This document provides information on estimating serum cholesterol levels, including: - What cholesterol is and how it is produced in the body - Symptoms of high cholesterol and how it can lead to atherosclerosis and heart disease - Methods for estimating total cholesterol using the Zak's method or enzymatic method - Classification and functions of lipoproteins such as LDL, HDL, VLDL - Desirable cholesterol levels according to NCEP-ATP III guidelines - Protocol for estimating total cholesterol and HDL cholesterol levels

1. Estimation of Serum Cholesterol
Dr. Azad Alam Siddiqui
Assistant Professor
Department of B.Voc(MMDT)
Km. Mayawati Govt.GirlsPG College
Badalpur, G.B. Nagar (U.P.)

2. What Is Cholesterol?
We may associate cholesterol with fatty
foods, but most of the waxy substance is
made by our own bodies. The liver
produces 75% of the cholesterol that
circulates in our blood. The other 25%
comes from food. At normal levels,
cholesterol actually plays an important
role in helping cells do their jobs. But
cholesterol levels are precariously high
in more than 100 million Indians.

3. Symptoms of High Cholesterol
High cholesterol does not cause any
symptoms. But it does cause damage
deep within the body.
Over time, too much cholesterol may lead
to a build-up of plaque inside the arteries.
Known as atherosclerosis, this condition
narrows the space available for blood
flow and can trigger heart disease. The
good news is high cholesterol is simple to
detect, and there are many ways to bring
it down.

4. ESTIMATION OF TOTAL
CHOLESTEROL

5. • At the end of this practical, you should be able to:
◉Explain role of cholesterol and lipoproteins in health and disease
◉State the desirable levels of total cholesterol and different
lipoproteins
◉State the principle of cholesterol estimation & HDL estimation,
◉interpret the results obtained and correlate them with the clinical
findings

6. Steroid and Sterol

7. Cyclopenttaannooperhyddrroophenanthrene
ring
Steroid

8. Cyclopenttaannooperhyddrroophenanthrene
ring
Sterol
Cholesterol

9. Phytosterol
s
Zoosterol
s
Ergostero
l◉Sitosterol
◉Sigmasterol
◉Fungi
◉ProtozoaCholestero
l
Sterol

10. Functions of Cholesterol

11. In plasma
membrane◉Membrane fluidity
◉Lipid rafts
Precursor molecule
of:
◉Steroid hormones
◉Vitamin D
◉Bile Acids
Nerve conduction
Signal
transduction
Vitamin D

12. Cholesterol Synthesis
From Acetyl
CoA;HMG CoA
Reductase
Mainly in
Liver ER and
Cytosol
Transporte
d to
periphera
l tissues
Before transport, cholesterol is esterified to form cholesterol
esters

13. Lipoproteins

14. Lipoprotein
Apo-lipoprotein+ LipidLipoprotein

15. Classification of Lipoproteins
• Lipoproteins can be classified-
• Based on density- They are separated by ultracentrifugation. depending upon the floatation
constant (SF), five major groups of lipoproteins have been identified that are important
physiologically and in clinical diagnosis.
• (i) Chylomicron- Derived from intestinal absorption of triacylglycerol and other lipids;
transport lipids absorbed from the intestine to adipose, cardiac, and skeletal muscle tissue,
where their triglyceride components are hydrolyzed by the activity of the lipoprotein lipase,
allowing the released free fatty acids to be absorbed by the tissues. when a large portion of the
triglyceride core has been hydrolyzed, chylomicron remnants are formed and are taken up by
the liver, thereby also transferring dietary fat to the liver. density is generally less than 0.95
while the mean diameter lies between 100- 500 nm

16. Classification of Lipoproteins
Based on density (contd.)
(ii) Very low density lipoproteins (VLDL), derived from the liver for the export of
triacylglycerol; The VLDL particles mainly carry triglycerides to the tissues. VLDL is
similar to LDL cholesterol, but LDL mainly carries cholesterol to the tissues instead of
triglycerides. density lies between 0.95- 1.006 and the mean diameter lies between 30-80
nm.
(iii) Intermediate density lipoproteins (IDL) are derived from the catabolism of VLDL,with a
density ranging intermediate between VLDL and LDL lipoproteins i.e. ranging between
1.006- 1.019 and the mean diameter ranges between 25- 50nm.

17. Classification of Lipoproteins
Based on density (contd.)
iv) Low-density lipoproteins (LDL), representing a final stage in the catabolism of
VLDL; transport cholesterol from liver to other tissues, density lies between 1.019-
1.063 and mean diameter lies between 18-28 nm
(iv) High-density lipoproteins (HDL), involved in cholesterol transport from
peripheral tissues to liver and also in VLDL and chylomicron metabolism. Density
ranges between 1.063-1.121 and the mean diameter varies between 5-15 nm.

18. Apo-B48 (Structural)
Apo-E (Binds to Apo-E receptor)
Apo-C2 (Activates LpL)
Apo-A1
(Structural)
Apo-B100
(Structural, Binds to
LDL-Receptor)
Apo-E
Apo-C2
Apo-B100

19. Lipoprotein Lipase
Apo C2
Apo E
Dietary fats and Cholesterol

20. Endogenous Fats and Cholesterol
Lipoprotein
Lipase
Hepatic
Lipase
Apo C2
Apo E
Apo B-100

21. ◉ After conversion to bile acids
◉ Directly into the bile
◉ Exfoliation of cells
Excretion of Cholesterol

22. Ultra-centrifugation Electrophoresis
Separation of Lipoproteins

23. Hyperlipidemias
and Hypercholesterolemia

24. Why is hyperlipidemia dangerous?

25. Causes of Hyperlipidemia
 Type 2 Diabetes Mellitus
 Hypothyroidism
 Nephrotic syndrome
 Alcoholism
 High carbohydrate intake
 Glycogen storage disorders
 Cushing syndrome
 Diet
 Obesity
 Pregnancy
 Acute hepatitis

26. Hypocholesterolemia

27. Hypocholesterolaemia can be caused by several things:
• Anaemia (a low amount of red blood cells)
• Hyperthyroidism
• Malnutrition, or a lack of food
• Liver disease
• The body being unable to absorb food (malabsorption)
• Rare genetic conditions, such as hypobetalipoproteinaemia and
abetalipoproteinemia
• Tangier disease

28. Desirable Levels

29. NCEP-ATP III Guidelines
Total Cholesterol
< 200 mg/dL Desirable
200-239
mg/dL
Borderline high
> 240 mg/dL High
HDL Cholesterol
< 40 mg/dL Low
≥ 60 mg/dL High
Tri-Acyl Glycerol
< 150 mg/dL Normal
150-199
mg/dL
High
200-499
mg/dL
Hypertriglyceridemic
>500 mg/dL Very highLDL Cholesterol
< 70 mg/dL In patients with
risk factors
< 100 mg/dL Optimal
160-189
mg/dL
High
>190 mg/dL Very high

30. Laboratory Estimation

31. Lipid
profile
◉ Total Cholesterol, Tri-Acyl Glycerol, LDL and
HDL
◉ Fasting sample for Tri-Acyl Glycerol estimation
◉ Tests should be repeated on a different occasion
◉ Friedwald equation:
Total Cholesterol = HDL + LDL + VLDL
VLDL = TAG/5
Not valid if TAG > 400 mg/dL

32. Protocol for Total
Cholesterol
Estimation
1. Zak’s Method
2. Enzymatic method

33. Zak’s Method
•The proteins present in the serum sample are first precipitated by adding
Ferric chloride- Acetic acid reagent. The protein free filtrate is treated with
conc. H2SO4.
Cholesterol
↓dehydration
Cholesta-3-5-diene (2 molecules)
↓oxidation
Bis cholesta-3-5-diene (1 molecule)
↓sulphonation
Liberman-Burchard reaction
Monosulphonic Acid
derivatives (Green colour)
Salkowski reaction
Disulphonic Acid
derivatives (Red colour)
Fe3+

34. 1. Take 0.1 mL serum,add 9.9 mL of FeCl3-CH3COOH mixture and
mixthoroughly with glass
rod.2. Centrifuge at 2000 rpm for 10
min
3.
Take 3 test tubes and mark them as B, S and
T
Blank Standard
Supernatant ------ ------
4. Mix well and keep in water bath at 50-60°C for 10 min. Cool to
room temperature and measure the OD at 540 nm.
Test
5 mL
FeCl3-CH3COOH 5 mL 4.9 mL
------ 0.1 mL
3 mL 3 mL
------
Standard
(200 mg/dL)
------
Conc.H2SO4 3 mL

35. Enzymatic Method
(CHOD/POD method)
Cholesterol ester Cholesterol + Fatty
acid
Cholesterol +
O2
Cholest-4-en-3-one +
H2O2
H2O2 + 4-AP + Phenol 2H2O + Quinone-
imine
◉ Absorbance of Quinoneimine at 510 nm is directly proportional to
the concentration of cholesterol in serum
CE hydrolase
Cholesterol
Oxidase
Peroxidase

36. Assay Procedure:
• Blood drawn from patients fasting for 12 to 16 hours.
• Mix well and incubate for 10 min at 370C and measure the absorbance of
standard and test sample against the reagent blank at 510nm.
Pipette into tubes marked Blank Standard Test
Working Reagent 1.O ml 1.O ml 1.O ml
Double distilled water 20µl --------------------- --------------------------
Serum sample ----------------------- --------------------- 20µl
Standard (200mg/dl) ----------------------- 20µl ---------------------------

37. • Calculation:
Total serum cholesterol in mg/dl = --------------------- Χ concentration of
standard
Result: ----------mg/dl
Absorbance of Test
Absorbance of standard

38. Protocol for HDL-
Cholesterol
Estimation

39. ◉To 1 mL serum, add 0.1 mL Phosphotungstate reagent and 50 µl
MgCl2 solution. Centrifuge at 2500 rpm for 10 minutes.
◉ LDL, VLDL and chylomicrons are precipitated by polyanions in
the presence of metal ions to leave HDL in solution.
◉Collect the supernatant and estimate cholesterol by the total
cholesterol method.

40. Precautions

41. ◉Glacial acid is extremely volatile, irritant and corrosive to
mucous membrane. Mouth pipetting of acetic acid should be
strictly avoided.
◉No mouth pipetting of H2SO4
◉
H2SO4
containing solution should be handled carefully and
contact with skin should be avoided
◉Always add acid to water
◉Standard precautions should be followed for handling serum.