Estimation of serum cholesterol

Biochemistry practical, that explains the estimation of total serum cholesterol using an enzymatic reagent.

1. ESTIMATION OF SERUM
TOTAL CHOLESTROL

2. LIPID PROFILE
Total Cholesterol Levels
LDL levels –Low density lipoprotein cholesterol
HDL levels -High density lipoprotein cholesterol
Triglyceride levels
Done in Fasting state

3. CHOLESTEROL:
Cholesterol is a important sterol synthesized in tissues
liver and intestinal cells
Approx. three fourth is synthesize in the body and and
one fourth originates from dietary intake
In plasma, both forms are transported in lipoproteins.

4. STEROID VS STEROL

5. TYPES OF CHOLESTEROL:
LDL
Carries cholesterol to the Peripheral Tissues
It can be deposited and causes Peripheral vascular diseases,
atherosclerosis.
The Bad Cholesterol
HDL
Transports cholesterol from peripheral tissues to the liver for
excretion.
Good Cholesterol

6. FUNCTIONS OF CHOLESTROL:
Synthesizes:
Vitamin D.
Steroid hormones: Testosterone, estrogen,
progesterone, aldosterone, cortisol.
Bile
Membrane component -Cholesterolplays has a role
inmembranefluidity but it's mostimportantfunction
is in reducing the permeability of thecell membrane.
Nerve signal conduction

8. SOURCES:
Food of animal origin
Synthesized by Liver

10. REAGENT–Enzymatic Reagent
Cholesterase
Cholesterol oxidase
Peroxidase
Phenol
4-aminophenazone
Conc. of Standard Solution: 200mg/dl
Sample : Serum

11. PRINCIPLE:
Cholesterol esters are hydrolyzed to free
cholesterol by cholesterase.
The free cholesterol produced is oxidized by
cholesterol oxidaseto produce H
2O
2.
H
2O
2couples with 4-aminophenazone and phenol
to yield Quinoamine(chromogen) by peroxidase.

12. REACTION:
CholestrolEster + H
2O
Cholestrol+ Fatty Acid
Cholesterase
Cholestrol Cholestrol+ H
2O
2
H
2O
2+ Phenol + 4 Aminophenazone Quinoamine
Cholesterol Oxidase
Peroxidase
Quinoamineis Chromogenic

13. EQUIPMENT
Spectrophotometer
Micropipette
•Standard Solution
•Reagent
•Absorption of
Unknown

14. PROCEDURE:
1. Take 3 cuvettes, label them as
Blank (B), Standard (S), Test (T)
B S T

16. B S T
STANDARD
SOLUTION
SAMPLE

17. 540nm =
Absorbance of
Known.S

18. 540nm =
Absorbance of
Unknown.
T

19. PROCEDURE:
Label three cuvettes as B -Blank, S -Standard, T-test.
Add 1ml of enzyme reagent in all three of them
Add 10 l of Standard Solution in S cuvette
Add 10 l of Serum (sample) in T cuvette
Allow them to stand for 10 min at 25˚C
Place them in spectrophotometer and adjust the wavelength to
540nm
Observe the absorbance

20. Results:
Conc. of Sample = Absorbance of Unknown
Absorbance of Known
Conc. of
Standard
Solution
x

21. OBSERVATION AND CALCULATION:
BLANK 'B' STANDARD 'S' TEST 'T'
ENZYMATICREAGENT 1ml 1ml 1ml
STANDARD SOLUTION -
10 l -
SAMPLE SOLUTION - - 10 l

22. INTERPRETATION:
Desirable: < 200mg/dl (adults)
Borderline: 200 -239 mg/dl
High Risk: > 240 mg/dl

23. CAUSES OF HYPERCHOLESTEREMIA:
Diabetes mellitus
Hypothyroidism
Nephrotic syndrome
Obstructive Jaundice
Familial hypercholesterolemia
Also depends on age, race, geographical distribution, food
consumption and lifestyle.

24. CONSEQUENCES OF HYPERCHOLESTEREMIA:
Coronary Artery Disease –Atherosclerosis
Gall Stones : Precipitation and accumulation of cholesterol in
gall bladder leads to formation gall stones

25. Atherosclerosis