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This method involves using an analog of glucose called 2-deoxyglucose (2DG) to measure the rate of glucose uptake into muscle cells in response to insulin.
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Radioactivity Questions 2005 Exam
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. • 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.
80. Principle of the Method 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
The Stock! • 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.
81. Which 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
82-84 Simple dpm, moles & vol • What is the volume of 2DG solution in the vial? • How many dpm are contained in 1 µl of the 2DG solution? • What is the concentration of 2DG in the vial?
85-87 Radioactive Solutions • We need to make up 1 ml of 50 mM glucose with tracer at 500 dpm/nmol. You dissolve 9 mg of glucose in 1 ml water to make a 50 mM solution. • How many dpm SHOULD be taken from the 2DG vial to make this glucose solution the desired specific activity? • What is the APPROXIMATE ratio of 2DG molecules to glucose molecules in the above 2DG/glucose mixture. • 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?
0.5 ml cells of suspension at 20,000 cells/ml 10,000 cells DISCARD 2DG/glucose spin incubate Final 1 ml 1 mM 2DG/glu COUNT
88-90. 2DG Uptake • APPROXIMATELY how much 2DG/glucose is trapped in the cells in Tube #4? • What is the approximate rate of glucose uptake in Tube #4 (in nmol/min/10,000 cells)? • In Tube #4, what percentage of the total dpm put into the tube has been taken up by the cells?
91-95. Tube 3 Scenarios • Performing the centrifugation step when the clock showed 21 minutes • Doubling the specific activity of the 2DG/glucose mixture • Counting the cell pellet in the scintillation counter for twice as long • 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) • Adding insulin insead of buffer X (ie, making it identical to Tube #5)
