Download - Estimation of Serum Cholesterol and HDL
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Estimation of Serum Cholesterol & HDL
Ashikh Seethy
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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
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Sterol and Steroid
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Steroid:
Cyclopentanoperhydrophenanthrene ringCyclopentanoperhydrophenanthrene ring
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Sterol:
Cyclopentanoperhydrophenanthrene ringCyclopentanoperhydrophenanthrene ring
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SterolsPhytosterols Zoosterols Ergosterol
◉ Sitosterol◉ Sigmasterol
◉ Fungi◉ Protozoa
Cholesterol
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Functions of Cholesterol
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In plasma membrane
◉ Membrane fluidity◉ Lipid rafts
Precursor molecule of:
◉ Steroid hormones◉ Vitamin D◉ Bile Acids
Nerve conduction
Signal transduction
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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
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Lipoproteins
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Lipoprotein
Apo-lipoprotein+ LipidLipoprotein
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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
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Dietary Fats and Cholesterol
Lipoprotein Lipase
Apo C2
Apo E
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Endogenous Fats and Cholesterol
Lipoprotein
LipaseHepatic
Lipase
Apo C2
Apo E
Apo B-100
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◉ After conversion to bile acids
◉ Directly into the bile
◉ Exfoliation of cells
Excretion of Cholesterol
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Separation of Lipoproteins
Ultra-centrifugation Electrophoresis
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Why is hyperlipidemia dangerous?
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Hyperlipidemias
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Causes of HyperlipidemiaSecondary:◉ Type 2 Diabetes Mellitus◉ Hypothyroidism◉ Nephrotic syndrome◉ Alcoholism◉ High carbohydrate intake◉ Glycogen storage disorders◉ Cushing syndrome
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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 +/– +++ +++ +/– +/–
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↓LpL or Apo CII
↓FamilialHyperchylomicronemia(Type I)◉ Autosomal Recessive
◉ Elevated Triglycerides◉ Eruptive Xanthomas
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Defect in LDL-Receptor
↓FamilialHypercholesterolemia(Type IIa)◉ Autosomal Dominant
◉ Elevated LDL-C◉ Tendon Xanthomas
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Familial
Dysbetalipoproteinemia
(Type III
Hyperlipoproteinemia)
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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
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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 +/– +++ +++ +/– +/–
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Hypolipidemias
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Defect in Microsomal Triglyceride transfer Protein
↓Abeta-lipoproteinemia(Bassen-Kornzweig syndrome)
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Defect in ABCA1 or Apo-A1
↓Tangiers disease
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Desirable Levels
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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
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Laboratory Estimation
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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
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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+
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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
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Protocol for Total Cholesterol Estimation
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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
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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
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Protocol for HDL-Cholesterol Estimation
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◉ 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
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Precautions
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◉ 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.
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Question
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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.
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THANK YOU!