DEPARTAMENTO DE BIOQUÍMICA Y BIOLOGÍA MOLECULAR

Universidad de Córdoba

Grado de Veterinaria. 2019/20

Biochemistry. Laboratory work.

Laboratory work nº 3
QUANTITATIVE ANALYSIS OF GLUCOSE BY THE GLUCOSE OXIDASE METHOD

INTRODUCTION
Blood is an organic, viscous and reddish tissue, which is composed of blood cells (erythrocytes, leukocytes and
platelets) suspended in blood plasma. Plasma, which represents the extracellular matrix, constitutes 55% of blood fluid
and is mostly water containing dissipated proteins, glucose, mineral ions, hormones, etc.
Blood is unstable and without precautions, it could coagulate and hemolysis altering its composition.
The zymogens cascade is activated without the presence of anticoagulants (sodium oxaloacetate, EDTA, heparin, etc.)
that ensure blood clotting. When the blood clot is made, plasma without clotting proteins is liberated, called serum. The
most of the clinical analysis use the serum instead of blood or plasma because it is very easy to obtain and conserve (-20
ºC). Moreover, the serum is lacking of substances, which could interfere with the analysis.
Carbohydrates are the biological molecules more abundant in the environment and are essential for the organisms
because are the major source of fuel for metabolism, being used both as an energy source and in biosynthesis. In plants,
carbohydrates are the first biomolecules synthetised during the photosynthesis and the main metabolic pathways start with
previous production of glucose. In animals, the main pathway involved in the glucose production is the gluconeogenesis
(generation of glucose from certain non-carbohydrate carbon substrates) and glycogenolysis (breakdown of liver glycogen
stores).
The glucose level in blood is regulated by the coordination of different hormones (insulin, adrenaline, glucagon, etc.).
Hyperglycemia (high blood sugar), hypoglycemia (low blood sugar) and the variability of the glucose levels are
important problems in clinical, which are related to an increase in the morbidity and mortality. In healthy animals, an
increase of glucose in blood appears after feeding, with a stressful or exciting situation or even with the general
anesthesia. However, the increase can be also related to diseases (diabetes mellitus, liver injury, etc.) or metabolic
disorders (overactive adrenal glands, thyroid or pituitary, hyperadrenocorticalism, etc.).
In contrast, hypoglycemia is observed in insulin-secreting tumors, hypopituitarism, dysfunctions of the adrenal glands,
conditions that interfere with their absorption, starvation, etc.
Therefore, the accurate glucose determination and quantification methods are very important and provide the
veterinarian with an important evaluation tool.

OBJECTIVE
The main objective of this practice is to become familiar with the analytical techniques used in the clinical laboratories
to determine blood glucose concentration, knowing its theoretical basis as well as to acquire the capability to determine
the solutions concentrations and dilution numbers.

THEORETICAL BASIS

Laboratory work nº 3: Quantitative analysis of glucose by the glucose oxidase method. Bioquímica Bilingüe. 1er curso del Grado en Veterinaria 1
 In contrast to other biomolecules (nucleic acids, proteins, etc.), glucose does not have an absorption spectroscopy,
which could determine the measurement by spectrophotometric analysis. However, there are other chemical methods and
enzymatic bioassays to determine the accurate glucose concentration.
The enzymatic bioassays are specific, reproducible, sensitive and fast to determine metabolites. In fact, the glucose
oxidase method is one of the methods used to determine glucose concentration in biological samples.
Glucose oxidase is an enzyme extracted from the growth medium of Aspergillus niger. Glucose oxidase catalyse the
oxidation of Beta D- glucose present in the plasma to D-gluconic acid with the formation of hydrogen peroxide. In the
second reaction, the hydrogen peroxide produced is then broken down to oxygen and water by a peroxidase enzyme.
Oxygen then react with an oxygen acceptor such as a mix of phenol and 4-aminoantipyrine which itself converted to a
coloured compound (quinonimine), the amount of which can be measured colorimetrically at 505 nm. The second reaction
is called Trinder Reaction. The pink intensity color is proportional to the glucose concentration present in the samples.

The glucose oxidase method is an indirect method, therefore glucose concentration is determined by comparing with a
standard curve of a known amounts of glucose solution. Each group will prepare a standard curve and determine glucose
concentration in serum samples (porcine or bovine).

MATERIALS
• Distilled water
• 0.2 % (p/v) Commercial glucose solution in distilled water
• Serum
• Work Solution: 10 U/ml glucose oxidase; 5 mM phenol; 0.4 mM 4-aminoantipyrine; 1 U/ml peroxidase; 100 mM
potassium phosphate pH 7.5.
• Test tubes and eppendorfs
• 5 ml glass pipette
• Propipette
• Micropipettes p200 and p1000 with the pipet tips required.
• Spectrophotometer fixed at 505 nm.
• Plastic cuvettes
• Rinse bottle

Laboratory work nº 3: Quantitative analysis of glucose by the glucose oxidase method. Bioquímica Bilingüe. 1er curso del Grado en Veterinaria 2
 PROTOCOL
To elaborate the standard curve, each group will prepare the next glucose solutions in 6 labeled test tubes (A-F). For
that, the student will make dilutions of the 0.2% (p/v) commercial glucose in distilled water.

0.2 % (p/v) Commercial standard glucose dilutions

Reagents A B C D E F
Distilled water (ml) 4.95 4.9 4.8 4.7 4.6 4.5
Commercial standard glucose 0.05 0.1 0.2 0.3 0.4 0.5
0.2 % (p/v) (ml)
Mix with vortex

Determine the commercial glucose concentration (mg/dl) in each test tube prepared above and complete the next
table:

A B C D E F
Glucose concentration (mg/dl)

Follow the instructions given in the below table. The final volume of each tube is 2.5 mL. Do not forget that the zero
and the serum samples do not have standard glucose:

Nº TUBE
Reagents 0 1 2 3 4 5 6 Diluted Diluted
serum serum
sample I sample II

Work solution (ml) 0.9 0.9 0.9 0.9 0.9 0.9 0.9 0.9 0.9

Distilled water (ml) 0.1 --- --- --- --- --- --- --- ---

Standard solution (ml) --- 0.1 (de A) 0.1 (de B) 0.1 (de C) 0.1 (de D) 0.1 (de E) 0.1 (de F) --- ---

Diluted serum (ml) --- --- --- --- --- --- --- 0.1 0.1

Mix with vortex

Incubate for 20 minutes at RT (room temperature) (The colour is stable for 2 h after the 20 min incubation)
Record absorbance values at 505 nm. Do not forget to calibrate the spectrophotometer previously with the zero
absorbance

We will use only one plastic cuvette for all the measurements. Firstly, we will calibrate with the zero absorbance (0 test
tube). For the rest of measurements, we will measure from the lower (light colour) to the higher concentration (strong
colour) at 505 nm. Use the table below to annotate the absorbance values.

Laboratory work nº 3: Quantitative analysis of glucose by the glucose oxidase method. Bioquímica Bilingüe. 1er curso del Grado en Veterinaria 3
 Plot the standard curve in the graph paper (glucose concentration (mg/dL) in abscissa and A505 in ordinate). The
glucose concentration in the serum will be determined inserting the A505 in the standard curve. To calculate the final protein
concentration you should take into account the dilution factor.
The normal glucose values in animal serum are:

Animal Glucose (mg/dl)

Dog 70-110
Cat 50-75
Horse 75-115
Cow 45-75
Sheep 50-80
Goat 50-75
Pig 85-150

Laboratory work nº 3: Quantitative analysis of glucose by the glucose oxidase method. Bioquímica Bilingüe. 1er curso del Grado en Veterinaria 4
 SURNAME, NAME: DNI: Signature:
Date: Group:

BIOQUÍMICA. Bilingüe. 1º Curso Grado en VETERINARIA

Laboratory work Nº 3: Quantitative analysis of glucose by the glucose oxidase method

[Standard glucose]
Nº Tube A505 nm
mg/dl
1. Complete the table and plot the standard curve in the graph
0 0
paper (glucose concentration (mg/dL) in abscissa and A505 in
ordinate). Explain how you calculate the glucose concentration 1 0.066
in one of the test tubes. 2 0.133
3 0.305
4 0.406
5 0.640
6 0.752
Diluted Serum
0.386
Sample I
Diluted Serum
0.580
Sample II

2. Determine the final glucose concentration (mg/dl) in the serum samples. Explain how you calculate these numbers indicating
which number corresponds with each animal.

Laboratory work nº 3: Quantitative analysis of glucose by the glucose oxidase method. Bioquímica Bilingüe. 1er curso del Grado en Veterinaria 5