Estimation of Serum Cholesterol

by
Cholesterol Oxidase Method
Structure of Cholesterol
Serum/Plasma Cholesterol
Biomedical Importance of Cholesterol
Sources of Serum Cholesterol
 Serum Cholesterol Estimation

• Fluorometric
• Spectrophotometric
Chemical method
Biochemical/enzymatic method
 Enzymatic Method
for Cholesterol Estimation
 History

1967---Tonks
1974—Alian et al.
 Principle

Cholesterol esterase
Cholesterol Esters ---------------------------→ Cholesterol + Fatty Acids

Cholesterol oxidase
Cholesterol + O2 ------------------------→ Cholesten-3-one + H2O2

Peroxidase
2 H2O2 + 4-aminoantipyrine + Phenol -----------→ Quinoneimine + 4 H 2O
Reaction Scheme
Requirements: Equipments

Incubator
Requirements: Equipments

Centrifuge Machine
 Requirements: Equipments

Colorimeter
Requirements: Reagents

Cholesterol Estimation Kit

Requirements: Serum
 Preparation

• Preparation of Reagents
• Preparation of Standards
• Preparation of Blank
• Preparation of Serum
Method
 Parameters of Analysis

•Wave Length : 530 mm

(the wavelength scanning range is 500-546 mm)
•Optical Pathway : 1 cm
•Temperature : 37°C
•Measurement Control : Against Reagent Blank
 Precautions

• Ingestion must be avoided.

• Contact with skin and eyes must be avoided.
• Material Safety Data Sheet must be taken care
of for additional information.
• The acetone-alcohol solvent is not allowed to
over-boil.
 Analytical Procedure

* Test condition * Calibration * Quality Control

Quality Control
 Specificity

• Cross Contamination
• Interferences in the Measurement
• Influence of Drugs on Clinical Laboratory
 Calculation

 By comparing with one standard

 By calculating from standard curve
Calibration Curve
 Limitation

Concentrated solution must be diluted

with 0.9% saline.
 Result

 Presentation of results
 Interpretation of results
 Performance Characteristics

• Reportable range
• Precision
 Standard Reference

• 3.6-5.2 mmol/L (normal)

• 5.3 -6.2 mmol/L (borderline high)
• 6.2 mmol/L (hypercholesterolemic).
 Advantages

• The stability of the Optical Density

Over a Period of Hours,
• The Stability of the Reagent for 2 or 3 Days,
• The High Degree of Reproducibility of the
Results.
 Accuracy

Linear regression analysis with different standard

cholesterol and standard serum will give the
accurate results.
Comment : Prescribed Food
Forbidden Food
 Case Study

Name of the subject : Sajid Anwar

Age : 28
Sex : Male
Serum cholesterol level : 120 mg/dL