Determining the Level of Glucose in Urine Sample using DOGPAP Assay through Spectrophotometer

DETERMINING THE LEVEL OF GLUCOSE IN URINE SAMPLE USING DOG-
PAP ASSAY THROUGH SPECTROPHOT0METER
INTRODUCTION:-
Glucose is one of the simplest forms of all carbohydrates belonging to
monosaccharides. Glucose in the form of foods rich in carbohydrates like grains,
starchy vegetables, dairy, whole fruit, etc. are body’s desired source of energy.
Since glucose is an important energy source for our body and unhealthy levels of
glucose due to disorders of carbohydrate metabolism can lead to serious
diseases conditions which can be treatable or even may lead to permanent
diseased condition. Level of glucose both in blood and urine is an important thing
to be monitored (Kathleen, 2017). Not having a normal level of glucose in urine is
a sigh of health problem. The most common of which is diabetes in which there
is an elevated level of glucose in urine. Diabetes is a condition in which body is
not being able to regulate glucose levels properly either due to insufficient
production of insulin or insulin resistance. To check the level of glucose in urine
is a simple and quick way to understand the abnormality of glucose in urine
(Dabra, 2018}. GOD-PAP assay is an enzymatic colorimetric method used to
detect the level of glucose in the urine samples. Glucose oxidase (GOD) oxidises
glucose present in the sample to form hydrogen peroxide. This hydrogen
peroxide reacts with phenol and 4-aminoantipyrine under catalysis of peroxidase
(PAP) to form a red coloured product Quinoneimine as indicator. The reading is
taken at (492 – 550 nm) in a spectrophotometer (Spectrum, n.d).
Glucose + 2 H2O + O2 Gluconic acid + H2O2
2 H2O2 +Phenol + 4-amino-antipyrine 4 H2O + Quinoneimine
A spectrophotometer is a device that measures the amount of light absorbed at
various wavelengths by the sample in terms of photons after it passes through
the sample. Considering the range of wavelength of source of light, it can be
classified into Ultraviolet spectrum, Visible spectrum and Infrared spectrum of
the electromagnetic spectrum.
Spectrophotometer is designed on the principle of photometry which states that
When a beam of light is of intensity I0 passes through a sample solution, a part of
it is reflected (Ir), a part is absorbed (Ia) and rest of the light is transmitted (It)
Thus, I0 = Ir + Ia + It
In case of any photometers Ir is kept constant by using identical cells and
moreover the readings of I0 and It are enough to determine the value of Ia. The
relationship between the amount of light absorbed and the concentration of the
substance is based on the two fundamental laws of photometry.
GOD
PAP

Beer’s Law
This law states that the amount of light absorbed is directly proportional to the
concentration of the solute in the solution.
Mathematical representation as,
Log10 I0/It = asc
as = Index of Absorbency
c = Concentration of Solution
Lambert’s Law
The Lambert’s law states that the amount of light absorbed is directly
proportional to the length or thickness of the solution under analysis.
Mathematical representation as,
A = Log10 I0/It = asb
A = Absorbance of test
as = Absorbance of standard
b = length / thickness of the solution
Log10 I0 / It = asbc
If b is kept constant then,
Log10 I0/It = asc
The absorbency index as is defined as
as = A/cl
c = concentration of the absorbing material in gm/liter.
l = distance traversed by the light in solution in cm.
Therefore,
The working principle of the Spectrophotometer is based on Beer-Lambert’s law
which states that the amount of light absorbed by a colored solution is directly
proportional to the concentration of the solution and the length of a light path
through the solution (Batra, 2018).