Practical Analysis Using Spectrophotometer By All Staff Members of Medical Biochemistry Department 2011-2012 1

Colorimetric estimation of serum cholesterol

Aim of practical session The aim of this practical session is to Estimate the concentration of cholesterol in a serum sample using a colorimetric method, Know the normal values of cholesterol in blood.

Medical importance of serum cholesterol determination The determination of serum Cholesterol is one of the important tools in the diagnosis of heart disease & stroke

Cholesterol is a fat substance that is found in all body cells. Introduction Cholesterol is a fat substance that is found in all body cells. The total Plasma cholesterol level = 140 – 200 mg %. About 50 % of these total are of exogenous origin and 50 % are synthesized in the liver.

Photometric measurements of serum cholesterol

Principle Cholesterol H2O2 Peroxidase Cholesterol Esterase Cholesterol esters free Cholesterol + fatty acids Cholesterol H2O2 Cholesterol oxidase Peroxidase H2O2 + Phenol + 4-amino antipyrine red colored product

Activity 1: Using a standard cholesterol solution to calculate the concentration of unknown one Materials required Automatic pipettes & test tubes. Cholesterol reagent Known cholesterol solution (Standard) Unknown cholesterol solution (Test) Distilled water A pump & goggles if available for lab safety.

For the Standard sample Procedure: Steps 1: Label 3 dry test tubes T For the test sample S For the Standard sample B For the Blank

Treat these tubes as shown in the following table: Step 2: Treat these tubes as shown in the following table: Sample Standard Blank ___ 10 µl Distilled water Cholesterol standard Test 1 ml reagent

Step 3: Pipette 10 ul of the given Test -Standard-DW into their corresponding dry test tubes 12

Step 4: for all ; Pipette 1ml of the cholesterol reagent into the previous 3 test tubes. 13

B S T B Step 5: Mix the contents of the tubes and let stand for 10 minutes at room temperature. 14

Step 6: Record the absorbance of both test & standard against the blank at 520 nm.

Step 7: Calculate the test concentration by the following equation: Concentration of serum cholesterol ( mg%) = Absorbance of Test ---------------------------- X Concentration of standard Absorbance of standard

Expected Values for total plasma cholesterol levels <200 mg/dl Normal Levels 200-240 mg/dl borderline values > 240 mg/dl High values

hypercholesterolemia Familial hypercholesterolemia Causes of hypercholesterolemia Familial hypercholesterolemia Obesity Hypothyroidism Obstructive jaundice Diabetes mellitus Coffee drinking cigarette smoking

Two types of Serum cholesterol LDL Bad" cholesterol HDL good" cholesterol HDL/LDL indicator of heart disease risk. 19

Several medicines can worsen cholesterol level: Thiazide diuretics Oral estrogens Some beta blockers Immunosuppressive agents

Types of serum lipoproteins

Complete Lipid profile Requires 12-18 hours fasting Total Cholesterol, TAG & HDL are measured directly (mg/dL) using colorimetric method. LDL is usually calculated : LDL = Total Cholesterol – HDL - (TAG/5)

Plasma Lipids Profile

THANK YOU High risk Normal 240 140-200 Cholesterol Above 200 Below 150 mg/dL TAG 160–189 mg/dL 100–129 mg/dL LDL Less than 40 mg/dL 40–59 mg/dL HDL THANK YOU