fatty acids (e.g.
acetate and propionate) from
the fermentation of soluble
fibers in the gastrointestinal
tract15. Also, fibers may increase
intestinal motility16, hinder
absorption of macronutrients
(which increases insulin
sensitivity and promotes
breakdown of fats)17. Intake of
fibers also increases satiety and
reduces the total energy
intake18.
Cholestyramine, on the other
hand, can also increase fecal
excretion of bile acids. This upregulates the hepatic LDL
receptors and lowers the plasma
LDL cholesterol19. The reduction
in LDL is coupled with an
increase in triglyceride in human

and most animals blood,

causing a mild
hypertriglyceridemia19. It is
because in these animals,
enterohepatic circulation of bile
acids down-regulates the
hepatic triglyceride synthesis,
while interference in this
circulation by cholestyramine
inhibits this down-regulation19.
However, from the results, rat
fed with cholestyramine showed
a drop in serum triglyceride
level, which was inconsistent
with the theory. This may be due
to the absence of such downregulation of hepatic triglyceride
synthesis by bile acids in this
specific species of rat19.

Causes and Drawbacks of

Hyperlipidemia
Hyperlipidemia is referred to
high blood lipid level3. There are
3 major types of hyperlipidemia:
(a) Hypertriglyceridemia, (b)
Hypercholesterolemia and (c)
Mixed hyperlipidemia20.
Causes of hyperlipidemia are
multi-fold, which involves
personal and family history of
cardiovascular diseases (CVD),
relevant medical history (e.g.
hypertension, diabetes and HIV
infection), lifestyle (exercise and
diet), smoking and alcoholism20.
Genetic factors of
hyperlipidemia include a single
dominant gene defect,
deficiency in lipoprotein lipase,
or other abnormalities in lipid
metabolism3. Diet is another key
factor for hyperlipidemia. Excess
saturated fat intake can raise
the blood cholesterol level, while
a high carbohydrate diet can
cause elevated triglyceride in
blood3. Besides, alcohol can also
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BIOL2537 Laboratory in Nutritional Science

contribute to hyperlipidemia
owing to its energy-rich nature
and the increase in availability
of substrates for triglyceride
synthesis in liver3.
Hypertriglyceridemia can cause
pancreatitis (inflammation of the
pancreas), but the major
consequence of hyperlipidemia
is linked to an increased risk of
atherosclerosis (hardening of
arteries), which may result in
coronary heart diseases, stroke
and peripheral vascular
diseases. The pathology of
atherosclerosis is characterized
by atherogenesis3. When the
endothelial lining of arteries are
damaged from high blood
pressure, local injury, or
inadequate oxygen supply, the
permeability into subendothelial
space will increase, which
enables lipoproteins to pass
more easily. Since LDL and
triglyceride-rich lipoprotein
remnants are atherogenic, when
they enter the subendothelial
space, they undergo oxidation.
Oxidized LDL will be scavenged
by marcophages by expressing
the acetylated LDL receptors.
LDL will then become lipid-rich
foam cells, which undergo
apoptosis and produce a lipidrich extracellular medium. In
response to this process,
smooth muscle cells migrate
from the tunica media of the
arteries and proliferate. This
produces a connective tissue
matrix containing collagen,
elastin and proteoglycans, which
is known as plaque. Presence of
plaque in blood vessels can lead
to significant reduction in blood
flow, producing symptoms such
as angina or claudication. The

thin cap and a large lipid core of

plaque are also unstable and
susceptible to rupture. This
causes an acute occlusion of the
arteries. Owing to the blockage
in arteries, nutrients and oxygen
delivery to organs such as heart
and brain are hindered, causing
syndromes such as myocardial
infarction and stroke3.

Other Drug Treatments for

Hyperlipidemia
Many pharmacological agents
are available for treatment of
hyperlipidemia. For example, in
a bid to treat
hypercholesterolemia, apart
from cholestyramine, colestipol
also serves as a bile acid
sequestrant which lowers blood
cholesterol by binding with bile
acids irreversibly and upregulating LDL receptors21.
Other than bile acid
sequestrants, fibrates and HMGCoA reductase inhibitors can
also lower blood cholesterol by
the formation of derivative
clofibrate22 that decreases
cholesterol absorption and
inhibition of cholesterol
synthesis that reduces the
cholesterol content of
enterocytes and ACAT
(cholesterol acyltransferase)
activity23. Some emerging drugs
like synthetic saponins and 2azetidinones have been shown
to impair cholesterol absorption
by their potential interaction
with cholesterol transporter
molecules24,25.
Tetrahydrolipstatin (generic
name: orlistat) is a drug applied
to treat hypertriglyceridemia. It
works by inhibiting the activities
of gastric and pancreatic
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BIOL2537 Laboratory in Nutritional Science

lipases, which prevents dietary

triglyceride hydrolysis in the
intestinal lumen 26. Dose
response studies in humans
have shown that maximal
inhibition of fat absorption of
orlistat can be up to 30% of
dietary fat with regular meals
everyday27.

the pipetting up and down

process, leading to a leakage of
solution from the mouth of
cuvettes. Also, the reagent and
samples might not be well
mixed before the measurement
of absorbance. Thus,
improvement in experimenters
skills is necessary.

Possible Experimental
Errors and Improvement

Conclusion

Several experimental errors

might occur and contributed to
inaccurate results. First, the rats
used might show different
sensitivity to the experimental
diets, such as different rates of
digestion, absorption and
metabolism of nutrients or drug.
As only one rat was used for
each experimental diet, any of
the above biological variations
would greatly affect the
accuracy of results. Thus, more
rats should be used for each test
diet to minimize the effect of
biological variations.
Not only did the amount of rats
matter, the number of trials also
played an important role in
results accuracy. Reliability
would be lower if the number of
trials was too few. In this study,
only two trials were performed
in measuring the absorbance
values of sample assays, which
was insufficient to give reliable
results. As such, more trials
should be done by preparing
more serum sample sets.
Technical errors might occur
during the sample preparing
process too. For example, the
volumes of solution might not
be exactly the same as the
guideline given, since air
bubbles were produced during

The effects of fiber and

cholestyramine on serum
triglyceride and total cholesterol
levels in rats were determined.
It is concluded that increasing
fiber content in diet could lower
both serum triglyceride and
total cholesterol levels. Oral
administration of
cholestyramine also showed a
reduction in serum triglyceride
and total cholesterol.
Compared to cholestyramine, a
20 % fiber diet could decrease
the serum triglyceride level to a
greater extent; 20 % and 10 %
fiber diets also exerted a greater
reduction in serum total
cholesterol.

References
1. Lamarche B et al. (1996)
Triglycerides and HDLcholesterol as risk factors for
ischemic heart disease.
Results from the Qubec
cardiovascular study.
Atherosclerosis 119(2): 235
45.
2. National Center for Health
Statistics and the American
Heart Association. (1992)
Facts about cardiovascular
disease. Circulation 85: A103
(abstr).
3. Reckless JPD & Lawrence JM
(2003) Hyperlipidemia.
8

BIOL2537 Laboratory in Nutritional Science

Encyclopedia of Food
Sciences and Nutrition. 3183
92.
4. Brown L et al. (1999)
Cholesterol-lowering effects
of dietary fiber: a metaanalysis. Am J Clin Nutr 69:
30 42.
5. Expert Panel on Detection,
Evaluation, and Treatment of
High Blood Cholesterol in
Adults (1993) Summary of
the second report of the
National Cholesterol
Education Program (NCEP)
Expert Panel on Detection,
Evaluation, and Treatment of
High Blood Cholesterol in
Adults (Adult Treatment Panel
II). JAMA 269: 3015 23.
6. Sacks FM et al. (1996) The
effect of pravastatin on
coronary events after
myocardial infarction in
patients with average
cholesterol levels. N Engl J
Med 335: 1001 9.
7. Malmendier CL, Delcroix C &
Lontie J-F (1987) The effect of
combined fenofibrate and
cholestyramine therapy on
low-density lipoprotein
kinetics in familial
hypercholesterolemia
patients. Clinica Chimica
Acta 162(2): 221 27.
8. Fossati P & Prencipe L (1982)
Serum triglycerides
determined colorimetrically
with an enzyme that
produces hydrogen peroxide.
Clinical Chemistry 28: 2077
80.
9. Allain CC et al. (1974)
Enzymatic Determination of
Total Serum Cholesterol.
Clinical Chemistry 20: 470
5.
10. Eastwood MA, Passmore R

(1983) Dietary fiber. Lancet

2: 202 6.
11. Truswell AS (1995) Dietary
fibre and plasma lipids. Eur J
Clin Nutr 49 (suppl): S105
9.
12. Glore SR et al. (1994)
Soluble fiber and serum
lipids: a literature review. J
Am Diet Assoc 94: 425 36.
13. Kris-Etherton PM et al.
(1988) The effect of diet on
plasma lipids, lipoproteins,
and coronary heart disease. J
Am Diet Assoc 88: 1373
400.
14. Anderson JW, Tietyen-Clark
JT (1986) Dietary fiber:
hyperlipidemia, hypertension
and coronary artery disease.
Am J Gastroenterol 81: 907
19.
15. Nishina PM, Freedland RA
(1990) The effects of dietary
fiber feeding on cholesterol
metabolism in rats. J Nutr
120: 800 5.
16. Schneeman BO & Gallaher D
(1985) Effects of dietary fiber
on digestive enzyme activity
and bile acids in the small
intestine. Proc Soc Exp Biol
Med 180: 409 14.
17. Schneeman BO (1987)
Dietary fiber and
gastrointestinal function.
Nutr Rev 45: 129 32.
18. Blundell JE & Burley VJ
(1987) Satiation, satiety, and
the action of fiber on food
intake. Int J Obes 11(suppl): 9
25.
19. Groot PHE et al. (1992)
Effects of cholestyramine on
lipoprotein levels and
metabolism in Syrian
hamsters. Biochimica et
Biophysica Acta (BBA) Lipids and Lipid Metabolism
9

BIOL2537 Laboratory in Nutritional Science

1123 (1, 3): 76 84.

20. Park A (2009)
Hyperlipidemia. Medicine
37(9): 497 9.
21. Hofmann AF (1998) Bile
secretion and the
enterohepatic circulation of
bile acids. In: Feldman M,
Scharschmidt BF, Sleisenger
MH, editors. Sleisenger &
Fordtrans Gastrointestinal
and Liver Disease (6th
edition). Philadelphia, PA: WB
Saunders. p.937 48.
22. Angelin B, Einarsson K &
Leijd B (1979) Biliary lipid
composition during
treatment with different
hypolipidemic drugs. Eur J
Clin Invest 9: 185 90.
23. Miettinen TA (1991)
Inhibition of cholesterol
absorption by HMGCoA
reductase inhibitor. Eur J Clin
Pharmacol 40: S19 21.
24. Morehouse LA et al. (1999)

Comparison of synthetic
saponin cholesterol
absorption inhibitors in
rabbits: evidence for a nonstoichiometric mechanism of
action. J Lipid Res 40: 464
74.
25. Burnett DA et al. (1994) 2Azetidinones as inhibitors of
cholesterol absorption. J Med
Chem 37: 1733 6.
26. Hadvary P, Lengsfeld H &
Wolfer H (1988) Inhibition of
pancreatic lipase in vitro by
the covalent inhibitor
tetrahydrolipstatin. Biochem
J 256: 357 61.
27. Zhi J et al. (1994)
Retrospective populationbased analysis of the doseresponse (fecal fat excretion)
relationship of orlistat in
normal and obese
volunteers. Clin Pharmacol
Ther 56: 82 5.

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