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METHODS IN ENZYMOLOGY EDITORS-IN-CHIEF John N. Abelson Melvin I. Simon DIVISIONOFBIOLOGY CALIFORNIAINSTITUTEOFTECHNOLOGY PASADENA,CALIFORNIA FOUNDINGEDITORS Sidney P. Colowick and Nathan O. Kaplan Preface As these volumes were being completed, American Paul C. Lauterbur and Briton Sir Peter Mansfield won the 2003 Nobel Prize for medicine for discoveriesleadingtothedevelopmentofMRI. The Washington Post story on October 6, 2003 announced the accolade, noting: ‘‘Magnetic resonance imaging, or MRI, has become a routine method for medical diagnosis and treatment. It is used to examine almost all organs withoutneedforsurgery,butisespeciallyvaluablefordetailedexaminationof thebrain and spinal cord.’’ Unfortunately,the articleoverlooked thegrowing usefulnessofthistechniqueinbasicresearch. MRI, along with other imaging methods, has made it possible to glance inside the living system. For patients, this may obviate the need for surgery; for researchers, it becomes a noninvasive method that enables the model systemstocontinue‘‘doingwhattheydo’’withoutbeingdisturbed.Thevalue and potential of these techniques is enormous, and that is why these once clinicalmethodsarefindingtheirwaytothelaboratory. Authors have been selected based on research contributions in the area about which they have written and based on their ability to describe their methodologicalcontributionsinaclearandreproducibleway.Theyhavebeen encouragedtomakeuseofgraphicsandcomparisonstoothermethods,andto providetricksandapproachesthatmakeitpossibletoadaptmethodstoother systems. The editor wants to express appreciation to the contributors for providing theircontributionsinatimelyfashion,tothesenioreditorsforguidance,andto thestaffatAcademicPressforhelpfulinput. P. Michael Conn xiii Contributors to Volume 385 Articlenumbersareinparenthesesandfollowingthenamesofcontributors. Affiliationslistedarecurrent. David L. Alexoff (12), Department of Stuart Clare (8), Centre for Functional Chemistry, Brookhaven National La- Magnetic Resonance, Department of boratory,Upton,NewYork11973 ClinicalNeurology,JohnRadcliffeHos- pital,UniversityofOxford,Headington, Robert S. Balaban (15), Laboratory of OxfordOX39DU,UnitedKingdom Cardiac Energetics, National Heart, Lung, and Blood Institute, National Christian A. Combs (15), Light Micro- InstitutesofHealth,Bethesda,Maryland scopy Facility, National Heart, Lung, 20892 and Blood Institute, National Institutes ofHealth,Bethesda,Maryland20892 Nicolau Beckmann (14), Novartis Insti- tuteforBiomedicalResearch,Analytical AndrE´ Constantiensco (9), Laboratoire and Imaging Sciences Unit, CH-4002 de Biomecanique, Centre Hospitalier Basel,Switzerland Universitaire Hautepierre, Strasbourg, France MarkusBeu(13),ClinicofNuclearMed- Bruce M. Damon (2), Department of icine, University Hospital Du¨sseldorf, Radiology and Radiological Sciences, Du¨sseldorf40225,Germany Vanderbilt University Institute of Ima- Kevin J. Black (6), Departments of Psy- gingScience,Nashville,Tennessee37232 chiatry,Neurology,andRadiology,Wa- Carmen S. Dence (16), Department of shingtonUniversitySchoolofMedicine, Radiology,WashingtonUniversitySchool St.Louis,Missouri63110-1093 ofMedicine,St.Louis,Missouri,63110 Britton Chance (20), Eldridge Reeves Doris J. Doudet (10), Department of JohnsonUniversity,Philadelphia,Penn- Medicine/Neurology, Pacific Parkinson sylvania19104 Research Center, University of British Columbia,Vancouver,BritishColombia DelphineL.Chen(17),WashingtonUni- V6T2B5,Canada versity School of Medicine, St. Louis, Missouri63110 David J. Dubowitz (7), Department of Radiology, Center for Functional Mag- PhilippeChoquet(9),LaboratoiredeBio- netic Resonance Imaging, University of mecanique,CentreHospitalierUniversi- California,SanDiego,LaJolla,Califor- taireHautepierre,Strasbourg,France nia92093-0677 Bradley T. Christian (11), Department Guillaume Duhamel (9), Laboratoire ofNuclearMedicineandPositronEmis- Mixte, Universite Joseph Fourier, Neu- sion Topography, Kettering Medical roimagerie Fonctionelle et Metabolique, Center,Kettering,Ohio454229 Grenoble,France ix x contributors to volume 385 M. R. Gerasimov (18), Department of D. W.J.Klomp(3), DepartmentofRadi- Chemistry,BrookhavenNationalLabo- ology,MedicalFacultyoftheUniversity ratory,Upton,NewYork11973 of Nijmegen. Nijmegan 6500 HB, The Netherlands JohnC.Gore(2),DepartmentofRadiol- ogy and Radiological Sciences, Vander- Jonathan M. Koller (6), Department of bilt University Institute of Imaging Psychiatry,WashingtonUniversitySchool Science,Nashville,Tennessee37232 of Medicine, St. Louis, Missouri 63110- Emmaunelle Grillon (9), Laboratoire 1093 Mixte, Universite Joseph Fourier, Neu- RakeshKumar(1),DepartmentofNucle- roimagerie Fonctionelle et Metabolique, arMedicine,AllIndiaInstituteofMed- Grenoble,France icalSciences,NewDelhi110029,India* Robert J. Gropler (16), Department of RolfLarisch(13),ClinicofNuclearMed- Radiology,WashingtonUniversitySchool icine, University Hospital Du¨sseldorf, ofMedicine,St.Louis,Missouri,63110 Du¨sseldorf40225,Germany A.Heerschap(3),DepartmentofRadiol- Jean-LouisLeviel(9),LaboratoireMixte, ogy,MedicalFacultyoftheUniversityof Universite Joseph Fourier, Neuro- Nijmegen, Nijmegan 6500 HB, The imagerie Fonctionelle et Metabolique, Netherlands Grenoble,France PilarHerrero(16),DepartmentofRadi- ology,WashingtonUniversitySchoolof JeanLogan(12),DepartmentofChemis- try, Brookhaven National Laboratory, Medicine,St.Louis,Missouri,63110 Upton,NewYork11973 JamesHolden(10),DepartmentofMedi- cal Physics, University of Wisconsin, KathrynE.Luker(19),MallinckrodtIn- stituteofRadiology,WashingtonUniver- Madison,Wisconsin53706 sity School of Medicine, St. Louis, Jean-NoE¨l Hyacinthe (9), Laboratoire Missouri63110 Mixte, Universite Joseph Fourier, Neu- roimagerie Fonctionelle et Metabolique, Robert H. Mach (16), Department of Grenoble,France Radiology,WashingtonUniversitySchool ofMedicine,St.Louis,Missouri,63110 N.R.Jagannathan(4),DepartmentHead ofNuclearMagneticResonance,AllIn- Evan D. Morris (11), Indiana University diaInstituteofMedicalSciences,Ansari School of Medicine, Department of Nagar,NewDelhi110029,India Radiology,Indianapolis,Indiana46202 Suman Jana (1), Nuclear Medicine Divi- Hans-Wilhelm MU¨ller (13), Clinic of sion, Albert Einstein College of Medi- Nuclear Medicine, University Hospital cine,Bronx,NewYork10461 Du¨sseldorf,Du¨sseldorf40225,Germany M.Khubchandani(4),DepartmentofNu- Raymond F. Muzic, Jr. (11), University clearMagneticResonance,AllIndiaIn- Hospitals of Cleveland, Case Western stituteofMedicalSciences,AnsariNagar, Reserve University, Cleveland, Ohio NewDelhi110029,India 44106 *CurrentAffiliation:DepartmentofRadiology,DivisionofNuclearMedicine,Hospitalofthe UniversityofPennsylvania,Philadelphia,Pennsylvania19104-4283 contributors to volume385 xi Kiyoshi Nakahara (5), Department of Abraham Z. Snyder (6), Departments of Physiology, The University of Tokyo Radiology and Neurology, Washington School of Medicine, Tokyo 113-0033, UniversitySchoolofMedicine,St.Louis, Japan Missouri63110-1093 SusanneNikolaus(13),ClinicofNuclear M. G. Sommers (3), Department of Radi- Medicine, University Hospital Du¨ssel- ology,MedicalFacultyoftheUniversity dorf,Du¨sseldorf40225,Germany of Nijmegen, Nijmegan 6500 HB, The Joel S. Perlmutter (6), Departments of Netherlands Neurology, Radiology, Anatomy and PaulVaska(12),DepartmentofChemis- Neurobiology,andthePrograminPhy- try, Brookhaven National Laboratory, sical Therapy, Washington University Upton,NewYork11973 SchoolofMedicine,St.Louis,Missouri 63110-1093 A.A.Veltien(3),DepartmentofRadiol- DavidPiwnica-Worms(19),Mallinckrodt ogy,MedicalFacultyoftheUniversityof InstituteofRadiology,WashingtonUni- Nijmegen, Nijmegan 6500 HB, The versity School of Medicine, St. Louis, Netherlands Missouri63110 HenningVosberg(13),ClinicofNuclear W.K.J.Renema(3),DepartmentofRadi- Medicine, University Hospital Du¨ssel- ology,MedicalFacultyoftheUniversity dorf,Du¨sseldorf40225,Germany of Nijmegen, Nijmegan 6500 HB, The Netherlands Michael J. Welch (16), Department of Jean-ChristopheRichard(17),Washing- Radiology,WashingtonUniversitySchool ton University School of Medicine, ofMedicine,St.Louis,Missouri,63110 St.Louis,Missouri63110 Karmen K. Yoder (11), Indiana Univer- MarkusRudin(14),NovartisInstitutefor sity School of Medicine, Department of Biomedical Research, Analytical and Radiology,Indianapolis,Indiana46202 Imaging Sciences Unit, CH-4002 Basel, Switzerland H. J. A. in ‘t Zandt (3), Department of Daniel P. Schuster (17), Washington Radiology, Medical Faculty of the Uni- versityofNijmegen,Nijmegan6500HB, UniversitySchoolofMedicine,St.Louis, TheNetherlands Missouri63110 Sally W. Schwarz (16), Department of AnneZiegler(9),CenterHospitalierUni- Radiology,WashingtonUniversitySchool versitaire,NeuroimagerieFonctionelleet ofMedicine,St.Louis,Missouri,63110 Metabolique,Grenoble,France [1] PET inresearch andclinicalimaging 3 [1] Positron Emission Tomography: An Advanced Nuclear Medicine Imaging Technique from Research to Clinical Practice By Rakesh Kumar and Suman Jana Introduction Positron emission tomography (PET) is an advanced diagnostic imagingtechnique,whichcannotonlydetectandlocalize,butalsoquantify physiological and biochemical processes in the body noninvasively. The ability of PET to study various biological processes opens up new pos- sibilities for both fundamental research and day-to-day clinical use. PET imaging utilizes (cid:1)-emitting radionuclides such as 11C, 13N, 15O, and 18F, which can replace their respective stable nuclei in biologically active molecules. These radionuclides decay by positron emission. After being emitted from the nucleus, a positron will combine with a nearby electron through a process known as annihilation. Annihilation converts the mass of both particles into energy in the form of two antiparallel 511-keV (cid:2) rays. The PET detectors are arranged in a ring in order to detect these (cid:2) rays. At present, 2-deoxy-2-[18F]fluoro-d-glucose (18F-FDG) is the most commonly used positron-emitting radiopharmaceutical used for PET imaging. 18F-FDG is a radioactive analog of glucose and is able to detect altered glucose metabolism in pathological processes. Like glucose, FDG is transported into cells by means of a glucose transporter protein and begins to follow the glycolytic pathway. FDG is subsequently phosphorylated by an enzyme known as hexokinase to form FDG- 6-phosphate.1,2 However, FDG-6-phosphate cannot continue through gly- colysis because it is not a substrate for glucose-6-phosphate isomerase. As a result, FDG-6-phosphate is trapped biochemically within the cell. This process of metabolic trapping constitutes the basis of PET imaging of the biodistribution of FDG. Because there can be a manyfold increase or decrease in the glucose metabolism of diseased tissue as compared to normal tissue, it is easy to detect such differences in metabolism using PET.ThisChapterdiscussesgeneralaspectsofPET,includingdrugevalu- ation, biological functions evaluation, clinical applications, and future directionsof PET imaging. 1K.M.McGowanetal.,Pharmacol.Ther.66,465(1995). 2R.L.Wahl,J.Nucl.Med.37,1038(1996). Copyright2004,ElsevierInc. Allrightsreserved. METHODSINENZYMOLOGY,VOL.385 0076-6879/04$35.00 4 imaging in animal and human models [1] Tracers Position emission tomography uses radioisotopes of naturally occur- ring elements, such as 11C, 13N, and 15O, in order to perform in vivo imaging of biologically active molecules. Although there is no radioisotope of H that can be used for PET, many molecules can replace a hydrogen orhydroxylgroupwith18Fwithoutchangingitsbiologicalproperties.Flu- orine-18canalsobeusedasasubstituteinfluorine-containingcompounds such as5-fluorouresil (5-FU), asdemonstrated by Mintun et al.3 Most PET tracers utilize a radioisotope that has a short half-life and can be produced by a cyclotron (see Table I for a list of important tracers). However, there are some radiotracers, such as copper-62, that can be manufactured in a nuclear generator. Radiopharmaceuticals are produced after the radioisotope has been generated and substituted into the compound of interest. Because of the short half-lives of most PET tracers, sequentialscanning on thesame day is not usually possible. Drug Evaluation The development of new drugs presents many questions that must be addressed: is the drug sufficiently delivered to target of interest? How is normal tissue affected? At what dose is toxicity produced? How much drug is eliminated from both target tissue and normal tissue in relation to time? Does the drug affect target tissue in the predicted way? All of these questions can be answered by labeling the drug of interest with an appropriate radionuclide forPET imaging. In addition, mathematical kinetic modeling is necessary for all aspects of drug pharmacology and to measure physiological functions, such as TABLEI Positron-EmitterRadiotracers Radioisotope Half-life(min) Carbon-11 20.4 Fluorine-18 109.8 Nitrogen-13 10 Oxygen-15 2.03 Copper-62 9.7 Rubidium-82 1.25 Yttrium-86 14.7 3M.A.Mintunetal.,Radiology169,45(1988). [1] PET inresearch andclinicalimaging 5 tissue perfusion, metabolic rate, and elimination. Dynamic data can be collected in a specific biological organ or tissue by defining the region of interest and recording the radioactivity over time. Input functions can be calculated by measuring tracer concentration in arterial blood. By com- paringtheinputfunctionsandtimeactivitycurvesovertheorganortissue of interest with theoretical models, it is possible to calculate the metabolism of the applied drugs. When these drugs are not metabolized in the tissue, the calculation of drug concentration is very simple. However, most drugs will undergo some degree of metabolism to produce metabolites. If these metabolites do not include the original radionuclide, there is no problem regarding the calculation of drug metabolism. How- ever, if these metabolites also contain a radionuclide it can be difficult for PET to distinguish signals from the parent radiopharmaceutical and those from its metabolite. Blasberg et al.4 and Salem et al.5 have suggested a number of mathematical calculations in order to determine the parent contribution from total measuredradiotracer activity. Pharmacokinetic studies of new pharmaceuticals are greatly simplified by labeling these compounds with a PET tracer. It is possible to measure the time-dependent, in vivo biodistribution of a new drug labeled with PET tracer in one experiment, which can be subsequently compared to many animal experiments. Furthermore, the various effects of a drug on different biological processes, such as blood flow, tissue metabolism, and receptoractivation,canalsobedemonstratedinvivothroughPETimaging. Biological Function Evaluation Theoretically, any biological function can be studied in vivo using an appropriately labeled PET tracer molecule. However, at present, PET tracers, which are utilized most commonly, are small molecules, which canbe labeledby well-defined methods.Anotherimportant consideration isthattheconcentrationofanyPETtracermoleculeshouldbesignificantly higher at the target sites than in the background. This allows biological functiontobemeasuredbydeterminingtracerconcentrationoveraspeci- fied time interval and by drawing a region of interest over the specified target tissue or organ region. Physiological processes such as oxygen con- sumption, blood flow, and tissue metabolism can also be demonstrated invivousingPET tracers. In the body, binding of an activating molecule (agonist) to a biochemical structure (receptor) can activate many biological functions. The same 4R.G.Blasbergetal.,CancerRes.60,624(2000). 5A.Salemetal.,Lancet355,2125(2000). 6 imaging in animal and human models [1] process canbeblocked byanantagonist,whichmayhaveahigheraffinity for receptors as compared to the agonist.Receptors can be visualized and quantified by labeling receptor-binding substances (ligands) with PET tracers. Most receptors have several biochemically similar subtypes and are composed of multiple subunits. Identification of these subtypes and subunits requires specific ligands. The details of in vivo research of receptors and their clinical applications in the diagnosis of neurodegenerative and heart diseases arediscussed later. Clinical Applications Theresolutionofcomputedtomography(CT)andmagneticresonance imaging (MRI) is excellent for the visualization of both normal and diseased tissues. However,all disease processesstartwithmolecular and cel- lularabnormalities.Mostdiseaseprocessestakealongtimetoprogressto astagewheretheycanbedetectedbythesestructuralimagingtechniques. Infact,manydiseasesmayalreadybeinadvancedstagesbythetimethey aredetectedbyMRIorCT.However,theprincipleofPETistodetectthe alteredmetabolismofdiseaseprocessesandnotthealteredanatomy,such as CT and MRI. PET, as a functional molecular imaging technique, can alsoprovidehighlyaccuratequantitativeresultsandthereforecanbeused forvarious researchand clinical applications. Kumaretal.6discussedtheroleof18F-FDGPETinthemanagementof cancerpatients.Forthesepatients,PEThasbecomeimportantindiagnosis, staging, monitoring the response to treatment, and detecting recurrence. However, PET has also played an important role for both diagnostic and therapeutic purposes in patients with neurological and cardiological infections and inflammations, vasculitis, and other autoimmunediseases.7 PET in Oncology 18F-FDGisthemostwidelyusedradiotracerinoncology.Becauseglu- cosemetabolismisincreasedmanyfoldinmalignanttumorsascomparedto normalcells,PEThashighsensitivityandahighnegativepredictivevalue. It has a well-established role in initial staging, monitoring response to the therapy, and management of many types of cancer, including lung cancer, colon cancer, lymphoma, melanoma, esophageal cancer, head and neck cancer, and breast cancer (TableII). 6R.Kumaretal.,Ind.J.Cancer.40,87(2003). 7M.Schirmeretal.,Expr.Gerontol.38,463(2003).