MAKE BIOCHEMISTRY MANUAL

Q1. Write down the method and procedure of Serum Cholesterol

estimation (Total, HDL and HDL cholesterol). Also write down
the reference range.
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

Requirements/ reagents:
 10*75 ml test tube
 Serum samples
 Cholesterol reagent
 Pipits
 Spectrophotometer
 Refrigerated centrifuge
 Precipitating reagent (phospholungstic acid MgCl2)
 PIPES buffer

PRINCIPLE:
Cholesterol can be measured by a variety of methods. The most common of those currently in
use utilizes a series of enzymatic pathways to produce a measurable product.

The following series of reactions are widely used for point of care, automated and manual
method.

Theory/method:

Enzymatic method:
 Cholesterol CE hydrolase cholesterol + fatty acid
 Cholesterol + O2 Cholesterol oxidase- cholest-4-en-3-one +H2O2
  H2O2 + 4-AP + phenol peroxidase H2O2 + quinone-imine
 Absorbance of quinonimine at 510 nm is directly proportional to the concentration of
cholesterol in serum.

Zak’s Method:
The protein present in the serum sample are first precipitated by adding Ferric Chloride-Acetic
acid reagent. The protein free filtrate is treated with concentrated H2SO4.

Cholesterol
Dehydration
Cholesta-3-5-diene (2 molecules)
Oxidation
Bis cholesta-3-5-diene (1 molecule)
Sulphonation

Liberman-Burchard reaction
monosulphonic acid
derivatives (green color) Salkowski reaction disulphonic acid derivatives (red color)

REFERANCE RANGES
Total cholesterol

Your total cholesterol number is the overall amount of cholesterol found in your blood.

 Acceptable: Below 200 mg/dL (milligrams per deciliter)

 Borderline: 200 to 239 mg/dL

 High: 240 mg/dL or higher

High-density lipoprotein (HDL)

HDL is also called good cholesterol and helps protect you from heart disease. This type removes
excess cholesterol from your blood, helping to prevent buildup. The higher your HDL levels are,
the better.

 Acceptable: 40 mg/dL or higher for men and 50 mg/dL or higher for women

 Low: 39 mg/dL or lower for men and 49 mg/dL or lower for women

 Ideal: 60 mg/dL or higher

 TOTAL CHOLESTEROL ESTIMATION:
Directly linked to risk of heart and blood vessel disease.

Goal values:

 75-169 mg/dL for those age 20 and younger

 100-199 mg/dL for those over age 21

PROCEDURE:

This test may be measured any time of the day without fasting. However, if the test is drawn as
part of a total lipid profile, it requires a 12-hour fast (no food or drink, except water). For the
most accurate results, wait at least two months after a heart attack, surgery, infection, injury or
pregnancy to check cholesterol levels.

Cholesterol is a type of fat, found in your blood. It is produced by your body and also comes
from the foods you eat (animal products). Cholesterol is needed by your body to maintain the
health of your cells. Too much cholesterol leads to coronary artery disease. Your blood
cholesterol level is related to the foods you eat or to genetic conditions (passed down from other
generations of family members).

HIGH DENSITY LIPOPROTEIN (HDL)

High levels linked to a reduced risk of heart and blood vessel disease. The higher your HDL
level, the better.

Goal value:

 Greater than 40 mg/dL

PROCEDURE:

This test may be measured any time of the day without fasting. However, if the test is drawn as
part of a total lipid profile, it requires a 12-hour fast (no food or drink, except water). For the
most accurate results, wait at least two months after a heart attack, surgery, infection, injury or
pregnancy to check HDL levels.

HDL is a lipoprotein (a combination of fat and protein) found in the blood. It is called "good"
cholesterol because it removes excess cholesterol from the blood and takes it to the liver. A high
HDL level is related to lower risk of heart and blood vessel disease.
CALCULATION:

 Serum total cholesterol (mg/dL) = (T-B) / (S-B) * concentration of standard * dilatation

factor
 Serum total cholesterol (mg/dL) = (T-B) / (S-B) *200 mg/dL * dilatation factor
 Dilatation factor = (0.1/8)/(0.05/8) = 2