1. Practical Analysis Using SpectrophotometerBy
All Staff Members of
Medical Biochemistry Department 1

2. Colorimetric estimation of serum cholesterol

3. Aim of practical session
The aim of this practical session is to
Estimate the concentration of cholesterol in a serum sample using a colorimetric method,
Know the normal values of cholesterol in blood.

4. Medical importance of serum cholesterol determination
The determination of serum Cholesterol is one of the important tools in the diagnosis of
heart disease & stroke

5. Cholesterol is a fat substance that is found in all body cells.
Introduction
Cholesterol is a fat substance that is found in all body cells.
The total Plasma cholesterol level = 140 – 200 mg %.
About 50 % of these total are of exogenous origin and 50 % are synthesized in the liver.

7. Photometric measurementsof
serum cholesterol

8. Principle Cholesterol H2O2 Peroxidase
Cholesterol Esterase
Cholesterol esters free Cholesterol + fatty acids
Cholesterol H2O2
Cholesterol oxidase
Peroxidase
H2O2 + Phenol + 4-amino antipyrine
red colored product

9. Activity 1:
Using a standard cholesterol solution to calculate the concentration of unknown one
Materials required
Automatic pipettes & test tubes.
Cholesterol reagent
Known cholesterol solution (Standard)
Unknown cholesterol solution (Test)
Distilled water
A pump & goggles if available for lab safety.

10. For the Standard sample
Procedure:
Steps 1: Label 3 dry test tubes T
For the test sample S
For the Standard sample B
For the Blank

11. Treat these tubes as shown in the following table:
Step 2:
Treat these tubes as shown in the following table:
Sample
Standard
Blank
___
10 µl
Distilled water
Cholesterol
standard
Test
1 ml
reagent

12. Step 3: Pipette 10 ul of the given Test -Standard-DW into their corresponding dry test tubes12

13. Step 4: for all ; Pipette 1ml of the cholesterol reagent into the previous 3 test tubes.13

14. B S T B
Step 5: Mix the contents of the tubes and let stand for 10 minutes at room temperature. 14

15. Step 6: Record the absorbance of both test & standard against the blank at 520 nm.

16. Step 7: Calculate the test concentration by the following equation:
Concentration of serum cholesterol ( mg%) =
Absorbance of Test
X Concentration of standard
Absorbance of standard

17. Expected Values for total plasma cholesterol levels
<200 mg/dl
Normal Levels
mg/dl
borderline values
> 240 mg/dl
High values

18. hypercholesterolemia Familial hypercholesterolemia
Causes of
hypercholesterolemia
Familial hypercholesterolemia
Obesity
Hypothyroidism
Obstructive jaundice
Diabetes mellitus
Coffee drinking
cigarette smoking

19. Two types of Serum cholesterol
LDL
Bad"
cholesterol
HDL
good" cholesterol
HDL/LDL
indicator of
heart disease
risk. 19

20. Several medicines can worsen cholesterol level:
Thiazide diuretics
Oral estrogens
Some beta blockers
Immunosuppressive agents

21. Types of serum lipoproteins

22. Complete Lipid profile
Requires hours fasting
Total Cholesterol, TAG & HDL are measured directly (mg/dL) using colorimetric method.
LDL is usually calculated :
LDL = Total Cholesterol – HDL - (TAG/5)

23. Plasma Lipids Profile

24. THANK YOU High risk Normal 240 140-200 Cholesterol Above 200
Below 150 mg/dL
TAG
160–189 mg/dL
100–129 mg/dL
LDL
Less than 40 mg/dL
40–59 mg/dL
HDL
THANK YOU