1. 4-1
Radiotracers
• Introduction
• Design of a Radiotracer Experiment
▪ Molecule labeled at specific location
▪ Physical processes
• Applications and techniques
• Basic premise
▪ Radioactive isotope behaves the same as stable isotope
▪ Radioactive isotope easier to follow and detect
→ Dilution 10-6 to 10-12
▪ Chemistry of element monitored by isotope behavior
▪ Trace dynamic mechanisms
▪ Also used to evaluate isotope effect
→ Slight differences in kinetics due to isotopic mass differences
• Used in biology, chemistry
2. 4-2
Radiotracer experiments
• Basic assumptions of experiments
• radioactive isotopes behave as the stable isotope
▪ difference in masses can cause a shift in the reaction rate or
equilibria (the isotope effect)
▪ in most cases isotope effect does not significantly affect
radioisotope method
▪ Isotope effect related to square root of the masses
→ Largest in small masses (i.e., H)
* Not as reliable with H, C limited in intermolecular
reactions
• radioactivity does not change the chemical and physical properties
of the experimental system
▪ Need to consider amount of activity
▪ Biological effects limited in short term
▪ Limit physical effects (i.e., crystal damage, radicals)
▪ Limited impact of daughter
→ Different chemical form
3. 4-3
Radiotracer experiment
• biological studies there is no deviation from the normal physiological state
▪ Chemical compound level should not exceed normal concentration
▪ specific activity of tracer must be sufficient
→ Shorted lived isotopes better
• Chemical and physical form of the radionuclide compound same as
unlabeled
▪ Need to consider sorption to surfaces or precipitation
→ Radionuclide often in concentration below saturation
→ Precipitates due to presence of stable isotope
• radionuclide and the stable nuclide must undergo isotopic exchange
▪ Redox behavior and speciation
• Radiochemical purity
▪ Activity due to single isotope
• Only labeled atoms are traced
▪ Radioisotope due to compound not free isotope or other chemical
form
4. 4-4
Experimental considerations
• Suitable isotope
▪ Half-life
→Too short difficult to use
→Too long need to much isotope
▪ Decay mode
→Gamma eases experiments
▪ Availability
→Production method
→generator
6. 4-6
Labeled compounds
• Specifically labeled
▪ labeled positions are included in name of compound
▪ Greater than 95% of the radioactivity at these positions.
→ i.e., aldosterone-1, 2-3H implies that <95% of the tritium label
is in the 1 and 2 positions.
• Uniformly labeled
▪ compounds labeled in all positions in a uniform pattern.
→ L-valine-14C (U) implies that all carbon atoms in L-valine are
labeled with equal amounts of 14C
• Nominally labeled
▪ some part of the label is at a specific position
▪ no other information on labeling at other positions
→ cholestrol-7-3H (N) some tritium is at position 7, but may also
be at other positions
• Generally labeled
▪ compounds (usually tritium) with a random labeled distribution
▪ Not all positions in a molecule labeled
7. 4-7
Synthesis
• Labeled compounds include
▪ 14C
▪ 3H
• Carbon
▪ Need to consider organic reactions for labeling
▪ Biosynthesis
→ Photosynthetic
→ Microbial
• Hydrogen
▪ reduction of unsaturated precursors
▪ Exchange reactions
▪ Gas reactions
8. 4-8
Physical processes
• Location in a system
▪ Precipitation, sorption
→ Measure change in
solution
concentration
▪ Separations
→ Ratio of isotope in
the separation
process
* Ion exchange,
solvent
extraction
▪ Reaction mechanisms
→ Intermediate
reaction molecules
→ Molecular
rearrangements
9. 4-9
Isotope effects
• Based on kinetic differences or equilibrium differences
▪ 0.5 mv2
→ Mass is different
• Distillation
▪ Mass difference drives different behavior
• Effects can be seen approaching equilibrium
• Kinetic isotope effects are very important in the study of chemical
reaction mechanisms
▪ substitution of a labeled atom for an unlabeled one in a
molecule causes change in reaction rate for Z < 10
▪ change can be used to deduce the reaction mechanism
• change in reaction rate due to changes in the masses of the
reacting species due to differences in vibrational frequency along
reaction coordinate in transition state or activated complex
• Experimentally straightforward to measure the existence and
magnitude of kinetic isotope effects
10. 4-10
Biological experiments
• Autoradiography
▪ oldest method
▪ radioactive sample is placed on photographic emulsion
▪ After period of time film is developed
▪ precise location of the radioactive matter in sample is found
▪ autoradiography used to locate radionuclides in a sample or chromatogram
• Radioimmunoassay (RIA)
▪ sensitive method of molecules in biological samples
▪ based on the immunological reaction of antibodies and antigens
→ antigen or antibody labeled with a radiotracer
→ limited amount of antibody is available, antigen will compete for
binding sites
→ Start with a certain amount of radiolabeled antigen, any additional
antigen added will displace some the radiolabeled antigen
→ Measure activity of the supernatant
* amount of unbound antigen
→ mix the same amounts of antibody and radiolabeled antigen together
with unknown stable antigen sample
→ stable antigen will compete with the radiolabeled antigen for binding
sites on the antibody molecules.
• Some of the radiolabeled antigen will not be able to bind
• constructing a calibration curve that shows the amount of radioactivity present in
the supernatant after adding standard
11. 4-11
Biological experiments
• DNA analysis
▪ extract the DNA from a sample
▪ DNA is cut into pieces using enzymes that cut either side of a
repeated sequence
→ DNA mixture of segments of differing size
→ Electrophoresis is used to sort the fragments by size
▪ spatially separated fragments are allowed to react with
radiolabeled gene probes
▪ gene probes contain radiolabeled specific DNA fragments of
DNA bind only to DNA segments containing a nucleotide
sequence that is complementary to its own (matching strand
in the DNA double helix
▪ original DNA fragments identified by the radiolabeled DNA
that has reacted
▪ physical pattern the autoradiograph is pattern of the DNA
sequences and sizes
12. 4-12
Environmental and industrial
• Environmental processes
▪ Flow
▪ Dispersion
→In atmosphere
and hydrosphere
▪ Short lived isotopes
→Isolated from
other systems
14. 4-14
Industrial uses of Radiation
• Radiation
▪ Imaging
▪ Density
▪ Analysis
▪ Curing
Requires source, detector, data analysis, and
shielding
15. 4-15
Measurement with neutrons and
photons
• Radiography
• Tomography
• Density
▪ Tracers in wells
▪ Am/Be source (1 Ci to 0.1 Ci)
▪ 137Cs (around 1 Ci)
• Used in determining
▪ flow - industrial production
▪ moisture content -airplane maintenance
▪ images
16. 4-16
Uses in Medicine
• Radiology
▪ anatomical structure (x-rays)
• Nuclear Medicine
▪ analyze function
▪ therapy
• MRI
▪ 1H, 13C, 17O
Equipment
• Detectors
▪ gamma
▪ coordinated to produce images
• Isotopes
▪ Need to produce and purify
17. 4-17
Isotope Production
• Reactor produced
▪ n,g reaction
• Cyclotron produced
▪ p,x reactions
▪ PET radionuclides
• Generators
▪ long lived parent, short lived daughter
(99mTc from 99Mo)
▪ Ion exchange holds parent, daughter is eluted
• Natural
▪ 212Bi from natural decay chain
18. 4-18
Tools for Nuclear Medicine
• Hot Atom Chemistry
▪ formation of different molecule upon decay or
production
• Organic chemistry
▪ synthesis of labeled compounds
MoAb with ligand
complex which can pass through barriers
complex similar to biological molecule
▪ must be biologically active
• Medical
▪ metabolism
▪ diagnosis
▪ therapy
19. 4-19
Isotopes
Isotope Half-life Use
51Cr 27.7 days blood and spleen scan
59Fe 44.5 days Fe metabolism
67Ga 78.3 hours tumors and infections
75Se 119.8 days pancreatic scanning
99mTc 6.02 hours many uses
111In 67.3 hours blood, bone
123I 13.2 hours thyroid
131I 8.05 days thyroid
133Xe 5.25 days lung
186Re 89.3 hours bone pain
205Tl 73.5 hours blood, heart
20. 4-20
External Sources
• X-rays
▪ oldest use discovered in 1895
travel through soft tissue, attenuated by bone
▪ barium as contrast media
▪ tomography
Computerized axial tomography
• Radiotherapy
▪ kill tumor from outside
▪ intersection of a few beams
21. 4-21
Diagnostic Nuclear Medicine
• Obtaining medical images
▪ gamma rays can be used to produce image
1st used with thyroid with 131I (fission product, half-life
of 8 days)
Measure of uptake and metabolic activity
observed for hours (dose to high 3 rads/µCi, 1-10 µCi)
• Need to have isotope accumulate in a specific organ
• Spatial pattern of emissions gives a 3-D picture
▪ Collimated detector needed
▪ single energy g best for collimator
99mTc (140 keV)
22. 4-22
Positron Emission Tomography
• ß+ produces two 511 keV g
• Identify line where decay occurred
• Possible to reconstruct distribution
• Useful isotopes include:
Isotope Half-life
15O 2 minutes
13N 10 minutes
11C 20 minutes
18F 110 minutes
• PET shows dynamic events
▪ blood flow
▪ respiration (lung to brain)
23. 4-23
Therapeutic Nuclear Medicine
• Uses ionizing radiation to kill tissue
▪ radical production
• Oxygen effect
▪ O2 has a large electron affinity
O2 + e- --> O2
-
• High LET
▪ alpha particles
24. 4-24
Clinical Applications
• Endocrine System
▪ Thyroid - Adrenals
• Central Nervous System
▪ Brain - CFS
▪ Eye
• Musculoskeletal System
• Gastrointestinal System
▪ Stomach - Intestines
▪ Pancreas - Liver
• Cardiovascular System
▪ Dynamics -Disease
25. 4-25
More clinical applications
• Urinary system
• Hematopoietic system (Blood)
▪ First done by Lawrence in 1938 on leukemia
• Lymphatic system
• Tumors
26. 4-26
Thyroid
Anterior and posterior images
from whole body I-131
scintigram
30 mCi I-131 (sodium iodide)
600 rad to lung
imaging for papillary
carcinoma of the
thyroid
33. 4-33
Skeletal, error
• Tc-99m DTPA and Tc-
99m MDP
• The outer package was
labeled MDP, but was
really DTPA
• MDP is
• methylenediphosphon
ate
(contains C-P-C bonds)
34. 4-34
Liver
• 5.2 mCi Tc-99m sulfur colloid i.v. (SPECT)
• 1.8 rad to liver, 0.1 rad to whole body
41. 4-41
Isotope dilution analysis
• quantitative analysis based on measurement of isotopic abundance of a
nuclide after isotope dilution
• Direct dilution
▪ determine the amount of some inactive material in a system
▪ define unknown amount as x grams
▪ To the system with x grams of inactive A, add y grams of active
material A* of known activity D
▪ know the specific activity of the added active material, S1
▪ Change specific activity
▪ basic equation of direct isotope dilution analysis
▪ unknown amount x of material A given in terms of amount y of
added labeled material A* and the two measured specific activities
S1 and S2
42. 4-42
Example
• A protein hydrolysate is to be assayed for aspartic acid
▪ 5.0 mg of aspartic acid, having a specific activity of 0.46 Ci/mg
is added to hydrolysate
▪ From the hydrolysate, 0.21 mg of highly purified aspartic acid,
having a specific activity of 0.01 Ci/mg, can be isolated
• How much aspartic acid was present in the original hydrolysate?
• We say that
• x=number of mg aspartic acid in original hydrolysate
• y=5.0 mg
• S1= 0.46 Ci/mg
• S2=0.01 Ci/mg
43. 4-43
Inverse IDA
• simple variant on the basic direct IDA
▪ inverse IDA measure the change in specific activity of an unknown
radioactive material A* after diluting it with inactive A
▪ assume have q mg (where q is unknown) of a radioactive substance
A* whose specific activity is known
→ (i.e., Sq=D/q)
→ (Sq can be measured by isolating a small portion of A*,
weighing it, and measuring its activity)
▪ add r mg of inactive A to A* and thoroughly mix the A and A
▪ isolate and purify the mixture and measure its specific activity Sr.
▪ Sr=D/(q+r)