Lipid profile of blood-Normal value, significance and methods of estimation

Lipid profile of blood: Normal value, significance and methods of estimation

Introduction:

Lipids are organic substances that contain mostly carbon and hydrogen and some oxygen. A number of lipids also contain nitrogen and phosphorus. Lipids in general are insoluble in water and soluble in organic solvents like benzene, chloroform, carbon tetrachloride, etc.
Lipids form a heterogenous group. They could be simple, compound or derived lipids. Simple lipids are esters of fatty acids and glycerol, e.g. fats (like triglcerides). Compound lipids combine with other compounds, e.g. lipoproteins. Derived lipids are of various type like steroids and cholesterol.
Most lipids such as cholesterol and triglycerides are nonpolar and hydrophobic. These molecules first must be dissolved, to be transported by the blood. They are made water soluble by combining them with proteins. The molecules thus formed are called lipoproteins.
Lipoproteins vary in size, weight and density and have different functions. But essentially all of them act as transport vehicles, picking up of various types of lipids and delivering them at the required places.
Lipoproteins are of three types or classes.
- Low-density lipoproteins (LDLs)
- High-density lipoproteins (HDLs)
- Very low-density lipoproteins (VLDLs)

A lipid profile test usually measures: Total cholesterol (TC), HDL- cholesterol and Triglycerides (VLDLs)

Total cholesterol TC ——— less than 200mg/dl

HDL cholesterol ————– over 40mg/dl

LDL cholesterol ————– less than 130 mg/dl

Triglycerides —————— 10-190 mg/dl

Total lipids ——————— 400-800 mg/dl (average being 600mg/dl)

Total Cholesterol
- Normal desirable value ——- less than 200mg/dl preferably near 150mg/dl
- More than 15-200 mg/dl Risk of coronary artery disease begins to rise.
- Above 200 mg/dl à Chance of a heart attack doubles with every 50mg/dl once the total cholesterol level is more than 200 mg/dl
LDL
- Normal desirable value —– less than 130mg/dl
- High value of LDL ———— 130-159 mg/dl considered border line high.
- LDL, above 159mg/dl ——- It is considered very high. It increases the risk of developing coronary diseases.
HDL
- Normal desirable value —- over 40mg/dl
Triglycerides
- Normal values —- 10-190 mg/dl
- For the risk of coronary artery disease the ratio of TC to HDL is important.
- A ratio of 3: 1 may be acceptable but a ratio of 4:1 is considered high risk.
- A person with TC of 240mg/dl and LDL of 160 mg/dl is said to have high blood cholesterol and comes in a high-risk category of developing coronary artery disease.
- A person with a TC of 180 mg/dl and HDL of 60mg/dl has a ratio of 3:1 that is a risk ratio of 3.
  – A ratio above 4 is considered more risk for coronary artery disease.
Total lipids
Normal value is 400-800 mg/dl
Value increases in hyperlipidermia, diabetes mellitus and hypothyroidism.
Value decreases in fat malabsorption, liver disease, hyperthyroidism or severe infection.

For lipid profile, the plasma or serum specimens must be collected under minimum standard conditions.
Some of these are given below:
- The person should not have eaten for 12-14 hours before blood is taken. Hence, usually blood is taken in the morning.
- The last meal taken around 6.00 pm should not be heavy and should have moderate amount of fat.
- The person should not have lost or gained weight for about 2 weeks prior to the test.
- The person should not be taking any medication that is known to affect plasma lipids or lipoproteins.
- The person should not have taken any alcoholic beverage or drugs, 24 hours prior to the test.

For accuracy, precision, rapidity and practicality, various methods have been evoked.
1. Liberman-Burchard color reaction:
- Cholesterol is obtained by extraction of serum with chloroform which separates the lipid from protein.
- The chloroform solution of cholesterol is mixed with concentrated sulphuric acid (H₂SO₄) in acetic anhydride to produce a green color.
- This has a maximum absorption peak at 620nm.
- The cholesterol content is estimated by spectrophotometric analysis.
2.Pearson-Stern method:
- In this the Lieberman-Burchard reaction is applied directly to serum without an initial organic solvent extraction step.
- In this method, serum is mixed with paratoluene sulphonic acid that liberates cholesterol from lipoproteins for direct reaction with sulphuric acid in the presence of acetic anhydride and acetic acid.
3. Jung-Parekh method:
- It is a direct method involving two reagents:
- Ferric acetate: uranium acetate
- Sulphuric acid: ferrous sulphate reagents
- Ferric acetate-uranium acetate- A unique precipitating reagent that removes bilirubin with proteins, clears the serum of lipids and extractcholesterol without any need of solvents.
- Sulphuric acid- ferrous sulphate reagentà When acetate reagent is mixed with this, it produces a purple color with maximum absorbance at 560nm.
- The color is stable for at least an hour.

It includes 3 steps:
- Extraction of lipids from serum or plasma
- Separation of phospholipids and glucose
- Hydrolysis of isolated triglycerides
The enzymatic reactions are preferred as they are rapid, convenient and specific.
The method given below is called GPO method. The reagents required comes in a complete test kit with a standard.
In this method triglycerides are hydrolysed to glycerol by lipases.
The glycerol is determined by the indicator.
The indicator is quinoeimine formed from hydrogen peroxide, 4-aminoanti-pyrine and 4-chlorophenol.
Under the catalytic influence of peroxidase.

Required reagents:

Buffer and reagent solution. (Contains buffer, magnesium ions, ATP, lipases, peroxidases, glycerol kinases 4-chlorophenol).
Enzyme reagent (contains GPO and 4-aminoantipyrine)
Standard

Procedure to estimate blood triglyceride:

Working reagent is formed by mixing enzyme reagent with buffer reagent. Let it stand for at least 15 minutes at room temperature. For this test blood plasma or serum is required.
Take 3 test-tubes and label them as test(T), standard (S) and blank (B).
Add the contents as given.
- Test(T)= working reagent 1ml+ serum 10l.
- Standard (S)= working reagent 1ml + standard 10l.
- Blank (B)= working reagent 1ml
Incubate all at 37^oC for 5 minutes.
Measure the absorbance of test and the standard against reagent blank within 60 minutes.