Estimation of Blood Glucose

Objectives

• To understand the importance of measuring

blood glucose level.

• To understand the principles of enzymatic

estimation of glucose.
Introduction
Glucose is a simple sugar which is a permanent
and immediate primary source of energy to all
the cells in our body. Glucose is carried through
the bloodstream to provide energy to all cells in
the body. Cells cannot use glucose without the
help of insulin.
Glucose is the main sugar that the body makes
from the food in the diet (from protein, fat and,
in largest part, carbohydrate). Ingested glucose
is absorbed directly into the blood from the
intestine and results in a rapid increase in blood
glucose.
Introduction
Body tries to maintain a constant supply of glucose for our
cells by maintaining a constant blood glucose concentration.
Blood sugar levels are regulated by the hormones insulin
and glucagon which act antagonistically. These two
hormones are secreted by the islet cells of the pancreas, and
thus are referred to as pancreatic endocrine hormones.
When the blood glucose levels are high, insulin hormone
secreted which causing liver to convert more glucose
molecules into glycogen and when the blood glucose levels
are low, glucagon secreted and act on liver cells to promote
the breakdown of glycogen to glucose and increases the
blood glucose concentrations. Essentially blood glucose
levels determine the time of secretion of these hormones. 
Glucose Metabolism
Clinical Significance
Normal range of blood glucose is 70-100 mg/dL.
The blood glucose level is easily changed under the influence of some
external and internal factors such as body composition, age, physical
activity and gender.
Diabetes (high level of blood sugar): Is a disease related to the abnormal
metabolism of blood sugar and deficient insulin production. So blood sugar
levels are an important parameter for the study of diabetes. The level of
glucose circulating in blood at a given time is called blood glucose level.
The blood glucose level varies at different time on various part of the day.
In people with diabetes, the body does not produce enough insulin or does
not respond to insulin properly. The result is that sugar builds up in the
blood stream, damaging the body's organs, blood vessels and nerves. This
condition in which too much sugar in the blood stream is called
Hyperglycemia.
Hypoglycemia is a possible side effect of diabetes medications in which
blood glucose level drops below 70mg/dL.
Types of Diabetes
1. Type I Diabetes or Insulin-dependent Diabetes
Mellitus (IDDM)
This type of diabetes is less frequent, occurs before the
age of 15 years due to less production of insulin from ß
cells of langerhans (Pancreas).

2. Type II diabetes or Non-insulin-dependent Diabetes

Mellitus (NIDDM)
This type of diabetes is more frequent in population.
Occurs at middle age. Characterized by impaired
(decreased) β-cell function, increased endogenous hepatic
glucose output and insulin resistance in target tissues.
Blood Glucose Analysis
The blood glucose analysis is carried out to
measure the amount of blood glucose at the
time of sample collection. It is used to detect
both hyperglycemia and hypoglycemia and
consequently helping the diagnosis of
diabetes.
Methods for Estimation of Blood Glucose Level
The concentration of blood glucose can be estimated
colorimetrically by spectrophotometerically or by
blood glucose test strips.
Blood Glucose Test Strips
How do Diabetes Test Strips Work?
Regular testing with diabetes test strips is an important part of
successful diabetes management.
When the blood is placed on to the test strip, the blood reacts with a
chemical called glucose oxidase enzyme producing gluconic acid
(gluconate) from glucose in the blood.
At the other end of the test strip, the meter transfers a current to the
test strip. The test strip has electric terminals that allow the metre to
measure the current between terminals.
Principle of Estimation of Blood Glucose by
Glucose Oxidase Method

Glucose is oxidised by GOD (Glucose

oxidase) to gluconic acid and hydrogen
peroxide which in conjunction with POD
(peroxidase), reacts with chloro 4-phenol
and PAP (4-Amino-antipyrine) to form a
red quinoneimine. The absorbance of the
coloured complex, proportional to the
concentration of glucose in the specimen, is
measured at 500 nm.
Principle of Estimation of Blood Glucose by
Glucose Oxidase Method
Glucose oxidase
Glucose + O2 + H2O Gluconate + H2O2
POD
H2O2 + 4-aminophenazone + Chloro4-phenol Quinoneimine (red-violet)
+ 4H2O

Color intensity (red-violet) is directly proportional to glucose

concentration in the specimen
Procedure of Estimation of Blood Glucose
by Glucose Oxidase Method
Pipette into well Blank Standard Assay
identified test tube
Working Reagent μL 1000 μL 1000 μL 1000
Distilled Water μL 10
.Standard Glucose Sol μL 10
Specimen (Serum) μL 10

Mix the tube contents and then let stand for 10 minutes at 37˚C or 20
minutes at room temperature. Read absorbance at 500 nm (460-560)
against reagent blank in a spectrophotometer.
Calculation of Serum Glucose
Calculate the results as follows:
Abs (Assay) 100
Concentration of Glucose = X Con. St. Gluc. Sol. X
(mg/dL) Abs (Standard) (0.01) (0.01)
mg (Wt.) of mL of used serum
glucose in 10μL of in the test
used standard

Concentration of standard glucose solution (Con. St. Gluc. Sol.) =100mg/dL (100mg/100mL).
One dL (deciliter) = 100mL.

10μL for working of Standard Glucose Solution = 0.010mL (= 10μL/1000).

Glucose Wt. (mg) Vol. (mL)

100 100
X 0.01mL (10 μL) X = 100 X 0.01/100 =
X = 0.01 mg/0.01 mL
mL of used serum in the test = 10μL/1000 (= 0.010)
Thank You Very Much
For Paying Attention