Objectives:

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
◉ Describe various causes of dyslipidemias
◉ State the principle of cholesterol estimation & HDL estimation,
interpret the results obtained and correlate them with the clinical
findings
Sterol and Steroid
Steroid:

Cyclopentanoperhydrophenanthrene ring
Sterol:

Cyclopentanoperhydrophenanthrene ring
 Sterols
Phytosterols Zoosterols Ergosterol

◉ Sitosterol ◉ Fungi
◉ Sigmasterol
Cholesterol ◉ Protozoa
Functions of Cholesterol
In plasma membrane
◉ Membrane fluidity
◉ Lipid rafts

Precursor molecule of:

◉ Steroid hormones
◉ Vitamin D
◉ Bile Acids

Nerve conduction
Signal transduction
 Cholesterol Synthesis

Transported
From Acetyl CoA; Mainly in Liver to
HMG CoA Reductase ER and Cytosol peripheral
tissues

Before transport, cholesterol is esterified to form cholesterol esters

Lipoproteins
 Lipoprotein

Apo-lipoprotein+ LipidLipoprotein
 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-B100
Apo-E
Apo-C2
 Dietary Fats and Cholesterol

Lipoprotein Lipase
Apo C2

Apo E
 Endogenous Fats and Cholesterol

Apo C2
Lipoprotein Hepatic
Lipase Lipase
Apo E

Apo B-100
 Excretion of Cholesterol
◉ After conversion to bile acids

◉ Directly into the bile

◉ Exfoliation of cells
 Separation of Lipoproteins
Ultra-centrifugation Electrophoresis
Why is hyperlipidemia dangerous?
Hyperlipidemias
 Causes of Hyperlipidemia
Secondary:
◉ Type 2 Diabetes Mellitus
◉ Hypothyroidism
◉ Nephrotic syndrome
◉ Alcoholism
◉ High carbohydrate intake
◉ Glycogen storage disorders
◉ Cushing syndrome
 Causes of Hyperlipidemia
Primary:
Phenotype I IIa III IV V

Lipoprotein, Chylomicrons LDL Chylomicron VLDL Chylomicrons

elevated and VLDL and VLDL and VLDL
remnants

Triglycerides +++ N ++ ++ +++

Cholesterol + +++ ++ N/+ ++

(total)

LDL-cholesterol - +++ - - -

Atherosclerosis +/– +++ +++ +/– +/–

↓LpL or Apo CII
↓
Familial
Hyperchylomicronemia
(Type I)
◉ Autosomal Recessive
◉ Elevated Triglycerides
◉ Eruptive Xanthomas
Defect in LDL-Receptor
↓
Familial
Hypercholesterolemia
(Type IIa)
◉ Autosomal Dominant
◉ Elevated LDL-C
◉ Tendon Xanthomas
Familial
Dysbetalipoproteinemia
(Type III
Hyperlipoproteinemia)
 GPIHBP1
Deficiency

ApoA-V
Deficiency
Endothelial Cell Familial Hypertriglyceridemia
• Type IV hyperlipoproteinemia
Trudy M. Forte et al. J. Lipid Res. 2009;50:S150-S155 • Type V hyperlipoproteinemia
 Causes of Hyperlipidemia
Primary:
Phenotype I IIa III IV V

Lipoprotein, Chylomicrons LDL Chylomicron VLDL Chylomicrons

elevated and VLDL and VLDL and VLDL
remnants

Triglycerides +++ N ++ ++ +++

Cholesterol + +++ ++ N/+ ++

(total)

LDL-cholesterol - +++ - - -

Atherosclerosis +/– +++ +++ +/– +/–

Hypolipidemias
Defect in Microsomal Triglyceride transfer Protein
↓
Abeta-lipoproteinemia
(Bassen-Kornzweig syndrome)
Defect in ABCA1 or Apo-A1
↓
Tangiers disease
Desirable Levels
Total Cholesterol Tri-Acyl Glycerol

< 200 mg/dL Desirable < 150 mg/dL Normal

200-239 mg/dL Borderline high 150-199 mg/dL High

> 240 mg/dL High 200-499 mg/dL Hypertriglyceridemic

LDL Cholesterol >500 mg/dL Very high

In patients with HDL Cholesterol
< 70 mg/dL
risk factors
< 100 mg/dL Optimal < 40 mg/dL Low

160-189 mg/dL High ≥ 60 mg/dL High

>190 mg/dL Very high NCEP-ATP III Guidelines
Laboratory Estimation
 Enzymatic Method

CE hydrolase
◉Cholesterol ester Cholesterol + Fatty acid

◉Cholesterol + O2 Cholest-4-en-3-one + H2O2

Cholesterol
Oxidase

◉H2O2 + 4-AP + Phenol 2H2O + Quinone-imine

Peroxidase

◉Absorbance of Quinoneimine at 510 nm is directly proportional to the

concentration of cholesterol in serum
 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
Fe3+

Liberman-Burchard reaction Salkowski reaction

Monosulphonic Acid derivatives Disulphonic Acid derivatives
(Green colour) (Red colour)
 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
 Protocol for Total
Cholesterol Estimation
1. Take 0.1 mL serum, add 9.9 mL of FeCl3-CH3COOH mixture and mix
thoroughly 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 Test
Supernatant ------ ------ 5 mL
FeCl3-CH3COOH 5 mL 4.9 mL ------
Standard ------ 0.1 mL ------
(200 mg/dL)
Conc.H2SO4 3 mL 3 mL 3 mL

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.
Calculation:

Serum total cholesterol (mg/dL) =

(T-B)/(S-B) x Concentration of Standard x Dilution factor

Serum total cholesterol (mg/dL) =

(T-B)/(S-B) x 200 mg/dL x Dilution factor

Dilution factor = (0.1/8)/(0.05/8)

=2
Protocol for HDL-Cholesterol
Estimation
◉ LDL, VLDL and chylomicrons are precipitated by polyanions in the
presence of metal ions to leave HDL in solution.
◉ The cholesterol content of the supernatant is estimated by
employing the procedure of total cholesterol estimation.

◉ To 1 mL serum, add 0.1 mL Phosphotungstate reagent and 50 μL

MgCl2 solution. Centrifuge at 2500 rpm for 10 minutes.

◉ Collect the supernatant and estimate cholesterol by the total

cholesterol method
Precautions
◉ 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 any

contact with skin should be avoided

◉ Always add acid to water

◉ Standard precautions should be followed for handling serum.

Question
A 32 year old woman was hospitalized with an acute myocardial
infarction. Coronary angiography indicated the presence of >75%
stenosis in all the three coronary arteries

Family history revealed that her father and two of her five siblings also
had myocardial infarction at young age.

Laboratory investigation shows TAG-135 mg/dL.

Estimate Total Cholesterol and HDL Cholesterol. Calculate the LDL

Cholesterol level.
THANK YOU!