This document discusses the estimation of serum total cholesterol levels through a colorimetric enzymatic method. Cholesterol is esterified in the sample and oxidized to produce hydrogen peroxide which reacts with phenol and 4-aminophenazone in the presence of peroxidase to form a colored compound (quinoamine). The absorbance is read at 540nm and used to calculate cholesterol concentration compared to a standard. Elevated cholesterol is a risk factor for coronary artery disease and atherosclerosis.

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 - Cholesterol plays has a role
in membrane fluidity but it's most important function
is in reducing the permeability of the cell 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 oxidase to produce H2O2.
H2O2 couples with 4-aminophenazone and phenol
to yield Quinoamine (chromogen) by peroxidase.

12. REACTION:
Cholestrol Ester + H2O Cholestrol + Fatty Acid
Cholesterase
Cholestrol Cholestrol + H2O2
H2O2 + Phenol + 4 Aminophenazone Quinoamine
Cholesterol Oxidase
Peroxidase
Quinoamine is 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'
ENZYMATIC REAGENT 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