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Estimation of Serum Cholesterol & HDL
Ashikh Seethy
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 ringCyclopentanoperhydrophenanthrene ring
Sterol:
Cyclopentanoperhydrophenanthrene ringCyclopentanoperhydrophenanthrene ring
SterolsPhytosterols Zoosterols Ergosterol
◉ Sitosterol◉ Sigmasterol
◉ Fungi◉ Protozoa
Cholesterol
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
From Acetyl CoA; HMG CoA Reductase
Mainly in LiverER and Cytosol
Transported to
peripheral tissues
Before transport, cholesterol is esterified to form cholesterol esters
Lipoproteins
Lipoprotein
Apo-lipoprotein+ LipidLipoprotein
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
Dietary Fats and Cholesterol
Lipoprotein Lipase
Apo C2
Apo E
Endogenous Fats and Cholesterol
Lipoprotein
LipaseHepatic
Lipase
Apo C2
Apo E
Apo B-100
◉ After conversion to bile acids
◉ Directly into the bile
◉ Exfoliation of cells
Excretion of Cholesterol
Separation of Lipoproteins
Ultra-centrifugation Electrophoresis
Why is hyperlipidemia dangerous?
Hyperlipidemias
Causes of HyperlipidemiaSecondary:◉ Type 2 Diabetes Mellitus◉ Hypothyroidism◉ Nephrotic syndrome◉ Alcoholism◉ High carbohydrate intake◉ Glycogen storage disorders◉ Cushing syndrome
Causes of HyperlipidemiaPrimary:
Phenotype I IIa III IV V
Lipoprotein,
elevated
Chylomicrons
and VLDL
LDL Chylomicron
and VLDL
remnants
VLDL Chylomicrons
and VLDL
Triglycerides +++ N ++ ++ +++
Cholesterol
(total)
+ +++ ++ N/+ ++
LDL-cholesterol - +++ - - -
Atherosclerosis +/– +++ +++ +/– +/–
↓LpL or Apo CII
↓FamilialHyperchylomicronemia(Type I)◉ Autosomal Recessive
◉ Elevated Triglycerides◉ Eruptive Xanthomas
Defect in LDL-Receptor
↓FamilialHypercholesterolemia(Type IIa)◉ Autosomal Dominant
◉ Elevated LDL-C◉ Tendon Xanthomas
Familial
Dysbetalipoproteinemia
(Type III
Hyperlipoproteinemia)
Trudy M. Forte et al. J. Lipid Res. 2009;50:S150-S155
GPIHBP1
Deficiency
ApoA-V
Deficiency
Familial Hypertriglyceridemia• Type IV hyperlipoproteinemia• Type V hyperlipoproteinemia
Endothelial Cell
Causes of HyperlipidemiaPrimary:
Phenotype I IIa III IV V
Lipoprotein,
elevated
Chylomicrons
and VLDL
LDL Chylomicron
and VLDL
remnants
VLDL Chylomicrons
and VLDL
Triglycerides +++ N ++ ++ +++
Cholesterol
(total)
+ +++ ++ N/+ ++
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
< 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/dLIn patients with risk factors
< 100 mg/dL Optimal
160-189 mg/dL High
>190 mg/dL Very highNCEP-ATP III Guidelines
Laboratory Estimation
Enzymatic 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
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)
↓oxidationBis cholesta-3-5-diene (1 molecule)
↓sulphonation
Liberman-Burchard reaction Salkowski reactionMonosulphonic Acid derivatives Disulphonic Acid derivatives(Green colour) (Red colour)
Fe3+
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/5Not 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 mixthoroughly with glass rod.
2. Centrifuge at 2000 rpm for 10 min3. Take 3 test tubes and mark them as B, S and T
4. Mix well and keep in water bath at 50-60°C for 10 min. Cool to roomtemperature and measure the OD at 540 nm.
Blank Standard Test
Supernatant ------ ------ 5 mL
FeCl3-CH3COOH 5 mL 4.9 mL ------
Standard
(200 mg/dL)
------ 0.1 mL ------
Conc.H2SO4 3 mL 3 mL 3 mL
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 thepresence of metal ions to leave HDL in solution.◉ The cholesterol content of the supernatant is estimated byemploying the procedure of total cholesterol estimation.
◉ To 1 mL serum, add 0.1 mL Phosphotungstate reagent and 50 μLMgCl2 solution. Centrifuge at 2500 rpm for 10 minutes.
◉ Collect the supernatant and estimate cholesterol by the totalcholesterol method
Precautions
◉ Glacial acid is extremely volatile, irritant and corrosive to mucousmembrane. Mouth pipetting of acetic acid should be strictly avoided
◉ No mouth pipetting of H2SO4
◉ H2SO4 containing solution should be handled carefully and anycontact 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 myocardialinfarction. 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 alsohad myocardial infarction at young age.
Laboratory investigation shows TAG-135 mg/dL.
Estimate Total Cholesterol and HDL Cholesterol. Calculate the LDLCholesterol level.
THANK YOU!
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