1812
Semester 2, 2005
Page 1 of 6
Part I – Multiple Choice Questions
Questions on the use of RADIOACTIVITY to measure metabolic fluxes
Useful Information
1 nCi = 2,200 dpm = 37 Bq
1 µCi = 2.2 million dpm = 37 kBq
1 ALI (Annual limit of Intake) for 14C is 40 MBq
Measuring Glucose uptake using the radio-labelled 2-deoxyglucose method
You wish to measure the rate of glucose uptake into muscle cells in response to
insulin. You have read about a method that uses an analog of glucose called
2-deoxyglucose (2DG). 2DG differs from glucose in that it has an hydrogen instead
of a hydoxyl at the C-2 position. Cells take up 2DG and convert it into
2-deoxyglucose 6-phosphate (2DGP). The 2DGP cannot proceed down glycolysis
and is ‘trapped’ in the cells after uptake. This is because 2DGP cannot be made into
fructose 6-phosphate. It can, however, be made into glycogen.
In the method, cells are incubated in the presence of normal glucose containing a
TRACER amount of radioactively labeled 2DG. After a particular time, the
incubation is stopped so as to determine the amount of radioactivity that has become
trapped in the cells.
1812 A
Semester 2, 2005
Page 1 of 6
1812 A
80.
A
B
C
D
E
Semester 2, 2005
Page 2 of 6
For the 2DG method to work properly, which of the following must be TRUE?
It is important that hexokinase works faster on 2DG that it does on glucose
The presence of trapped 2DGP should not affect the normal metabolism of
glucose
Muscle glucose transporters should behave differently towards 2DG than they
do towards glucose
2DGP should be able to desphosphorylate back to 2DG
2DGP should be able to move freely in and out of the muscle cells
You purchase 50 µCi of [U14-C] 2-deoxyglucose. It arrives as a aqueous solution
contained in a 3 mm thick glass vial. The label on the vial is as shown in the picture.
[U-14C] 2deoxyglucose
50 Ci
0.2 mCi/ml
200 Ci/mol
81.
A
B
C
D
E
1812 A
Which statement is CORRECT?
To avoid exposure to beta-particles, the glass vial should be encased in a lead
container from now on
Beta-particles will not penetrate the glass and gloved hands
Drinking the entire contents will exceed your Annual Limit of Intake (ALI) for
radiation
Spilling the contents of this vial on your hands will require hospitalization
You should maintain a distance of at least one metre away from this vial
Semester 2, 2005
Page 2 of 6
1812 A
Semester 2, 2005
Page 3 of 6
82.
What is the volume of 2DG solution in the vial?
83.
How many dpm are contained in 1 µl of the 2DG solution?
84.
What is the concentration of 2DG in the vial?
You have a suspension of muscle cells in a suitable buffer (Buffer X) at a
concentration of 20,000 cells/ml. You set up some incubations in Eppendorf tubes as
shown in the table below. Each incubation contains a final volume of 1 ml and a final
concentration of 5 mM glucose. The glucose is associated with a tracer amount of
[U-14C]-2DG to give a (planned) final specific activity of about 500 dpm per nmol.
Some of the incubations contain insulin.
Tube
1
2
3
4
5
Cell suspension (ml)
0.5
0.5
0.5
0.5
0.5
Buffer X (ml)
0.5
0.4
0.4
0
0
200 ng/ml Insulin solution (ml)
0
0
0
0.4
0.4
2DG/glucose mixture (ml)
0
0.1
0.1
0.1
0.1
The tubes were set up was as follows:

Firstly, the cells, buffer and/or insulin were added to the tubes..The tubes were
then placed in a water bath at 30°C.

At time zero, the reaction in Tube #2 was started by adding 0.1 ml of the
2DG/glucose mixture.

At 1 min intervals, Tubes 3, 4 and 5 were started in a similar manner (ie, at a
clock time of 1, 2 and 3 min respectively).

At 5 min on the clock, Tube #2 was centrifuged for 30 seconds to form a pellet
of the cells. The supernatant was immediately aspirated (removed) and
discarded. The remaining pellet was counted for 14C for 10 minutes.

Tubes 4, 3 and 5 were treated in the same way when the clock read 7, 11 and
13 minutes respectively.
1812 A
Semester 2, 2005
Page 3 of 6
1812 A
Semester 2, 2005
Page 4 of 6
You decide that you will need 1 ml of the radioactive 2DG/glucose mixture.
You dissolve 9 mg of glucose in 1 ml water to make a 50 mM solution.
85.
How many dpm SHOULD be taken from the 2DG vial to make this glucose
solution the desired specific activity?
86.
What is the APPROXIMATE ratio of 2DG molecules to glucose molecules in
the above 2DG/glucose mixture.
87.
After YOUR ASSISTANT makes up the 2DG/glucose mixture, you count 10
and 20 µl aliquots and find that these contain 25,123 and 52,715 dpm
respectively. What is the ACTUAL specific activity of the mixture?
The results of the experiment are shown in the table below.
Tube
Incubation
dpm in cell
time (min)
pellet
1
0
30
2
5
4151
3
10
7330
4
5
22030
5
10
42732
88.
APPROXIMATELY how much 2DG/glucose is trapped in the cells in
Tube #4?
89.
What is the approximate rate of glucose uptake in Tube #4
(in nmol/min/10,000 cells)?
90.
In Tube #4, what percentage of the total dpm put into the tube has been taken
up by the cells?
1812 A
Semester 2, 2005
Page 4 of 6
1812 A
Semester 2, 2005
Page 5 of 6
The following information relates to Questions 91 - 95
Consider TUBE #3 and the following table of options.
Property
A
B
C
D
E
dpm in cell pellet
INCREASE
INCREASE
INCREASE
INCREASE
SAME
nmol 2DG/glucose in
INCREASE
INCREASE
INCREASE
SAME
SAME
INCREASE
INCREASE
SAME
SAME
SAME
INCREASE
SAME
SAME
SAME
SAME
cell pellet
rate of 2DG/glucose
uptake into pellet
(nmol/min)
rate of 2DG/glucose
uptake into pellet
(nmol/min/10,000 cells)
Which option BEST represents the pattern of changes that would result if Tube #3
was altered in the following ways:
91.
Performing the centrifugation step when the clock showed 21 minutes
92.
Doubling the specific activity of the 2DG/glucose mixture
93.
Counting the cell pellet in the scintillation counter for twice as long
94.
Doubling the volume of all additions (ie, 2 ml incubation volume, 1 ml cells,
0.2 ml 2DG/glucose mixture and 0.8 ml buffer)
95.
Adding insulin insead of buffer X (ie, making it identical to Tube #5)
1812 A
Semester 2, 2005
Page 5 of 6
1812 A
Semester 2, 2005
Page 6 of 6
PART II – Short answer question worth 6 marks
A kibitzing* colleague suggests that your results are not valid because a significant
amount of ‘free’ 2DG/glucose gets transferred into the scintillation vial. In other
words, they think that 2DG/glucose associated with the outside of the cells or solution
simply left with the cell pellet is interfering with your results. They also point out that
2DG/glucose uptake can still occur during the centrifugation and transfer steps.
Kibitz; to look on and offer intrusive comments and unwanted, usually meddlesome, advice to others.
*
.
They suggest the following improvements. Comment on each.
i)
“You should re-suspend the pellet of cells in 1 ml fresh buffer and centrifuge
them again before you count them.”
ii)
“You should formally ‘stop’ the reactions by adding something that will break
open the cells on demand (eg, strong acid).
iii)
“You should include incubations with dead cells as a control”
1812 A
Semester 2, 2005
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