School of Biomedical Sciences

LKS Faculty of Medicine

The University of Hong Kong

Biochemistry Practical Manual

for
IASM Block 2019-20

Testing for abnormal glucose metabolism

Practical Supervision
Dr Joanna WY HO

Technical Assistance
Ms Fan WY WONG

Tasks
1. In this practical you will estimate the glucose concentrations in the blood samples collected from
patients who had undergone OGTT. Using the spectrophotometric glucose oxidase method, you
will determine the glucose concentration in a series of:
a. diluted reference standards and then generate a calibration/standard curve of photometric
absorbance against glucose concentration;
b. patient plasma samples having an OGTT and plot the OGTT results on the graph paper
provided.

Experiments
Precaution: For safety, all biological specimens should be treated as biohazards and Universal
Precautions must be STRICTLY applied in handling samples exercised. Latex
gloves should be worn at all times to avoid exposure to the infectious agents. All
pipette tips and samples should be discarded as bio-hazardous waste. Read the
Universal Precautions notice for handling specimens before embarking on the
experiment.

Part One

Preparation of the Serial Dilutions of Glucose Solutions from a Glucose Stock

(Work in Eppendorf Tubes)

1. Label six Eppendorf tubes as follows: 1,2,3,4 and 5

2. Transfer 20 μL of the benzoic acid solution into tube 1 (this is the reagent blank), 2, 3, and 4
only. [NOT tube 5!]
3. Transfer 20 μL of the stock glucose solution into tube 5 and 4. Mix contents in tube 4 well
by pipetting up and down a few times.
4. Transfer 20 µL of solution from tube 4 into tube 3. Mix well by pipetting up and down a few 1
times.
5. Transfer 20 µL of solution from tube 3 to tube 2. Mix well by pipetting up and down a few
times.

HKU/FOM | IASM_GluMet_Fall2019
 By the end of the serial dilutions, you have now obtained the following samples with known
glucose concentration for the preparation of the glucose standards:

Eppendorf # 1 2 3 4 5
Glucose (mg/dL) 0 50 100 200 400

Part Two

Preparation of the Reaction Mixtures

(Work in Eppendorf Tubes)

1. Label FIFTEEN Eppendorf tubes from 1 – 15.

i. Glucose standards (tube 1 - 5) {all from PART ONE}:

# 1 for the reagent blank, # 2-5 for the glucose solutions of known concentrations.
ii. Patient samples (tubes 6 – 15):
# 6-10 for patient 1 (0, 30, 60, 90, 120min samples), # 11-15 for patient 2.

2. Add 1400 µL [dispense 700 µL TWICE] of chromogenic reagent to each of the labeled
empty tubes (tubes 1-15).
3. Pipette 10 µL each of the solutions from the five tubes in PART ONE into tubes 1-5.
4. Pipette 10 µL of the timed patient samples into vials 6 -15.
5. Vortex to mix. Ensure the cap is capped on well before vortexing.
6. Incubate for 15 minutes at room temperature.
7. (Ask the demonstrators to carry out this step for you) Transfer the samples onto a 96-well
plate and read the absorbance in a spectrophotometer at 500 nm against the reagent blank
after the incubation time.

Steps of calculation and date processing

1. From your subgroup results, plot
a. Plot one graph (the standard curve) showing OD values (ie., A500nm) against
concentration using the values from the reference standards. It should be a best fit line.
b. Obtain blood glucose values (mg/dL) of the patient samples from graph plotted in step.1a
[it is also recommended that to convert the patient’s glucose values to mmol/L (mM) as
well].
c. Plot one graph (the OGTT curves) showing patient’s glucose level (either in mg/dL OR
mM) against time points (0-120 minutes). It should have both patient’s trends compared
on a same graph.
2. From other subgroup in your PBL group, copy glucose values obtained in step.1b for the
other patient(s). Plot patient’s values to the OGTT curve as instructed in step.1c.

Discussion: Use the information in the reference sheet provided to interpret your results. Discuss your
findings and draw a conclusion (50 words or less). Complete ONE lab report form per PBL group at the
end of practical session.
2

HKU/FOM | IASM_GluMet_Fall2019