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 àDilution to §Chemistry of element monitored by isotope behavior §Trace dynamic mechanisms §Also used to evaluate isotope effect àSlight differences in kinetics due to isotopic mass differences Used in biology, chemistry

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 àLargest 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 àDifferent chemical form

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 àShorted lived isotopes better Chemical and physical form of the radionuclide compound same as unlabeled §Need to consider sorption to surfaces or precipitation àRadionuclide often in concentration below saturation àPrecipitates 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 Experimental considerations Suitable isotope §Half-life àToo short difficult to use àToo long need to much isotope §Decay mode àGamma eases experiments §Availability àProduction method àgenerator

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4-6 Labeled compounds Specifically labeled §labeled positions are included in name of compound §Greater than 95% of the radioactivity at these positions. ài.e., aldosterone-1, 2- 3 H 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. àL-valine- 14 C (U) implies that all carbon atoms in L-valine are labeled with equal amounts of 14 C Nominally labeled §some part of the label is at a specific position § no other information on labeling at other positions àcholestrol-7- 3 H (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

4-7 Synthesis Labeled compounds include § 14 C § 3 H Carbon §Need to consider organic reactions for labeling §Biosynthesis àPhotosynthetic àMicrobial Hydrogen §reduction of unsaturated precursors §Exchange reactions §Gas reactions

4-8 Physical processes Location in a system §Precipitation, sorption àMeasure change in solution concentration §Separations àRatio of isotope in the separation process *Ion exchange, solvent extraction §Reaction mechanisms àIntermediate reaction molecules àMolecular rearrangements

4-9 Isotope effects Based on kinetic differences or equilibrium differences §0.5 mv 2 àMass 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

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 àantigen or antibody labeled with a radiotracer à limited amount of antibody is available, antigen will compete for binding sites àStart with a certain amount of radiolabeled antigen, any additional antigen added will displace some the radiolabeled antigen àMeasure activity of the supernatant *amount of unbound antigen àmix the same amounts of antibody and radiolabeled antigen together with unknown stable antigen sample à 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

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 àDNA mixture of segments of differing size àElectrophoresis 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

4-12 Environmental and industrial Environmental processes §Flow §Dispersion àIn atmosphere and hydrosphere §Short lived isotopes àIsolated from other systems

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4-14 Industrial uses of Radiation Radiation §Imaging §Density §Analysis §Curing Requires source, detector, data analysis, and shielding

4-15 Measurement with neutrons and photons Radiography Tomography Density §Tracers in wells §Am/Be source (1 Ci to 0.1 Ci) § 137 Cs (around 1 Ci) Used in determining §flow- industrial production §moisture content-airplane maintenance §images

4-16 Uses in Medicine Radiology §anatomical structure (x-rays) Nuclear Medicine §analyze function §therapy MRI § 1 H, 13 C, 17 O Equipment Detectors §gamma §coordinated to produce images Isotopes §Need to produce and purify

4-17 Isotope Production Reactor produced  n,  reaction Cyclotron produced §p,x reactions §PET radionuclides Generators §long lived parent, short lived daughter ( 99m Tc from 99 Mo) §Ion exchange holds parent, daughter is eluted Natural § 212 Bi from natural decay chain

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

4-19 Isotopes IsotopeHalf-lifeUse 51 Cr27.7 daysblood and spleen scan 59 Fe44.5 days Fe metabolism 67 Ga78.3 hourstumors and infections 75 Se119.8 dayspancreatic scanning 99m Tc6.02 hoursmany uses 111 In67.3 hoursblood, bone 123 I13.2 hoursthyroid 131 I8.05 days thyroid 133 Xe5.25 dayslung 186 Re89.3 hoursbone pain 205 Tl73.5 hoursblood, heart

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

4-21 Diagnostic Nuclear Medicine Obtaining medical images §gamma rays can be used to produce image 1st used with thyroid with 131 I (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  best for collimator 99m Tc (140 keV)

4-22 Positron Emission Tomography ß + produces two 511 keV  Identify line where decay occurred Possible to reconstruct distribution Useful isotopes include: IsotopeHalf-life 15 O2 minutes 13 N10 minutes 11 C20 minutes 18 F110 minutes PET shows dynamic events §blood flow §respiration (lung to brain)

4-23 Therapeutic Nuclear Medicine Uses ionizing radiation to kill tissue §radical production Oxygen effect §O 2 has a large electron affinity O 2 + e - --> O 2 - High LET §alpha particles

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

4-25 More clinical applications Urinary system Hematopoietic system (Blood) §First done by Lawrence in 1938 on leukemia Lymphatic system Tumors

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

4-27 Thyroid papillary carcinoma of the thyroid status post total thyroidectomy 200 mCi I-131 sodium iodide Dose > 30 mCi requires hospitalization

4-28 Brain 20 mCi Tc-99m DTPA No activity

4-29 Brain 20 mCi Tc-99m DTPA Brain Activity

4-30 Skeletal 18.2 mCi Tc-99m MDP Only bone uptake, should have soft tissue, bladder and renal uptake

4-31 Skeletal Tc-99m MDP (Bone Study) In-111 labeled White Blood Cells (Sickle cell) No spleen uptake seen Tc-99m Sulfur Colloid (Marrow uptake)

4-32 Skeletal and Soft tissue Tc-99m pyrophosphate Electrical injury

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)

4-34 Liver 5.2 mCi Tc-99m sulfur colloid i.v. (SPECT) 1.8 rad to liver, 0.1 rad to whole body

4-35 Lung Xe-133 ventilatio n image

4-36 Lung 4.2 mCi Tc-99m MAA i.v. and 10.4 mCi Xe-133 gas by inhalation

4-37 Tumor 15 mCi F-18 fluorodeoxyglucose (FDG) 0.59 rad whole body

4-38 Tumor 14.8 mCi F-18 fluorodeoxyglucos e i.v

4-39 Tumor 11.0 mCi F-18 fluorodeoxyglucose (FDG) i.v

4-40 Tumor 10.8 mCi F-18 fluorodeoxyglucose i.v.

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, S 1 §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 S 1 and S 2

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 S 1 = 0.46  Ci/mg S 2 =0.01  Ci/mg

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 à(i.e., Sq=D/q) à(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 S r. § S r =D/(q+r)