1. Lipid profile test 52 By Dr. Sakar Karem Abdulla
Fifth Stage Student 52 By
Dr. Sakar Karem Abdulla
M.Sc. Clinical Biochemistry

2. Lipid profile test Total S. Cholesterol Total S. Triglyceride S. VLDL
S. LDL
S. HDL

3. LDL
LDL are formed from VLDL and carry cholesterol to peripheral tissue.
Approximately 60% of LDL is transported back and endocytosed into the liver.
The remaining 40% of LDL is carried to extrahepatic tissue.
The excess LDL particles uptake by macrophages near the endothelial cells of arteries through SR-A1 and SR-A2.

5. HDL
HDL are the most dense lipoproteins, HDL involved in transport of Ch from cells to the liver in a process known as reverse Ch transport.
HDL synthesized in both hepatic and intestinal cells as nascent particles which in outer layer contain phospholipids, free Ch, apolipoproteins and very low levels of TG or Ch in the core.

6. Reverse cholesterol transport
ABCA1(ATP-binding cassette transporter) transport free cholesterol from cells lining the blood vessels to APOA1 in nascent HDL.
Nascent HDL acquires the enzyme LCAT(lecithin cholesterol acyltransferase) from the circulation which convert Ch to Ch ester and become mature HDL.
Mature HDL transport cholesterol to liver by two mechanisms:

7. Reverse cholesterol transport
Direct pathway of hepatic Ch. Uptake in which mature HDL bind to scavenger receptor(SR-B1) on the liver which recognize APOA1 as ligand.
Indirect pathway of hepatic Ch uptake in which mature HDL interact with TG rich apoB particle (VLDL) to exchange Ch with TG in VLDL by Ch ester transfer protein(CETP), VLDL convert to LDL and bind to LDL-receptor on the liver.

9. Causes of low plasma HDL
Primary causes include:
Familial hypo alpha lipoproteinemia
Tangier disease
Familial LCAT deficiency
Fish-eye disease

10. Causes of low plasma HDL
Secondary causes include:
Smoking
Obesity
Diabetes mellitus
Chronic kidney disease
Certain drugs e.g: B-blocker

11. Reagents R1precipitant:phosphotungstic acid, magnesium chloride
R2 Buffer: phosphate buffer, chloro-4- Phenol, sodium cholate, preservative
R3 Enzyms: cholesterol oxidase (CO), cholesterol esterase (CE), Peroxidase (POD), 4 – Amino-antipyrine
R4 Standard: Cholesterol 200mg/dl
Reagent preparation: add the content of vial R3 into vial R2, mix gently until complete dissolution.

12. Principe of HDL test
non-HDL cholesterol fractions + α- cyclodextrin + Mg+2 + dextran SO4 → water-soluble complexes (precepitate)
HDL-cholesteryl esters → HDL-cholesterol + free fatty acid(by cholesteryl esterase)
HDL-cholesterol + O2 → cholestenone + H2O2 (cholesterol oxydase)
2H2O2 + phenol + 4-aminoantipyrine → qunoneimine dye + 4H2O (by peroxidase)

13. Wait for 10 mint and then centrifuge
HDL test procedure
Assay
Standard
Serum
500 µl /
Precipitate reagent
50 µl
Wait for 10 mint and then centrifuge
Working reagent
1000 µl
1000µl
Supernated part of serum
10 µl
Mix and incubate 5 mint in room temperature then read absorbance at 500 nm.

14. Calculation S.HDL=Abs.Assay/Abs.standard x conc.standard x 1.1
S.VLDL=S.TG / 5
S.LDL= S.Ch – (S.VLDL+S.HDL)