Estimation of Blood Glucose and Cholesterol: 

1.  Estimation of Blood Glucose:

 

§  Method: Glucose oxidase and peroxidase (GOD-POD)

§  Specimen: Blood should be collected in a sodium Fluoride anti-coagulated vial to its antiglycolytic activity.

§  Principle: In presence of glucose oxidase (GOD) enzyme glucose is converted into gluconic acid and hydrogen peroxide (H2O2). After that this hydrogen peroxide (H2O2) in presence of peroxidase (POD) enzyme, oxidatively couples with 4-amino antipyrine and phenol to produce red quinonimine dye. The intensity of this red color complex is directly proportional with the concentration of glucose present in blood. The absorbance of this coloured dye is measured at 505 nm (500-550)

β – D glucose + O₂ + H2O           ----------->           Gluconic acid + H2O2

H2O2 + 4-amino antipyrine + Phenol      -------------->       Quinonimine dye (red) + H2O

§  Requirement:

i.                    Fluoride vial

ii.                  Glucose GOD-POD reagent

iii.                Test-tube

iv.                Colorimeter or Semiauto-analyser

§  Procedure:

	Test/Sample	Standard	Blank
Working Reagent	1ml	1ml	1ml
Standard		0.01ml
Serum/Plasma	0.01ml

 

§  Incubation: We incubated the assay mixture for 15 min at 37°C or 30 min at room temperature (25-30°C). After incubation, the absorbance of the test and standard will be measured against reagent blank at 505 nm.

§       Calculation: Glucose in (mg/dl) = OD of Sample / OD of Stand. x Conc. Of Stand. (100)

§  Advantages: -- It is specific for glucose estimation.

-          This method is sensitive and simple.

-          It is a cheap method.

§  General Parameter: i. Reaction type – Endpoint

ii. Linearity is up to 500mg/dl

* If the sample value is 500mg/dl, dilute the sample with water, and repeat the assay.

§  Expected Value:

*Random blood glucose test – 79-160mg/dl

*Fasting blood glucose test – 70-110mg/dl

*Postprandial blood glucose test – Under 140mg/dl

 

2.  Estimation of Blood Cholesterol:

 

§  Method: Cholesterol oxidase and peroxidase (CHOD-POD)

§  Principle: Cholesterol esters are hydrolyzed by cholesterol Esters (CE) to give free cholesterol and fatty acids. Cholesterol oxidase (CHOD) oxidizes the 3-OH group of free cholesterol to liberate Cholest-4-en-3-one and Hydrogen peroxide (H2O2). In presence of peroxidase (POD), Hydrogen peroxide couples with 4-amino antipyrine, and phenol to produce Red quinonimine dye. The absorbance of cholesterol dye is measured at 505nm (490-510nm) and its proportion to the amount of cholesterol concentration in the sample.

 

Cholesterol esters     ------------->      Cholesterol + Fatty acids

 

Cholesterol + O2     --------------->     Cholest-4en-3-one + H2O2

 

H2O2 + Phenol + 4-amino antipyrine     --------------->    Quinonimine dye (red) + H2O

 

§  Procedure:

	Test	Standard	Blank
Reagent -1	1000μl	1000μl	1000μl
Reagent-2	10μl
Serum/Plasma			10μl

 

§  Requirement: i. Cholesterol Reagent

ii. Test-tube

iii. 1cm Cuvettes

iv. General laboratory equipment

v. Colorimeter or semi-auto analyzer

vi. Heparin or EDTA vial

§  Incubation: We will incubate the assay mixture for 10-15 min at 37°C. After incubation the absorbance of the test and standard we will measure against the reagent blank at 505nm.

§  Calculation: Cholesterol in (mg/dl) = OD of Sample / OD of Stand. x Conc. Of Stand.(200)

 

§  Linearity: The assay is linear between 10mg/dl and 600mg/dl. If the results obtained were greater than the linearity limit, we should dilute the sample to ½ with NaCl 9mg/L and multiply the result by 2.

§  Reference values:  *Desirable --- < 200mg/dl

     * Borderline to high risk --- 200-239mg/dl

*High risk --- > 240 mg/dl

§  Clinical Significance: Cholesterol is the main lipid found in the blood, bile, and brain tissue. It is the main lipid associated with arteriosclerotic vascular diseases. It is required for the formation of steroids and cellular membranes. The liver metabolizes the cholesterol and it is transported in the blood serum by lipoproteins. Increased levels are found in hypercholesterolemia, hyperlipidemia, hypothyroidism, uncontrolled diabetes, nephrotic syndrome, and cirrhosis. Decreased levels are found in malabsorption, malnutrition, hyperthyroidism-anemia, and liver diseases.