Lipid profile:
HDL & LDL cholesterol
HDL-cholesterol (HDL-C):
Clinical significance:
The main role of high density lipoproteins (HDL) in lipid
metabolism is the uptake and transport of cholesterol from
the peripheral tissues to the liver by a process known as
reverse cholesterol transport.
Low HDL-cholesterol levels are strongly associated with
increased risk of coronary heart disease (CHD).
Therefore, measurement of HDL-cholesterol helps to define the
risk of ischemic heart disease (IHD) in patients with raised total
cholesterol level. HDL-cholesterol is often reduced in patients
with IHD.
While increased plasma HDL-cholesterol is negatively
correlated with the incidence of IHD and has a protective
effect.
Ischemic heart disease: heart disease that results from
decreased oxygen supply to the heart muscle
(myocardium) because of narrowing of a coronary artery
by atherosclerosis.
Laboratory measurement of HDL-cholesterol:
-Specimen collection:
• Blood Specimen should be collected after 12-14 hours
fasting (at least 12 hours).
• Use serum or plasma. For plasma, use EDTA.
• Centrifuge and remove serum or plasma from blood cells
as soon as possible (otherwise within 2 hours).
• HDL- cholesterol is stable in the specimen for 1-3 days at
2 - 8 °C.
Reagents:
• Reagent 1 (R1): Precipitant
Phosphotungstic acid (PTA), MgCl2
• Reagent 2 (R2): HDL-cholesterol standard 100 mg/dl.
• Working Reagent (WR): (R1 + R2) of total cholesterol
measurement.
Principle of the test:
Low density lipoproteins (LDLs), very low density lipoproteins
(VLDLs) and chylomicrons in the specimen (serum or plasma)
are precipitated by addition of phosphotungstic acid (PTA) and
magnesium chloride (MgCl2).
After centrifugation, a supernatant is obtained which contains
high density lipoproteins (HDLs) from which the HDL-cholesterol
can be determined using the reagent for total cholesterol
measurement (Total Cholesterol Enzymatic Reagent).
Manual Procedure:
2 steps
1- Precipitation:
Put 500µl of the specimen in a test tube, then add 50 µl of
the precipitant (PTA).
Pipette into Test tube
Specimen (serum or plasma) 500 µl
Precipitant (PTA) 50 µl
Mix well, let stand for 10 minutes at room temperature.
Centrifuge for 15 minutes at 3500 - 4000 RPM, then use the
supernatant for determination of HDL- cholesterol in the
next step.
2-determination of HDL-cholesterol by reagent for total
cholesterol measurement:
Pipette into well identified test tubes Blank standard test
Total cholesterol reagent 1ml 1ml 1ml
HDL-cholesterol standard 25 µl
Supernatant 25 µl
• Mix well, let stand for 10 minutes at room temperature.
• Record absorbance at 500 nm.
• Color is stable for 1 hour.
Calculation:
HDL-cholesterol =
Concentration of HDL-cholesterol standard = 100 mg/dl
A = absorbance in nm, measured by spectrophotometer.
Expected value:
< 40 mg/dl: Low
≥ 60 mg/dl: High
LDL cholesterol (LDL-C):
Clinical significance:
- Increased plasma LDL- cholesterol is positively correlated
with the incidence of IHD.
Estimation of LDL- cholesterol:
- LDL cholesterol is calculated by Friedewald formula, using
the initial routine investigations (total cholesterol ,triglyceride
(TG) and HDL cholesterol):
LDL-C = Total cholesterol - (VLDL-C + HDL-C)
VLDL-C is equal to one fifth of triglyceride: VLDL-C = TG/5
Friedewald formula
LDL-C = Total cholesterol - (VLDL-C+ HDL-C)
VLDL cholesterol = TG/5
LDL-C = Total cholesterol - [(TG / 5) + HDL-C)]
The unit used for all parameters is mg/dl
This formula is not valid if triglyceride concentration is ≥
400 mg/dl
Expected values:
Optimum (recommended level): < 100 mg/dl
Near or above optimum: 100 - 129 mg/dl
Borderline high: 130 -159 mg/dl
High: 160 -189 mg/dl
Very high: ≥ 190 mg/dl