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!