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MODULARISED MEDICAL

CURRICULUM
Stage 3 Semester 1
BIOCHEMISTRY PRACTICAL
2014/2015
School of Biomolecular and Biomedical Science &
School of Medicine and Medical Science,
University College Dublin,
Belfield,
Dublin 4.

TITLE

: ANALYSIS OF PLASMA
LIPOPROTEINS

Name
Student id

Title:
Analysis of Plasma Lipoproteins
Aim:

1. To carry out a biochemical analysis of the cholesterol and triglyceride profile of


the serum prepared from three donor volunteers and
2. To make a clinical assessment of the results with respect to risk of development of
cardiovascular disease.
Methodology:
Determination of total cholesterol in plasma or serum
1. A reagent solution which contains phosphate buffer, 4-aminophenazone, phenol,
3,4- dichlorophenol, dydroxyethoxy-n-alkanes, cholesterol, esterase, cholesterol
oxidase, peroxidase was prepared..
2. Two solutions as below were pipetted into 2 different cuvette(1 cm light path)
respectively.
Solution 1
1 ml Reagent solution.

Solution 2
10 l plasma
1 ml Reagent solution.

3. Each solution was mixed and incubated for 20 minutes at room temperature.
4. The absorbance of sample against blank (within 10 minutes) at 500nm was read
5. The concentration of cholesterol in the plasma was calculated in unit mmol/l.
c= 14.3 x A 500nm sample

Determination of high-density-lipoprotein (HDL) in plasma


1. Precipitating solution was prepared by mixing phosphotungstic acid 0.55 mmol/l
and magnesium chloride MgCl2 solutions at 25 mmol/l.
2. 0.5 ml of plasma and 1ml of precipitating solution were then pipetted into a
centrifuge tube. It was mixed and let stand for 10 minutes at room temperature.
By doing this chylomicrons, very-low-density-lipoproteins (VLDL) and lowdensity-lipoproteins (LDL) were precipitated.
3. The solution was centrifuged for 10 minutes at 4000 rpm. Centrifugation leaves
only the HDL in the supernatant, their cholesterol content was then determined
enzymatically.
4. After centrifugation, the supernatant was aspirated off into a small tube. The
cholesterol in this supernatant was then measured as follow:
Pipette into test tube

Solution 1

Solution 2

Distilled water
Supernatant
Reagent solution

0.1ml
1ml

0.1ml
1ml

5. Each solution was mixed and incubated for 20 min at room temperature.
6. The absorbance of sample was measured against blank at 500nm within 10 min.
7. The concentration of HDL- cholesterol was measured in mmol/l
c= 4.65 x A500nm sample

Determination of triglycerides in serum or plasma


1. The standard glycerol solution was prepared at 2.28 mmol/l as standard solution.
2. The following reagents were mixed to form the chromogen reagent:
Reagent 1
Reagent 2
Pipes buffer pH 7.5 Lipoprotein lipase
ca1400 U/l
50mmol/l
Glycerokinase
ca 1000 U/l
ESPAS
Glycerol-3-phosphate oxidase ca 5000 U/l
1 mmol/l
Peroxidase
440 U/l
4-amino-antipyrine
0.7 mmol/l
ATP
0.30 mmol/l

3. Each solution is prepared as below. The solutions are then mixed and left to stand
for 20 minutes at room temperature.

Chromogen reagent
Standard
Plasma

Blank
1 ml
-

Standard
1 ml
10 l
-

Sample
1 ml
10 l

4. The absorbance was read at 546nm.


5. The concentration of triglycerides in serum or plasma is calculated as follow:
Absorbance of sample x concentration of standard
Absorbance of standard

Results and self-assessment questions:


1. Having experimentally determined the levels of:

a) Total cholesterol (mmol/l)

b) HDL cholesterol (mmol/l)

c) Total triglyceride (mmol/l)

6. The level was used to determine the relationships using equation below:
d)

LDL cholesterol(mmol/l)= total cholesterol- HDL cholesterol- Triglyceride


2.2

e)
VLDL cholesterol (mmol/l) = [Total cholesterol] [HDL cholesterol] [LDL cholesterol]

f) The percentage ratio of HDL cholesterol relative to total cholesterol was


calculated, remembering a value of 20% is desirable.
Ratio(%) = [HDL cholesterol] x 100
[Total cholesterol]

2. The results for 3 donors serum sample relative to the value in Table 3 below were

compared. The clinical assessment of the data with respect to the donors risk of
development of cardiovascular disease were made.
Table 3: Normal, Borderline and High Risk Distribution for Plasma Lipoprotein
Cholesterol and Plasma Triglyceride
HDL

LDL

Category

Normal
Borderline
High Risk

Total Plasma
Cholesterol
mmol L-1
5.1<
5.1 to 6.2
>6.2

Plasma
Triglyceride
mmol L-1
1.68<
1.69 to 2.23
2.24 to 4.4

Males

Female

>1.28
1.27 to 1.03
1.02<

>1.53
1.52 to 1.15
1.14<

2.5 to 3.3
3.4 to 4.1
>4.2

3. A middle aged man (47 years) presents to your medical clinic with high

cholesterol using the pin-prick test, his total cholesterol 8.5 mmol L-1. His
triglyceride level was
subsequently determined to be 7.0 mmolL-1 and his
glucose determined as 10.5 mmolL-1.
a) Suggest ways in which you would advise this patient.
Treatments are advised since the man has high risk distributions for plasma
lipoprotein, cholesterol, and plasma triglyceride and glucose level. Medicine such

as statin and insulin should be given. Besides, the patient should include Omega-3
fatty acids in his diet to lower the triglycerides level. Soluble fiber can lower the
absorption of cholesterol in the intestine, thus increasing patients fiber intake can
be another option. Moreover, patients should be advised to do more exercise. A
diet low in fat, salt and sugar and high in fiber and with plenty of fruit and
vegetables should be taken to reduce blood glucose level. Exercise can raise HDL
and lowering triglycerides at the same time. Other advices such as quit smoking if
your patient smoke and watch the alcohol should be given.
b) Would you recommend a full biochemical analysis of the profile of cholesterol
i.e as carried out in your practical? If yes explain?
Yes, the patient has high risk distribution for cholesterol with the value of
8.5mmol L-1 while the borderline is 6.2mmol l-1. Thus, a full biochemical
analysis should be carried out to increase the accuracy of the measurement. This
is because pin-prick test only measure total cholesterol. A full understanding of
your cholesterol profile requires measurements of HDL, LDL, and triglycerides as
well. Second, the results need combination with other risk factors such as family
history, nutritional habits, age, and gender to fully understand the risk for
cardiovascular disease.Treatment can be carry out before the condition get worst.
c) Suggest some initial queries you would make to the individual with respect to his
lifestyle.
I would need to enquire more about the family history of premature heart disease,
medical history such as pre-existing heart disease or already had a heart attack,
hypertension, diabetes mellitus. Enquiry included smoking and drinking alcohol
habit should be included as well.

d) When would you recommend therapeutic intervention?


At least two or preferably three fasting lipoprotein measurements provide the
most accurate baseline. Sampling can be done on consecutive days. However,
metabolic stability is required before initiating cholesterol-lowering drug therapy
in patients who have had a recent coronary event.

e) Suggest what types of therapeutic drugs might be used and the biochemical basis
of their action.
Statin drugs
works by blocking the enzyme linked to the liver's
cholesterol production, HMG-CoA (3-hydroxy-3methyglutaryl COA) reductase, thus inhibiting the liver's
ability to produce LDL. This causes an increase in the
number of the LDL receptors on the surface of liver
cells, resulting in more cholesterol being removed from
the bloodstream and a reduction in risk for highcholesterol related diseases.
Nicotinic acid

Nicotinic acid reduces the plasma levels of both VLDLs and


LDLs by inhibiting hepatic VLDL secretion, as well as
suppressing the flux of FFA release from adipose tissue by
inhibiting lipolysis. In addition, nicotinic administration
strongly increases the circulating levels of HDLs.

Cholestyramine These compounds are nonabsorbable resins that bind bile acids
or
colestipol which are then not reabsorbed by the liver but excreted. The
(resins)
drop in hepatic reabsorption of bile acids releases a feedback
inhibitory mechanism that had been inhibiting bile acid
synthesis. As a result, a greater amount of cholesterol is
converted to bile acids to maintain a steady level in circulation.
Additionally, the synthesis of LDL receptors increases to allow
increased cholesterol uptake for bile acid synthesis, and the
overall effect is a reduction in plasma cholesterol.
Ezetimibe

Discussion

Ezetimibe functions to reduce intestinal absorption of


cholesterol, thus effecting a reduction in circulating cholesterol.
The drug functions by inhibiting the intestinal brush border
transporter involved in absorption of cholesterol.

Conclusion