Outline of Today’s lecture
1. Discuss the different types of cholesterol
2. Identify healthy cholesterol levels
3. Clinical significance of cholesterol
4. Principle of cholesterol estimation
Objectives
To determine serum cholesterol level
Master how to determine the conc. of
cholesterol in serum.
Understand the clinical significance of
cholesterol
What is cholesterol?
Cholesterol, from the Greek chole- (bile) and
stereos (solid) followed by the chemical suffixol for an alcohol, is an organic chemical
substance classified as a waxy steroid of fat
Types of Cholesterol
LDL Cholesterol-”bad” Cholesterol
HDL Cholesterol-”good” Cholesterol
Triglycerides
Which is the most important?
– LDL
– HDL & Triglycerides are a close 2nd and 3rd
LDL—The “bad” Cholesterol
Contributes to build up of fat deposits in
arteries
Decreases blood flow to
the heart
Want this to be low?
– Lowered by diet, exercise, and most
medications
HDL—The “good” Cholesterol
Helps carry “bad” cholesterol away from
arteries to liver
The higher the better!
How can you raise your HDL?
– Exercise
– Some medications help
– Eating properly may help
DETERMINATION OF SERUM / PLASMA
CHOLESTEROL
Cholesterol is distributed in all the cells of the
body, especially in nerve tissue. It occurs in
animal fats but not in plant fats.
it is widely distributed throughout the body,
especially in the blood, brain, liver, kidneys, and
nerve fiber myelin sheaths, and it is essential
component of cell membrane development and
production of bile acids, adrenal steroids, and
sex hormones.
Cyclopentanophenanthrene Nucleus
Structure of Cholesterol
Cholesterol is essential for life to make strong
cell membrane and hormones. The body
manufacturers about 1000 mg of cholesterol
daily.
Too much cholesterol can cause fatty plaques
on arterial walls, which narrows the artery. This
condition known as atherosclerosis.
The build up of fat on the vessel walls can
occlude and in some cases totally block the flow
of blood to organs like the heart. When
atherosclerosis occur in heart vessels, also
known as coronary heart disease, chest pain
and heart attacks can result .
Acute myocardial Infarction
The highest source of cholesterol comes
from organ meats (e.g., liver) and eggs,
but other common sources are any type of
meat, poultry, cheese, and butterfat. The
recommended daily amount of cholesterol
is 300 mg for the average person .
The cholesterol level in serum is
dependent on age, sex, geographical
/cultural region and nutrition.
At birth, the cholesterol level is usually
below 100 mg/dl(2.6 mmol/l).
Levels
increase
slowly
throughout
childhood and generally throughout life.
This increase is greater in men than in
women during the reproductive years.
A- Raised levels may occur in:
Diabetes mellitus.
Nephrotic syndrome.
Myxoedema.
Late pregnancy.
Liver disease.
Cholesterol is sometimes deposited in the
skin as yellow nodules or xanthemas and
in arteries (artheroma). However,
B- Low levels occur in:
Severe Infection
Severe Anemia.
Massive Liver Cell Damage.
Xanthomas
Types of Samples
• Plasma
Water + solids (e.g. glucose, urea, albumin, fibrinogen)
No cells
• Serum
Serum = plasma – clotting factors
Requirements
Requirements:
Automatic pipettes
Tips
Cuvettes
 Spectrophotometer
Specimen:
Serum or plasma
Working Reagent
Mes buffer pH 6.5
Phenol
3,5-Dichlorophenol
4-Aminoantipyrine
Cholesterol Esterase
Cholesterol Oxidase
Peroxidase
75 mM
6 mM
0.2 mM
0.5 mM
≥500 kU/L
≥300 kU/L
≥1200 kU/L
Standard soln. of Cholesterol = 200 mg/dl
Preparation of Cuvettes
Procedure
Blank
Standard
Sample
Cholesterol Esterase
Cholesterol esters + H2O
Cholesterol + Fatty acid
Cholesterol + O2 + H2O Cholesterol Oxidase
Cholestenone + H2O2
Peroxidase
2H2O2 + 4-Aminophenazone + phenol
Coloured quinonic derivative + 4H2O
Procedure
.
Reagents
Blank
Sample
Standard
Sample (ml)
-
10
-
Standard (ml)
-
-
10
Working reagent (ml)
1
1
1
Procedure
Add the reagents and sample as directed
Mix and let stand at room temperature for 10 min.
Read the absorbance at 505 nm (495-550).
Zero the spectrophotometer with the blank reagent.
Read the absorbance's of the standard and samples
within 1 hr
Enter the absorbance's readings and calculated the
concentration of the sample.
Calculations:
Concentration of cholesterol in the
sample(s) =
Absorbance of the Sample X Conc. of the Std.
Absorbance of the Standard
50 – 200 mg/dl
Clinical interpretation
Abnormal ???
Level mg/dL
Level mmol/L
<200
<5.2
200–240
5.2–6.2
> 240
> 6.2
Desirable level
corresponding to lower
risk for heart disease
Borderline high risk
High risk