Practical 6: Determination of Serum Cholesterol Level

Objective:
To determine the serum cholesterol level

Introduction:
Cholesterol is a waxy, fat-like substance which is found in all cells of the body. A human
body needs some cholesterol to make hormones, and substances which help to digest foods.
Besides, a human body makes all the cholesterol it needs. However, cholesterol also is found
in some of the food that an individual eat. Cholesterol travels through the bloodstream in
small packages called lipoproteins (lip-o-PRO-teens). These packages are made of fats on the
inside and proteins on the outside. The structure of cholesterol is shown in the below
diagram.

The structure of cholesterol

There are two kinds of lipoproteins which carry cholesterol throughout the body. They are
known as low-density lipoproteins (LDL) and high-density lipoproteins (HDL). Having
healthy levels of both types of lipoproteins is very important. A LDL cholesterol is also
called as a “bad” cholesterol. A high LDL level leads to a buildup of cholesterol in the
arteries. Low-density lipoprotein (LDL) is one of the five major groups of lipoprotein. These
groups are arranged from least dense to most dense accordingly, chylomicrons, very low-
density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), low-density lipoprotein
and high-density lipoprotein (HDL). In addition, lipoproteins transfer lipids around the body
in the extracellular fluid. They can be sampled from the blood and allow the fats to be taken
up by the cells of the body by receptor-mediated endocytosis. Lipoproteins are complex
particles composed of multiple proteins which transport all fat molecules around the body
within the water outside cells. They are typically composed of 80-100 proteins which are
organized by a single Apo lipoprotein B for LDL and the larger particles. A single LDL
particle is about 220-275 angstroms in diameter transporting 3,000 to 6,000 fat molecules,
varying in size according to the number and mix of fat molecules contained within. The lipids
carry cholesterol, phospholipids, and triglycerides amounts of each vary considerably. A
HDL cholesterol is also called as “good” cholesterol. This is because it carries cholesterol
from other parts of the body back to the liver. Liver removes the cholesterol from the body.
Moreover, high-density lipoproteins (HDL) is one of the five major groups of lipoproteins.
They are typically composed of 80-100 proteins per particle which organized by one, two or
three Apo lipoprotein A1 and more as the particles enlarge picking up and carrying more fat
molecules) and transporting up to hundreds of fat molecules per particle. Unlike the larger
lipoprotein particles which deliver fat molecules to cells, HDL particles remove fat molecules
from cells which want to export fat molecules. The fats carried include cholesterol,
phospholipids, and triglycerides, amounts of each are quite variable.

Additionally, cholesterol can both be produced by the body itself and obtained from food
sources. As cholesterol plays an important part in the human body, its main function is
hormone production. Cholesterol produce hormones such as estrogen, testosterone,
progesterone, aldosterone and cortisone. Besides that, cholesterol also produce Vitamin D
and bile. Cholesterol produces bile acids which help in digestion and vitamin absorption.
Cholesterol plays a very important part in both the creation and maintenance of human cell
membrane. High cholesterol levels are result of modifiable and non-modifiable risk factors.
Two major risk factors which are diet and exercise, are highly modifiable, because something
can be done to change these risk factors and reduce the likelihood of having high cholesterol.
Limiting intake of fat in the diet helps to manage cholesterol levels. It is helpful to limit foods
that contain cholesterol such as egg yolks, meat and cheese, saturated fats which are found in
some meats, dairy products, and chocolate, baked goods, and deep-fried and processed foods
and Trans fats which are found in some fried and processed foods. Moreover, abnormal
cholesterol levels may also be secondary because of diabetes, liver or kidney disease,
polycystic ovary syndrome, pregnancy and other conditions which could to increase levels of
female hormones and underactive thyroid gland. Drugs that increase LDL cholesterol and
decrease HDL cholesterol are progestin, anabolic steroids and corticosteroids. Lipids are fats
which are either absorbed from food or synthesized by the liver. Although all lipids are
physiologically important, triglycerides and cholesterol contribute to disease. The primary
function of triglycerides is to store energy in adipocytes and muscle cells.

In exogenous lipid metabolism, over 95% of dietary lipids are triglycerides and the rest are
phospholipids, free fatty acids (FFAs), cholesterol, and fat-soluble vitamins. Dietary
triglycerides are digested in the stomach and duodenum into monoglycerides (MGs) and free
fatty acids (FFAs) are digested by gastric lipase, emulsification from vigorous stomach
peristalsis, and pancreatic lipase. Furthermore, dietary cholesterol esters are de-esterified into
free cholesterol by these same mechanisms. Monoglycerides, free fatty acids, and free
cholesterol are then solubilized in the intestine by bile acid micelles, which shuttle them to
intestinal villi for absorption. Once absorbed into enterocytes, they are reassembled into
triglycerides and packaged with cholesterol into chylomicrons which is known as the largest
lipoproteins. Chylomicrons transport dietary triglycerides and cholesterol from within
enterocytes through lymphatic into the circulation. In the capillaries of adipose and muscle
tissue, Apo protein C-II (Apo C-II) on the chylomicron activates endothelial lipoprotein
lipase (LPL) to convert 90% of chylomicron triglycerides to fatty acids and glycerol, which
are taken up by adipocytes and muscle cells for energy use or storage. Cholesterol-rich
chylomicron remnants then circulate back to the liver, where they are cleared in a process
mediated by Apo protein E (Apo E). In endogenous lipid metabolism, lipoproteins
synthesized by the liver transport endogenous triglycerides and cholesterol. Lipoproteins
circulate through the blood continuously until the triglycerides which contain are taken up by
peripheral tissues or the lipoproteins themselves are cleared by the liver.

Moreover, the factors that stimulate hepatic lipoprotein synthesis generally lead to elevated
plasma cholesterol and triglyceride levels. The low-density lipoproteins (LDL), the products
of VLDL and IDL metabolism are the most cholesterol-rich of all lipoproteins. About 40% to
60% of all LDL are cleared by the liver in a process mediated by Apo B and hepatic LDL
receptors. While the rest are taken up by either hepatic LDL or no hepatic non-LDL
receptors. Hepatic LDL receptors are down-regulated by delivery of cholesterol to the liver
by chylomicrons and by increased dietary saturated fat, they can be up-regulated by
decreased dietary fat and cholesterol. Non hepatic scavenger receptors, most notably on
macrophages, take up excess oxidized circulating LDL not processed by hepatic receptors.
Monocytes are rich in oxidized LDL migrate into the sub endothelial space and become
macrophages. These macrophages then take up more oxidized LDL and form foam cells
within atherosclerotic plaques. The size of LDL particles varies from large and buoyant to
small and dense. Small, dense LDL is rich in cholesterol esters, because it is associated with
metabolic disturbances such as hypertriglyceridemia and insulin resistance, and is
atherogenic. The increased atherogenicity of small, dense LDL derives from less efficient
hepatic LDL receptor binding, leading to prolonged circulation and exposure to endothelium
and increased oxidation. High-density lipoproteins (HDL) are initially cholesterol-free
lipoproteins that are synthesized in both enterocytes and the liver. HDL metabolism is
complex, but the role of HDL is to obtain cholesterol from peripheral tissues and other
lipoproteins and then transport it to where it is needed most other cells, other lipoproteins and
the liver. Its overall effect is anti-atherogenic. Efflux of free cholesterol from cells is
mediated by ATP-binding cassette transporter A1 (ABCA1), which combines with Apo
protein A-I (Apo A-I) to produce nascent HDL. Free cholesterol in nascent HDL is then
esterified by the enzyme lecithin-cholesterol acyl transferase (LCAT), producing mature
HDL. The blood HDL levels may not completely represent reverse cholesterol transport.

Principle:

The cholesterol esters of the sample are hydrolysed by Cholesterol ester hydrolase (ChEH). 4
–Cholesten-3-one and H2O2 are then formed from the released free Cholesterol by
Cholesterol oxidase (ChOD). A measurable Red quinonimine derivative which absorbance
light at 505nm is formed from Hydrogen peroxide (H2O2) and 4-aminoantipyrine in the
presence of Phenol and peroxidase (POD).

Cholesterol esterase

Cholesterol ester + H2O Cholesterol + Fatty Acids

Cholesterol oxidase

Cholesterol + O2 4- Cholesten-3-one + H2O2

peroxidase

2 H2O2 + Phenol + 4-Aminoantipyrine Red quinone + 4 H2O

Reference Range:
Serum: 2.8 – 5.2 mmol/l (109 – 202 mg/dl)

Reagents:
Reagent (R1)

Pipes buffer, pH: 6.90 50 mmol/L

Phenol 24 mmol/L

Sodium Cholate 0.5mmol/L

4-Aminoantipyrine 0.5 mmol/L

Cholesterol esterase 180U/L

Cholesterol oxidase 200 U/l

Peroxidase 1000 U/l

Apparatus:

Centrifuge, Spectrophotometer, sample tubes and cuvettes, Pipettes and Pipette tips, beaker,
water bath (Temperature: 37°C), and Rotator

Materials:

Distilled Water, Biochemistry Control N Lot: 3578, expiry date: 2017 – 01 (2.52 mmol/L),
and Human Blood Samples

Procedures:
1) The human blood sample was centrifuged at 4000rpm around 10 minutes.

2) The spectrophotometer was set at 505 (480 – 520) nm.

3) The solutions were prepared as below:

 Blank Standard Sample
Working reagent 1 ml 1 ml 1 ml
Distilled water 10 ul
Standard 10 ul
Sample 10 ul

4) The solutions were mixed and incubated for 5 minutes in the water bath at 37°C
temperature.

5) The solutions were pipetted into cuvettes.

6) The spectrophotometer was adjusted to 0 using the blank.

7) The absorbance (A) was measured for standard and sample.

8) The concentration of sample was calculated by using the formula below:

A sample ____ X C standard = C sample

A standard

No Sample Absorbance (OD Value) Final Concentration

Reading 1 Reading 2 reading (mmol/L)
1 Blank 0 0 0 0
2 Standard 0.033 0.037 0.035 2.52
3 Sample 0.08 0.05 0.065 4.68
Results:

= A1 sample × 2.52

A1 standard
= 0.065 × 2.52

0.035

= 4.68 mmol/L

Discussions:

1) Atherosclerosis is a disease in which plaque builds up inside the arteries. Arteries are
blood vessels which carry rich oxygen blood to the heart and other parts of the body. Plaque
is made up of fat, cholesterol, calcium, and other substances found in the blood. Besides that,
plaque hardens and narrows the arteries over time. This limits the flow of rich oxygen blood
to the organs and other parts of the body. Atherosclerosis can lead to serious problems such
as heart attack, stroke, or even death. Moreover, atherosclerosis can affect any arteries in the
body, including arteries in the heart, brain, arms, legs, pelvis, and kidneys. As an aftermath,
different diseases may develop based on which arteries are affected. Certain traits, conditions,
or habits may raise the risk for the disease. These conditions are known as risk factors. Some
risk factors, such as lack of physical activity, smoking, and an unhealthy diet can be
controlled but other factors such as age and a family history of heart disease cannot be
controlled. In addition, some people who have atherosclerosis have no signs or symptoms.
They may not be diagnosed until after a heart attack or stroke. Besides medicines and medical
procedures, lifestyle changes is the main treatment to live a healthier life.

2) Hypercholesterolemia means the cholesterol level in the blood is higher than the normal
reference range.

Conclusion:

The result shows a normal range of serum cholesterol level. Therefore, the sample is taken
from a normal patient.

Reference:

http://www.healthline.com/