Homepage http://www.pet.au.dk
The Second Year
The Positron Emission Tomography Center of the Aarhus University Hospital system was inaugurated October 20, 1993 as a joint activity supported by the County Council of Aarhus, the Medical School of Aarhus University, and the Medical Research Council of Denmark. Therefore, administratively the center is both a department of Aarhus General Hospital and a laboratory of the Institute of Experimental Clinical Research of Aarhus University. The center's mission is to explore the pathophysiology of disease mechanisms by means of tomography studies in vivo. The center is also the seat of the MRC (Denmark) chair of brain research.
The center occupies the bottom floor of the neurology house of Aarhus General Hospital, including its 18/9 MeV Scanditronix negative ion cyclotron and the Siemens/CTI ECAT Exact HR47 positron emission tomograph, generously awarded by the Karen Elise Jensen Foundation in 1993.
Professor and neurologist Johannes Jakobsen, Cand.Med., Dr.Med., directed the center until September 1994 when the current director, Albert Gjedde, Cand.Med., Dr.Med., was appointed. The center was licensed for general operation October 13, 1994 and received further licenses for production and delivery of radioactive pharmaceuticals in November and December of 1994.
Therefore the year 1996 was the second full year of operation of the PET Center. The year witnessed a steady increase in the number of active projects, the number of patients examined clinically, the number of researchers and staff associated with the center, the number of collaborators in Aarhus, the rest of Denmark and abroad, the number of papers published from the center, and the funds awarded for research. It is particularly gratifying that it has been possible to involve many new research partners from the Aarhus University Hospital system. The number of examinations of patients and volunteers rose 40% to 920, of which 285 served clinical purposes and 635 research purposes, a 30:70 split which is in agreement with the original plans for the center. The split was approximately 40:60 between studies of the heart and studies of the brain.
The center currently has active collaborations with laboratories in Denmark, Germany, Canada, USA, Australia, and Japan. Details of these developments are given in the subsequent reports, divided into sections covering Mapping of the Active Brain, Monoamine Neurotransmission, Brain Perfusion and Metabolism, Regulation of Myocardial Blood Flow, the Physiology and the Pathophysiology of the Liver, Early Detection of Malignancies, Technology Research (Radiochemistry and Neuroimaging), and Productivity.
Again in 1996, the Aarhus PET Center wishes to acknowledge the generous support received from authorities, funding agencies, and colleagues around the world. This support has enabled the center to carry on and expand in this time of budgetary problems experienced by all sectors of the Danish health care system. Half of the support of the center arises from the Research Initiative of Aarhus University Hospitals, a joint venture between the County Council of Aarhus and Aarhus University. The other half is assembled from numerous sources, including the Medical Research Council of Denmark, the Danish Heart Association, the Novo Nordic Foundation, the Parkinson Society, the Danish Medical Association, and several altruistic private funds. The PET Center is very thankful for this support which is detailed in connection with the individual projects summarized in the Annual Report. In general, the PET Center wishes to thank the McConnell Brain Imaging Center at the Montreal Neurological Institute and its Director, Professor Alan C. Evans, for the continuing support of the technological development at the Aarhus Center.
Dansk resumé:
Århus Universitetshospitals PET-center har nu tilbagelagt det andet hele arbejdsår. Centret blev oprettet 20. oktober 1993, men tilladelser til at fremstille, udlevere og anvende radioaktive lægemidler blev først indhentet med udgangen af 1994. Centret var oprindeligt normeret med otte ansatte, men idag arbejder mere end 30 personer med tilknytning til centret. Centret udfører såvel klinisk som eksperimentelt arbejde, og det eksperimentelle arbejde udføres på frivillige patienter og raske og på forsøgsdyr.
Fra begyndelsen var det tanken, at PET-centret skulle tilfredsstille et stort behov for samarbejde mellem institutioner i Århus, Danmark og udlandet, og samarbejdet har da også været vejledende for PET-centrets udvikling. Nær ved et halvt hundrede projekter er afviklet, igangværende eller under igangsættelse, i samarbejde med alle sygehusene i Århus Universitetshospital, Århus Universitet, Ålborg Universitetscenter, Odense Universitet, Danmarks tekniske Universitet, Danmarks farmaceutiske Højskole, universitetet i Lund, Københavns Universitet og Rigshospitalet, NeuroSearch A/S, universitetet i Uppsala, Hammersmith Hospital i London, Oxford University, Johns Hopkins University i Baltimore, University of West Virginia i Morgantown, Montreal Neurological Institute, og universiteterne i Matsuyama og Osaka i Japan, samt Brisbane i Australien. Af samme grund beskæftiger PET-centret et stort antal Ph.D.studerende og fellows fra en række lande, herunder Danmark, Tyskland, Storbritannien, Holland, Belgien, USA, Canada og Japan. På mange måder udtrykker denne udvikling et klart ønske fra PET-centrets side om at fungere som en egentlig forskeruddannelsesinstitution i samarbejde med Århus Universitets Institut for Eksperimentel Klinisk Forskning. PET-centrets arbejdssprog er derfor helt naturligt blevet engelsk, hvorfor denne årsrapport anvender dette sprog, med danske sammenfatninger.
Dette arbejde har naturligvis ikke kunnet lade sig gøre uden bidrag fra nær og fjern. Det største enkelte bidrag kommer fra Århus Amtsråd gennem det såkaldte forskningsinitiativ, som i sin visdom har bestemt, at moderne sundhedsvidenskab ikke lader sig praktisere uden offentlige tilskud, også fra amterne. Dette bidrag udgør ikke mindre end 60% af PET-centrets indtægter. Af de resterende 40% stammer en fjerdedel fra Statens sundhedsvidenskabelige Forskningsråd, som støtter centret med en million kroner om året, og de resterende 3 millioner kroner stammer fra offentlige og private fonde i ind og udland. PET-centrets drifts og lønningsbudget er nu af størrelsesordenen 10 millioner kroner om året, hvortil kommer værdien af centrets infrastruktur og overhead, som udgør ca. 5 millioner kroner, som fortrinsvis dækkes af Århus Amtsråd direkte gennem Århus Kommunehospital. Centret er disse bidragydere taknemmelige for de glimrende arbejdsbetingelser, som centret oplever i sit daglige arbejde.
Arbejdet er naturligvis heller ikke muligt uden en motiveret og engageret stab, som jeg hermed vil give min dybfølte tak for det store arbejde i 1996. Denne tak skal også rettes til administrationen for Århus Kommunehospital, ledelsen af Århus Universitetshospital, Århus Amtsråd og Statens sundhedsvidenskabelige Forskningsråd.
Albert Gjedde, Aarhus PET Center
Table of Content
Localization of a Cerebral Center for Timing of Sound and Speech
Cognitive Activation in Alzheimer's Disease
Advanced Processing of Tomographic Images
Monoaminergic Neurotransmission
Monoamine Transporters in Brain: Basic Research
Synthesis, Release, and Binding of Dopamine in Living Brain
Regulation of Dopamine Metabolism Estimated by PET
Brain Metabolism and Blood Flow
Regulation of Oxygen Consumption in Mammalian Brain In Vivo
Measurement of Cerebral Blood Flow by Dynamic MRI Bolus Tracking
Effect of 30% Stable Xenon Inhalation on Regional CBF Measured by PET
Myocardial Blood Flow and Metabolism
Rate-Limiting Steps in Myocardial Glucose Metabolism
Myocardial Perfusion, Forearm Flow and Morphology of Resistance Arteries in Patients with Syndrom X
Liver Physiology and Pathophysiology
Role of Liver Innervation, Studies in Anaesthesized Pigs
Early Detection of Cholangiocarcinoma in Patients with Primary Sclerosing Cholangitis
PET Scanning of Osteogenic Sarcomas
Aziridines in the Synthesis of 11C and 18F-Labelled Amino Acids
Solid Phase Supported Reaction of N.C.A. H11CN with Arabinose: A Simplified Synthesis of 1-11C Glucose
Rapid Synthesis and Purification of 11C-Labelled Benzyl Iodides using a C-18 Solid Phase Matrix
The Use of Vented Sterile Filters in the Automation of Preparative HPLC Injections
Choice of Chromatographic Methods or the Routine Analysis of Partially Acetylated 2-18F Fluorodeoxyglucose
Cyclotron, PET-camera, and computer facilities
PET Imaging of Living Laboratory Animals: Studies on Swine
Diagnostic Use of PET-Scanning
Teaching and Conference Participation
Pathophysiological Seminars 1996
The Positron Emission Tomography Center engaged 32 individuals in 1996. Some researchers, although not formally attached to the PET Center, carried out their research at the Center. The last of the PET Center's eight permanent positions was filled by Flemming Hermansen in 1996. Two nuclear medicine technologists, Bente Lund and Helle Nørholm were promoted to other positions. Twentyfour positions were covered by grant and other support, or through collaborations. Thus, in 1996 the PET family consisted of the following people:
Academic Staff
Dirk Bender, Ph.D. (Radiochemistry)
Antony D. Gee, Ph.D. (Radiochemistry)
Albert Gjedde, C.Med., Dr.Med. (Neuroscience)
Søren B. Hansen, C.Sc., Ph.D. (Medical Physics and Computer Sciences)
Flemming Hermansen, C.Med. (Clinical Physiology and Nuclear Medicine)
Susanne Keiding, C.Med., Dr.Med. (Internal Medicine)
Donald F. Smith, Ph.D., Dr.Med. (Neuropsychopharmacology)
Ex Officio Staff
K. A. Jessen, C.Sc., Lic.Sc. (Chief, Medical Physics)
Technical Staff
Jytte Brask, secretary
Gloria Stocks Gee, nuclear medicine technologist
Helle Larsen, nuclear medicine technologist
Vikie F. Larsen, nuclear medicine technologist
Bente Lund, nuclear medicine technologist
Torben Lund, cyclotron and tomograph operations
Palle Monefeldt, secretary
Niels Nielsen, cyclotron and tomograph operations
Helle Nørholm, nuclear medicine technologist
Ilse Rasmussen, nuclear medicine technologist
Students and Fellows
Flemming Andersen, C.Sc., graduate student in computer sciences
Niels Buus, C.Med., fellow in cardiology
Morten Bøttcher, C.Med., graduate student in cardiology
Erik Hvid Danielsen, C.Med., graduate student in neurology
Nicholas Gillings, B.Sc., M.Phil., graduate student in radiochemistry
Koichi Ishizu, M.D., Ph.D., fellow in nuclear medicine
Peter Johannsen, C.Med., graduate student in neurology
Mette Marie Madsen, medical student
Frank Mirz, C.Med., fellow in audiology
Peter Høst Poulsen, C.Med., graduate student in neurosurgery
Barbara Ravnkilde, C.Psych., graduate student in neuropsychology
Anders B. Rodell, C.Sc., graduate student in computer sciences
Klaus Roelsgaard, C.Med., graduate student in hepatology
Thomas Sølling, C.Sc., fellow in computer science
Poul Videbech, C.Med., fellow in psychiatry
Leif Østergaard, C.Sc., C.Med., graduate student in neuroradiology
The aim of the auditory study is to demonstrate significant change in cerebral blood flow of a cerebral center of timing during specific auditory stimulations. In the first study, known auditory pathways and active cortical areas were explored (1). Five subjects listened passively to seven different auditory signals which covered a broad spectrum of audible sounds: pure tone (1000 Hz, 65 dB SPL), pure tone pulse trains (with different frequencies and durations), broad band noise, music and signal words. The second study was initiated to find a specific area in the brain that subserves time discrimination in auditory perception. To test the existence of such an area, the study included auditory stimulation of five subjects with narrow band noise, standardized speech and single word pairs with different durations of the first phoneme to vary its semantic and orthographic characteristic. Most stimulations required attention to different time patterns of auditory stimuli. PET-images were performed using Hann-filter (18mm FWHM), 50 seconds emission scans, 50,000 cps after injection of 500 MBq H215O, and t-statistic maps created after pixel-by-pixel subtraction of PET-volumes (2).
The results demonstrated significant increases in cerebral blood flow in temporal lobes bilaterally during passive listening to auditory stimuli. Activation occurred in the superior temporal gyrus on both sides, representing the primary auditory cortex. The stimuli of the second study elicited generally extensive activation in the frontal and parietal areas bilaterally. Temporal lobe activations were less conspicuous. The right inferior frontal area was mainly activated during the tasks involving time discrimination, additionally an increase in cerebral blood flow occurred in the middle frontal and inferior parietal areas which are known to participate in attention to different sensory modalities (3, 4, 5).
The results of the two projects subserve the hypothesis of localized cortical areas involved in interpretation of time factors in auditory signals. Furthermore, the involvement of the temporal area was confirmed during simple auditory stimulation.
Acknowledgements:
Supported by MRC Denmark (12-1633 and 12-1634), grants from "Handelsgartner Ove Villiam Buhl Olesen og ægtefælle Edith Buhl Olesens Mindelegat", and“"Forskningsfondet til studier vedrørende Tunghørhed og Døvhed"”and Japan Society for the Promotion of Science.
Dansk resumé:
De to studier havde til formål med PET at identificere cerebrale centre, der aktiveres ved specifikke auditive stimuli. Projekt I omfattede 5 forsøgspersoner, som passivt lyttede til et bredt spektrum af enkle lyde. Som forventet fandtes en generel aktivering af g. temporalis sup. bilateralt (primær hørecortex). Projekt II, der også omfattede 5 forsøgspersoner, indeholdt stimulering med kompliceret sammensatte lyde, der blev genereret m.h.p. at finde et akustisk tidsintegrationscenter. Der resulterede en aktivering af højre g. frontalis inf., som vi antager har indflydelse på opfattelsen af tidsfaktorer i akustiske stimuli. Samtidig fandtes udbredte aktiveringer i velkendte opmærksomhedsområder (insula, g.cinguli ant., lobul. parietalis inf. dxt. og g. frontalis medius dxt.). Figur 1-6 viser repræsentative udsnit af undersøgelsen.
Referencer:
1.Wyler AR, Ray MW, Brain and Language. 1986, 27: 195-98.
2.Worsley KJ, Evans AC, Marret S, Neelin P. J Cereb Blood Flow and Metab. 1992, 12:900-18.
3.Mesulam MM, Ann Neurol. 1981, 10:309-325.
4.Corbetta M, Miezin FM, Dobmeyer S. et al. Science 1990, 248:1556-59.
5.Pugh KR, Shaywitz BA, Shaywitz SE, et al. NeuroImage. 1996, 4:159-173.
Mapping cerebral blood flow changes with Positron Emission Tomography (PET) in response to nociceptive input is a technique to obtain better understanding of the underlying neurophysiological mechanisms related to pain perception. A number of recent studies have described structures within the human forebrain which show increased blood flow during painful stimulation (e.g. Talbot et al. 1991; Jones et al. 1991; Coghill et al. 1994; Casey et al. 1994, 1996; Hsieh et al. 1995, 1996). Thus, there is substantial evidence to suggest that a distinct network of neurons participate in the processing of painful stimuli. We wished to use the PET technique (H215O) to evaluate the cerebral processing of painful stimuli during specific conditions and manipulations.
The aim of this first pain study at the PET Center in Aarhus was to determine if the primary somatosensory cortex (MI/SI) participated in discrimination between painful heat stimuli at different intensities. Furthermore, we wished to compare the brain activation pattern caused by painful tonic heat to the pattern caused by painful phasic heat. Ten healthy subjects participated. The subtraction of the maps of the low from the high tonic painful heat demonstrated significant activity in contralateral MI/SI cortex and ipsilateral cerebellum and subsignificant activity in contralateral secondary somatosensory cortex, anterior cingulate gyrus, thalamus, anterior insula and supplementary motor area. The subtraction of the maps of the low from the high phasic heat revealed a significant blood flow decrease in the contralateral middle temporal gyrus. Blood flow increases below statistical significance were noted in contralateral anterior cingulate and bilateral anterior insula, contralateral supplementary motor area, and ipsilateral putamen and cerebellum.
In conclusion, this study showed the existence of discrete activation sites which may subserve the perceptual distinction between painful phasic and tonic heat. The primary sensorimotor cortex is involved in this grading. The brain activation pattern caused by painful tonic heat was similar, but not identical to that caused by painful phasic heat.
We are presently initiating new PET studies to investigate the effect of other stimulus related factors and the modulation of neurogenic pain and allodynia. Moreover, brain mechanisms related to endogenous pain regulatory mechanisms like hypnosis and placebo-effects will be examined.
Acknowledgements:
MRC Denmark (12-1633, 12-1634). The study was supported by the Foundation for Neurology Research, Institute for Experimental Clinical Research, University of Aarhus and grants from the Danish (12-1633, 12-1634) and Canadian (SP-30) Medical Research Councils.
Dansk resumé:
Positron-Emission-Tomografi (PET) er for nærværende den bedste teknik til at studere ændringer i blodgennemstrømning som markør for neuronal aktivitet i den menneskelige hjerne. PET har været anvendt til at visualisere cerebral aktivitet udløst med eksperimentelle smertefulde påvirkninger hos raske forsøgspersoner (Talbot et al. 1991; Jones et al. 1991; Coghill et al. 1994; Casey et al. 1994, 1996; Derbyshire et al. 1994). En samlet gennemgang af disse undersøgelser antyder, at et netværk af strukturer bliver aktiveret ved smerte: SI, SII, insula, thalamus og anterior cingulus.
Vi har ønsket at beskrive den cerebrale aktivitet udløst af forskellige smertefulde påvirkninger for at opnå en bedre forståelse af den basale neurofysiologi, der ligger til grund for “"smerteopfattelse”". I den første undersøgelse blev der anvendt to niveauer af smertefulde varmepåvirkninger på 10 raske forsøgspersoner. PET-undersøgelsen viste, at SI og cerebellum blev aktiveret ved denne forskel. Endvidere kunne der i en række andre strukturer observeres aktivitet lige under grænsen for signifikans. Vi konkluderede, at SI indgår i det cerebrale netværk, der er aktiv ved diskrimination af forskellige smertefulde påvirkninger.
Det er fortsat nødvendigt at identificere stimulus-relaterede forhold, der indvirker på det cerebral aktiveringsmønster. Dernæst vil det være muligt at beskrive mere komplekse smertefysiologiske processer såsom endogen modulation af neurogene smerter og allodyni samt indvirkning af psykologiske faktorer på smerteopfattelsen.
Referencer:
Svensson P, Johannsen P, Jensen TS, ArendtNielsen L, Nielsen J, Jørgensen HS, Gee AD, Baarsgaard SH, Gjedde A. Cerebral representation of graded painful phasic and tonic heat in man: a positron emission tomography study. In: Jensen TS et al. (eds). Precedings of 8th World Congres on Pain. IASP Press (in press).
Svensson P, Johannsen P, Jensen TS, ArendtNielsen L, Nielsen J, Stødkilde H, Hansen S, Gee T, Gjedde A. Cerebral processing of graded phasic and tonic noxious heat stimuli in man: a positron emission tomography study. IASP, Vancouver 1996.
On the basis of the acetylcholin hypothesis, i.e the claiming that cognitive deficits in Alzheimer's disease primarily relate to cholinergic deficits, the dementia project was initiated to develop a PET test of cognitive activation as a basis for future pharmacological PET activation studies of cognitive function in Alzheimer's disease.
Sustained and divided attention to two different sensory stimulations (red/black reversing checkerboard and vibrotactile stimulation) were chosen as the cognitive tasks. The study included 16 healthy elderly volunteers and 16 volunteers with Alzheimer's disease. During scanning the subjects were to attend to and detect an expected, but non-occurring change in frequency of one stimulus (sustained attention task) or two stimuli (divided attention task). Five 3D PE-tomograms of regional cerebral blood flow (rCBF) were acquired in single 40 s frames. PET volumes were aligned to the subject's MR brain image and to Talairach co-ordinates. After normalization, the rest conditions were subtracted from the activated conditions and a voxel-by-voxel t-statistic map was calculated.
The control subjects (mean age 63.7; range 51- 73 years) were scanned during 1995 while the data analysis was completed during spring 1996 (1,2,4). The results showed that the attention tasks significantly activated the right inferior parietal lobule (Brodmann area 40) and the right middle frontal gyrus (Brodmann area 45) (Fig. 1), while the anterior cingulate gyrus was sub-significantly activated. A simple sustained attention task more strongly activated the parietal area while a divided attention situation favored the frontal sites. The results supports the hypothesis of Mesulam (Ann. Neurol. 1981; 10:309-325) of a distributed network participating in attention functions in which the parietal attention site is a "poly-modal"”attention site incorporating sensory input from several different sensory modalities.
During 1995 and 1996, 16 patients with Alzheimer's disease were included in the study after a thorough dementia work-up (3) establishing the diagnosis according to ICD-10 criteria. The mean age of the patients was 67.9 (range 55- 75 years) with a mean “"Mini-Mental" State Examination score of 19.2 (range 10 -24).
Using the non-quantitative non-dynamic PET data a statistical comparison of the rest conditions was made between patients and age-matched control subjects. This evaluation revealed significant rCBF deficits in the patients in the temporo-parietal region, most prominently on the left side, extending caudally and medially to the left inferior longitudinal fascicle in the direction of the left hippocampus. A significant reduction was also seen in the right middle frontal gyrus while on the left side the reduction was sub-significant (Fig. 2). The sites of rCBF reductions suggest a functional connectivity similar to the one previously described in non-human primates and is an indirect evidence of the same cortical connections in the human brain (5,6).
The activation analysis of the patients performing the attention task has not yet been completed, but preliminary results shows a different activation pattern in the patients. In the tasks with simple sensory stimuli without attention, the patients significantly activated medial frontal structures. These areas are not activated in the healthy control subjects. This indicates, that Alzheimer patients exposed to simple sensory stimuli recruit alternative neural pathways to process the simple input to the brain (7).
Based on the technical experiences from the dementia project a methodological activation study has been carried out to optimize scanning parameters (injected dose and data acquisition in relation to the activation task) in the activation studies (8,9).
Figure 1. Attention sites in healthy subjects: right inferior pariental lobule (to the left) and right middel frontal gyrus.
Figure 2. Brain areas with significant reduction of cerebral blood flow in Alzheimer's disease.
Acknowledgement:
Grant support MRC, Denmark: 12-1633 and 12-1634.
Dansk resumé:
Demens-PET-aktiveringsundersøgelsen bygger på den kolinerge hypotese, der postulerer, at kognitive deficit ved Alzheimers sygdom primært skyldes kolinerg dysfunktion. Seksten Alzheimer patienter og 16 aldersmatchende raske kontrolpersoner blev PET skannet, mens de udførte forskellige opmærksomhedsopgaver. Der er undersøgt to forskellige typer opmærksomhed v.h.a. to forskellige sensoriske stimulationer; visuel stimulation og vibration af højre hånd. Analysen af data fra de raske personer er afsluttet i 1996 og viser signifikant aktivering af et område i højre isse og pandelap (Fig. 1). Dette resultat støtter en hypotese fremsat af M. Mesulam om opmærksomhedsfunktionernes organisation i hjernen, og kaster nyt lys på hjernens bearbejdning af forskellige typer af opmærksomhed. En statistisk sammenligning af ændringer i hjernens blodgennemstrømning (rCBF) mellem raske og syge viste signifikant nedsat rCBF i isse- og pandelapperne (Fig. 2). Analysen af Alzheimer patienternes aktiveringsmønster ved opmærksomhedsopgaverne er endnu ikke afsluttet. En foreløbig analyse af patienternes hjerneaktivering ved stimulation uden opmærksomhed viser signifikant aktivering af de midterste dele af pandelappen. Dette ses ikke hos raske og tyder på at selv simple opgaver giver anledning til aktivering af et alternativt kompenserende netværk af nerveceller hos patienter med Alzheimers sygdom.
Referencer:
Johannsen P, Jakobsen J, Bruhn P, Hansen SB, Gee A, Gjedde A. Attention on different sensory modalities weights frontal and parietal activations differently in elderly normal controls measured with PET. NeuroImage 1996; 3: S186.
Johannsen P, Jakobsen J, Bruhn P, Hansen SB, Gee A, Gjedde A. Attention on different sensory modalities weights frontal and parietal activations differently in elderly normal controls measured with PET. Annual meeting of the Danish Neurological Society, April 1996. Acta Neurol. Scand.1996; In Press. 3rd price in the“"Mogens Fog lecture competition"”at the Annual Meeting of Danish Neurological Society, April 1996
Johannsen P, Ehlers L, Abelskov KE, Gulmann NC, Christensen T, Gjedde A, Jakobsen J. Klinik for demensudredning- 110 konsekutive patienter. Ugskr Læger 1997, 159: 1246-1251.
Johannsen P, Jakobsen J, Bruhn P, Hansen SB, Gee A, Stødkilde-Jørgensen H, Gjedde A. Cortical sites of sustained and divided attention normal elderly humans. Submitted for NeuroImage. January 1997.
Johannsen P, Jakobsen J, Gjedde A. Relative CBF deficits in Alzheimer's disease evaluated statistically. 3rd International Conference on Functional Mapping Human Brain, Copenhagen May 19-23, 1997.
Johannsen P, Jakobsen J, Gjedde A. A statistical evaluation of flow deficits in Alzheimer patients using PET. BRAIN97, Baltimore, Maryland. June 15-19 1997. Submitted
Johannsen P, Jakobsen J, Bruhn P, Gjedde A. Sustained and divided attention sites in Alzheimer's disease. 3rd International Conference on Functional Mapping Human Brain, Copenhagen May 19-23, 1997.
Johannsen P, Hansen SB, Østergaard L, Gjedde A. Dose and data acquisition optimization in 3D O-15-water activation studies using the ECAT Exact HR-47 positron emission tomograph. 3rd International Conference on Functional Mapping Human Brain, Copenhagen May 19-23, 1997.
Johannsen P, Hansen SB, Østergaard L, Gjedde A. Optimization of H215O dose and data acquisition in 3D activation studies using an Ecat Exact HR PET camera. BRAINPET97. Bethesda, Maryland, June 20-22 1997.
The purpose of the study of cerebral blood flow in patients with major depression is to integrate clinical and neuropsychological examinations with PET measurements, to understand brain function in patients with affective disorder. The hypothesis is, that an abnormal function in the loop between the prefrontal cortex and the basal ganglia can account for some of the symptoms of the disorder, including the psychomotor retardation and reduced cognitive function, whereas other symptoms such as anxiety are associated with abnormalities in the limbic system.
The patients were selected such that they fulfill the DSM-IIIR criteria of major depression and have a score on the Hamilton depression scale at least greater than 17. They are compared to controls matched for age, sex and education. We expect to examine 50 patients and 50 controls. So far 34 examinations (17 patients and 17 controls) have been succesfully completed in 1996. Further recruitment of subjects for the project is planned in 1997.
The project is carried out in co-operation with the Department of Biological Psychiatry, The Psychiatric Hospital in Århus (chief Professor Raben Rosenberg) by dr. Poul Videbech and neuropsychologist Barbara Ravnkilde.
Acknowledgements:
MRC Denmark (12-1633 and 12-1634)
Dansk resumé:
Formålet med projektet "Hjernens blodgennemstrømning hos deprimerede patienter"”er at integrere kliniske og neuropsykologiske undersøgelser med PET-scanninger for at studere hjernefunktionen hos patienter med affektiv sygdom. Hypotesen er, at abnorm funktion i nervebanerne mellem præfrontal cortex og basal ganglierne kan betinge til visse af symptomerne ved sygdommen- specielt psykomotorisk hæmning og reduceret kognitiv funktion, hvorimod andre symptomer såsom angst er associerede med abnormiteter i det limbiske system.
Patienterne er udvalgt, således at de opfylder DSM-IIIR-kriterierne for major depression, og har mindst en score på Hamilton-depressionsskalaen på 17. De sammenlignes med kontroller, der matchede for alder, køn og uddannelse. Vi håber på at kunne komme til at undersøge 50 patienter og 50 kontroller. Indtil videre er der udført 34 undersøgelser (17 patienter og 17 kontroller). Vi regner med fortsat at rekruttere patienter og kontroller til studiet i 1997.
Projektet er et samarbejde med Afdeling for Biologisk Psykiatri, Psykiatrisk Hospital i Aarhus (leder: Professor Raben Rosenberg) og dr. Poul Videbech og cand.psyk. Barbara Ravnkilde.
Referencer:
P Videbech: MR findings in Patients with Affective Disorder. A metaanalysis. Acta Psychiatr Scand 1997, accepted for publication.
In 1996, the PET Center had three ongoing imaging research projects for graduate studies. The purpose of M.Sc. Flemming Andersen's graduate study project is to test the hypothesis that it is possible to create and visualize a digital 3D atlas of the localization of receptor systems in healthy and diseased brains in vivo, in order to enable the correlation and integration of neuroanatomical and functional information about the brain.
The graduate study of M.Sc. Anders Bertil Rodell aims to improve the planning and simulation of skull and brain surgery by means of 3D computer graphics, based on the invention of new computer based tools. Pre-visualization of the image gives these tools the power to survey and simulate the surgical task on a computer screen.
The development in 1996 was targeted at the creation of a framework which will be the basic for further development of visualization and analysis applications. The framework uses a shared-memorybased interprocess-communication system for accessing and sharing of image, segments and color information and controlling different applications. This is embedded in an image server WALNUT, shared resources is managed and delegated to the clients. This is done without user interaction, and the different clients behave as one multipurpose application but with better performance and ease of use.
The main client application so far is WROI (see figure on next page); a 2D image analysis program with basic segmentation features such as ROI's, isocontour and thresholding on MR, PET and CT, histogram analysis and image fusion. Other clients are an advanced studybrowser (filebrowser) and a 3D visualisation program in progress.
Analyses of PET data also implies modelling of time activity curves measured in areas of the brain. Since April 96 Thomas Sølling has been working on developing a user friendly program that can use the most common models for analyses. This work has been extended to a joint venture project with Johns Hopkins Hospital in USA, where Thomas Sølling has been studying different techniques for modelling binding potential. The purpose of the modelling of the measured time activity curves for regions of interest in the brain is to gain knowledge of the rate constants which makes it possible to describe the kinetics of the brain. These rates constants opens the possibility for diagnosing diseases in the brain but also to discover different compounds influence on the activity of the brain.
Acknowledgements:
Flemming Andersens graduate study is supported by The Medical Research Council program grant for neurological studies to Aarhus General Hospital.
Anders Rodells graduate study is supported by a collaboration between Aarhus University Hospitals, Aalborg University, and Aalborg General Hospital.
Dansk resumé:
PET Centret har i 1996 har 3 forskningsprojekter indenfor billedbehandling og -analyse. M.Sc. Flemming Andersens projekt er en test af den hypotese at det er muligt at opbygge og visualisere digitale 3D atlas af receptor systemer for den syge og raske hjerne in vivo, og derved integrere og visualisere neuroanatomisk og funktionel information. M.Sc. Anders Bertil Rodell udvikler et system til forbedring af planlægning og simulering af kranie og hjernekirurgi ved brug af 3D computergrafik. Dette sker ved udvikling af nye teknikker og programmer.
I 1996 er der blevet udviklet en fælles basis for udvikling af analyseprogrammer til brug i forskellige projekter. Dette er baseret på en server applikation WALNUT, som via shared memory interprocess-kommunikation giver klienterne mulighed for at dele ressourcer. Herved opnås at forskellige applikationer fremstår samlet, hvilket sker transparent for brugerne, som kan drage fordel af bedre ydeevne og standardiserede værktøjer.
Til aftestning af konceptet er udviklet client applikationen WROI (se figur 1); et 2D billedbehandlingsprogram til segmentering (ROI'er, isokonturer, thresholding mm.), histogramanalyse og sammenlægning af forskellige billedmodaliteter.
Analyse af PET-data indbefatter blandt andet modellering af aktivitetskurver målt i områder af hjernen. Siden april 1996 har M.Sc. Thomas Sølling været beskæftiget med udvikling af et brugervenligt program, som kan benytte de mest anvendte modeller til analyse. Arbejdet er udvidet til et joint venture projekt med Johns Hopkins Hospital i USA, hvor Thomas Sølling har været i sammenlagt fem måneder i 1996, hvor han har studeret forskellige modelleringsteknikker. Formålet med modellering af aktivitetskurver for hjernen er at få et større kendskab til konstanter, hvorved hjernens kinetik kan beskrives. Disse konstanter kan give mulighed for bl.a. diagnosticering af sygdomme samt måle forskellige stoffers indvirken på hjernens aktivitet.
Figur 1: Viser et eksempel på brug af applikationen WROI til segmentering af PET, MR og CT billeder.
Monoamines (dopamine, noradrenaline and serotonin) are neuro-transmitters that have been implicated in several neuropsychiatric disorders such as depression, anxiety, schizophrenia and drug addiction. This project aims at developing new radiotracers for use in studying monoaminergic processes by PET in the living brain.
Basic research on dopaminergic drugs carried out at the PET Center during 1996 included studies with 11C-amphetamine analogs1,2 and a 11C-dopamine transporter ligands. The mechanism of action of amphetamine and cocaine in the CNS is generally attributable to their interactions with dopaminergic neurotransmission. It is now well established that cocaine exerts its main effect by the blockade of presynaptic dopamine transporters/reuptake sites (DAT's) resulting in elevated dopamine (DA) concentrations in the synapse. In contrast, consensus as to the mechanism of action of amphetamine (AMP) derivatives on dopaminergic neurons is more unclear. Proposed mechanisms of action include: blockade of catecholamine uptake sites, stimulation of dopamine release from storage vesicles into the cytoplasm by alkalinisation of storage vesicles, inhibition of monoamine oxidase (MAO) activity, and exchange diffusion at the DAT (ie release of DA from the DA transporter). Of the proposed mechanisms of action, the exchange diffusion theory3 seems to be the most consistent with reported experimental findings. The theory states that AMP induced DA release is due to a reversal of the normal direction of DA transport via the DAT. AMP binds to the DAT extracellularly and is transported across the cell membrane where AMP is released. The transporter protein subsequently binds to intracellular DA which is then transported across the cell membrane to the extracellular space.
According to the above theory, cocaine and amphetamine act at the same site on the transporter. The fact that DAT blockers abolish amphetamine induced DA release is compelling evidence in favor of this theory. However, preliminary studies in our laboratory, using the non-invasive in vivo autoradiographic technique Positron Emission Tomography (PET), have indicated that amphetamine and cocaine analogs bind at two different sites. We wished to determine whether 1) methamphetamine (MAMP) exhibited saturable binding in the striatum and 2) if methamphetamine and cocaine analogs bind to the same site on the dopamine transporter. Methamphetamine labelled with the positron emitting radionuclide 11C (t1/2 20 min) was displaced from pig striatum by 0.5 mg/kg non-radioactive methamphetamine. Scatchard analysis showed a BMAX of 6 nmol/ml and a Kd of 10 nmol/ml for methamphetamine. In contrast, striatial 11C-methamphetamine binding was not displaceable by 0.5 mg/kg of the potent dopamine reuptake blocker WIN 35-428. In crossover experiments using the labelled cocaine congener 11C-X, specific striatal 11C-X binding was markedly reduced by 0.5 mg/kg WIN 35-428 but not 0.5 mg/kg methamphetamine. These experiments indicate that 1) methamphetamine acts at a different striatal binding site than cocaine, and 2) methamphetamine binding is saturable. Work is in progress to further characterise the nature of the "amphetamine receptor"’.
Basic research on serotonergic receptors carried out at the PET Center during 1996 focussed mainly on the relatively fast-acting antidepressant drug venlafaxine. The brain binding kinetics and distribution of venlafaxine,4 labelled with 11C in the O-methyl position,5 was studied by PET after intravenous injection in anesthetized pigs. In addition, venlafaxine's action on serotonin (5-HT) uptake was studied in vitro in blood platelets obtain from humans or pigs. Venlafaxine resembled imipramine, paroxetine and citalopram in causing a dose-dependent inhibition of 5-HT uptake in blood platelets from pigs and humans. Venlafaxine-derived radioactivity entered the living brain readily and showed higher binding potentials in diencephalic and telecephalic regions than in cerebellum. Acute administration of an antidepressant drug (i.e. imipramine, citalopram or paroxetine) enhanced the distribution and altered the binding of venlafaxine in certain brain regions. The findings show that [11C]venlafaxine has a relatively low binding potential and lacks of specificity for the 5-HT transporter in living brain. It may, however, be of value for estimating the cerebral level of venlafaxine in patients treated with the antidepressant drug.
Acknowledgements:
This work was supported by "Dr.med.vet. Axel Thomsen og hustru Martha Thomsen, født Haugen-Johansens legat", "Hjerne-Sagen -Landsforeningen for Apopleksi- og Afasiramte"”, and The Institute of Experimental clinical Research at Aarhus University.
Dansk resumé:
Monoaminer er hormoner der frigøres fra nervecellerne og påvirker de omkringliggende celler. Forstyrrelser i frigørelsen af monoaminer synes at bidrage til forekomsten af mange hjernelidelser, såsom depression, angst, skizofreni og stofmisbrug. Vi har udført en serie undersøgelser af monoaminfrigørelse v.h.a. PET skanning af hjernen hos grise. Noget af vores arbejde drejer sig om virkningen af misbrugsstofferne kokain og amfetamins påvirkning af dopaminsystemet i hjernen. Disse stoffer synes at interferere med overførelsen af dopamin via cellemembranen. Vi ønsker at kunne afgøre præcist hvilke membranprocesser der er ansvarlige for disse forandringer.
Vi har også undersøgt graden hvorpå det antidepressive stof venlafaxine (Trade name Effexor) virker via frigørelsesområder for serotonin i hjernen. Målet med disse undersøgelser er, at udvikle holdbare metoder, for at afgøre hvorvidt serotoninsystemet er i uorden hos patienter med psykiatriske og/eller neurologiske lidelser. Dette forventes at kunne anvendes som en hjælp til diagnosticering af grundlaget for psykiske lidelser for bedre at kunne vælge den rigtige behandling.
Referencer:
1. Gee A.D., Gillings N., Smith D., Inoue O., Kobayashi K. and Gjedde A.: ‘The effect of amine pKa on the Transport and Binding of Amphetamine Analogs in the Pig Brain’: In ‘Quantification of Brain Function Using PET’, Academic Press (1996), 42-46.
2. Gillings N.M and Gee A.D. ‘-The Synthesis of ß,ß-Difluoro[N-methyl-11C]methamphetamine for the Investigation of the Transport and Binding of Biogenic Amines’ -J. Labelled. Compd. Radiopharm, 35: 142-143.
3. Fischer J.F. and Cho A.K. ‘-Chemical Release of Dopamine from Striatal Homogenates: Evidence for an Exchange Diffusion Model’ -J.Pharmacol.Exp.Ther. 192, 642-653 (1979).
4. Smith D.F., Gee A.D., Danielsen E., Hansen S.B. and Gjedde A. ‘Binding of [O-methyl-11C]Venlafaxine in Living Pig Brain: Weak Displacement by Serotonin Reuptake Inhibitors’ -Nucl. Med. Biol. -Accepted
5. Gee A.D., Smith D.F. and Gjedde A. ‘The Synthesis of [O-methyl-11C]Venlafaxine: A Non-Classical, Fast-Acting Antidepressant’ -J. Labelled. Compd. Radiopharm., In press.
The dopamine theory of schizophrenia holds that dopamine release and binding in the striatum, and particularly the ventral striatum, is elevated in this disorder, perhaps because of impaired innervation from the frontal and prefrontal cortex. Increased release and binding of dopamine would result in reduced ability of a neuroleptic to block dopamine receptors, as expressed in the drugs half-inhibition concentration.
The half-inhibition concentration (IC50 ) of a drug indicates its ability to inhibit the binding of a ligand to a receptor. We determined the apparent IC50 of the neuroleptic haloperidol towards the binding of tracer N-methylspiperone (NMSP) in caudate nucleus of healthy volunteers, patients with bipolar disease without psychotic episodes, patients with bipolar disease with a propensity for psychosis, and patients with schizophrenia. In patients with schizophrenia or psychotic propensity, the apparent IC50 of haloperidol was significantly elevated when compared to the apparent IC50 of haloperidol in healthy volunteers and nonpsychotic patients with bipolar disease. The increase may be due to increased plasma protein binding of the inhibitor, lower affinity of the neuroreceptors towards the inhibitor, or increased binding of unmeasured exo- or endogenous competitors of the same receptors:
Table 1: Average IC50 of Haloperidol in Psychosis SEM
| Diagnosis | State | Group | pl-[Haloperidol] [nM] | IC50 [nM] |
| Healthy | Control | I (24) | 6.8•±0.6 | 1.7•±0.5l |
| Bipolar Illness | Nonpsychotic Psychotic | II (7) III (7) | •7.6•±1.8 13.3•±3.9 | 1.0•±0.2 5.8•±3.7 |
| Schizophrenia | Drug-Naive Drug-Treated | IV (22) V (5) | 15.3•±3.7 26.1•±16.2 | 4.0•±0.6 4.6•±0.9 |
The association of the higher IC50 values with the presence of psychosis across different disease entities predicted a four-fold increased binding of dopamine. This predicted increase of dopamine binding in turn implied that the patients with psychotic propensity had the same dopamine binding after haloperidol blockade as the non-psychotic subjects before the haloperidol blockade, i.e., 5 pmol/cm3.
Table 2: Predicted Neurotransmitter Binding in Psychosis
| Groups | Trait | [DA] Ratio | Bmax [pmol/cm3] | SDA [%] | BDA [pmol/cm3] | SI [%] | SDA(I) [%] | BDA(I) [pmol/cm3] |
| I+II | No Psychosis | 0.33 | 19 | 25 | 5 | 83 | 5 | 1 |
| III+IV | Psychosis (Unmedicated) | 2.75 | 29 | 73 | 21 | 77 | 17 | 5 |
| V | Schizophrenia (Medicated) | 2.83 | 37 | 74 | 27 | 85 | 11 | 4 |
Bmax(40) is the maximum binding capacity at age 40 reported by Wong et al. (1997a,b). BDA(40) is the predicted dopamine binding at age 40, C and S its concentration and saturation. BDA(HAL)(40) is the predicted dopamine binding after neuroleptic blockade at age 40.
The prediction that patients with a propensity for psychosis may suffer from excessive dopamine release was tested in normal volunteers administered amphetamine, a competitor of dopamine transport which elicits symptoms reminiscent of psychosis. The healthy volunteers were given amphetamine and the binding of the radioligand raclopride, another competitor of dopamine binding to D2-like receptors, was determined before and after the ingestion of amphetamine. The density of the dopamine receptors was calculated by comparing the radioligand binding at high and low specific activity. At both high and low specific activity of the radioligand, amphetamine elicited a 70% surge of the predicted binding of endogenous dopamine, as shown in the accompanying raclopride binding curves:
By different methods, Laruelle et al. (1996) have shown, that patients with schizophrenia have significantly greater release of an endogenous competitor of raclopride binding than do normal individuals. The next phase of the current project is devoted to the description of the time course of the dopamine surge in normal individuals and patients with a propensity for psychosis.
Acknowledgements:
Supported by MRC Denmark (12-1633, 12-1634), MRC Canada (SP-30), US PHS (MH42821, DA09482), and NARSAD
Dansk resumé:
Sygdommen skizofreni forklares hyppigst med den såkaldte dopaminsyntese, ifølge hvilken en nedsættelse af aktiviteten i forpandelappen (præfrontalcortex) medfører en kompensatorisk forøgelse af aktiviteten i hjernens basale ganglier. De basale ganglier i hjernen menes at være ansvarlige for hjernens følsomhed for ydre og indre påvirkninger. Når denne følsomhed er forøget kan ufrivillige bevægelser og psykoser opstå på grund af manglende hæmning. Det første stadium i skizofreniens udvikling kan derfor være passivitet, som med tiden overlejres af en tendens til psykoser. I samarbejde med laboratorier i Canada og USA har vi vist, at visse narkotika, som virker psykosefremkaldende, netop forøger omsætningen, frigørelsen og bindingen af signalstoffet dopamin i hjernens basale ganglier.
Referencer:
Gjedde A, Leger G, Cumming P, Yasuhara Y, Evans AC, Guttman M, Kuwabara H (1993) Striatal L-DOPA decarboxylase activity in Parkinson's disease in vivo: Implications for the regulation of dopamine synthesis. J Neurochem 61: 1538-1541.
Cumming P, Kuwabara H, Gjedde A (1994) Kinetic Analysis of 6-[18 F]fluoro-L-dihydroxy-phenylalanine ([18F]DOPA) metabolism in the rat. J Neurochem 63: 1675-1682.
Cumming P, Venkatachalam TK, Rajagopal S, Diksic M, Gjedde A (1994) Kinetics of the cerebral uptake of alpha[14C]methyl-para-tyrosine in the rat. Synapse 17: 125-128.
Reith J, Benkelfat C, Sherwin A, Yasuhara Y, Kuwabara H, Andermann F, Bachneff S, Cumming P, Diksic M, Dyve S, Etienne P, Evans A, Lal S, Shevell M, Savard G, Wong D, Chouinard G, Gjedde A (1994) Elevated dopa decarboxylase activity in living brain of patients with psychosis. Proc Nat Acad Sci USA 91: 11651-11654.
Cumming P, Ase A, Diksic M, Harrison J, Kuwabara H, Laliberte C, Gjedde A (1995) Metabolism and bloodbrain clearance of L-3,4-dihydroxy-[3H]phenylalanine ([3H]DOPA) and 6-[18F]fluoro-L-DOPA in the rat. Biochem Pharmacol 50: 943-946.
Cumming P, Kuwabara H, Ase A, Gjedde A (1995) Regulation of DOPA decarboxylase activity in brain of living rat. J Neurochem 65: 1381-1390.
Gjedde A, Reith J, Wong DW (1995) Dopamine receptors in schizophrenia. Lancet 346: 1302-1303.
Kuwabara H, Cumming P, Yasuhara Y, Leger G, Guttman M, Diksic M, Evans AC, Gjedde A (1995) Regional striatal DOPA transport and decarboxylation activity in Parkinson's disease. J Nucl Med 36: 1226-1231.
Wong DF, Harris JC, Naidu S, Yokoi F, Marenco S, Dannals RF, Ravert HT, Yaster M, Evans A, Rousset O, Bryan RN, Gjedde A, Kuhar MJ, Breese GR (1996) Dopamine Transporters are Markedly Reduced in Lesch-Nyhan Disease in vivo. Proceedings of the National Academy of Sciences of the United States of America. 93: 5539-5543.
Gjedde A (1996) DOPA Decarboxylase. Movement Disorders. 11: 462-463.
Gjedde A, Reith J, Wong DF (1996) In Schizophrenia, some Dopamine D2-Like Receptors are still Elevated. Psychiatry Research: Neuroimaging 67: 159-161.
Wong DF, Young D, Wilson D, Meltzer CC, Gjedde A (1997a) Quantification of neuroreceptors in living human brain. III. D2-like dopamine receptors; theory, validation, and changes during normal aging. J Cereb Blood Flow Metab 17: 316-330.
Wong DF, Perlson GD, Tune LE, Young LT, Meltzer CC, Dannals RF, Ravert HT, Reith J, Kuhar MJ, Gjedde A (1997b) Quantification of neuroreceptors in living human brain. IV. Effect of aging on density elevations of D2-like receptors in schizophrenia and bipolar illness. J Cereb Blood Flow 17: 331-342.
At the PET Center at Aarhus University Hospitals an inquiry into the dopamine metabolism is taking place. The studies are focused on how the dopamine synthesis is regulated in healthy subjects and in patients with Parkinson's disease. An animal model has been developed for experimental investigations of the dopamine metabolism by PET.
In collaboration with the Doctors Poul Mogensen and Erik Dupont of the Dept. of Neurology, three groups of subjects are being investigated. One group is healthy. One group consists of patients with Parkinson's disease treated with L-DOPA for several years, and one group consists of untreated newly diagnosed patients with Parkinson's disease. In all groups, the regulation of the dopamine metabolism determined by PET with and without the influence of L-DOPA treatment.
Preliminary results will be presented at the BRAIN 97 XVIIIth International symposium on Cerebral Blood Flow and Metabolism, Baltimore Maryland USA in June 1997. These results shows no short term effect of the standard treatment with L-DOPA on the net influx rate constant ki in controls subject and patients with Parkinson's Disease in any region of the brain involved in dopamine production and hence rules out short-term up- or down-regulation of DOPA decarboxylase activity. The substantia nigra activity was unaffected by the presence of the disease. Ongoing analysis of data is focused on DOPA transport across the blood-brain-barrier, DOPA decarboxylase activity and storage of DOPA in the neurons.
Figure 1: The image shows the uptake of 18F-DOPA in the brain, horizontal section. On the color scale red indicates high activity and deep purple low activity. The normal uptake is shown to the right sidewith highest uptake in putamen and nu. Caudatus. In a case of Parkinson's disease to the left the uptake is reduced 85% in putamen and 30% in nu.Caudatus.
Figure 2: PET image showing uptake of 18F-DOPA in the young pig brain, coronal section. Maximum activity is seen in the striatum.
At the PET Center, we have established an animal model for determining 18 F-DOPA metabolism in the brain of young Yorkshire pigs and adult so-called minipigs. The results show that anesthetized pigs are appropriate experimental animals for PET imaging of DOPA decarboxylation in the living brain. The striatal DOPA decarboxylation rate was found to range between those of rats and humans. The results indicate, that the partial volume effect in the striatum is directly related to the size of the region. The results will be presented at BRAIN-PET-97, Washington, USA in June 1997. Determination of the DOPA metabolism in young pigs is finished and will be related to results from adult minipigs. These results will form the standard for future studies at the PET Center.
Future:
Animal research will focus on the regulatory effect of dopamine on DOPA decarboxylase and the effect of autoreceptors. In collaboration with Dr Peter Høst Poulsen, traumainduced changes in dopamine metabolism will be investigated. In collaboration with scientists from Odense, København, Lund, Stockholm, Upsala and Cambridge, the PET-Center will investigate metabolic and immunological aspects of transplanted fetal CNS-tissue in Parkinson's disease, using pigmodels.
Acknowledgements:
This work has kindly been financially supported by grants from: The Danish Parkinson society (Dansk Parkinson forening), Aarhus University Research Foundation (Århus Universitets forskningsfond), The Danish Medical Association Research Fund (Lægeforeningens Forskningsfond/Wacherhausen), Novo Nordic Foundation (Novo Nordisk Fonden) and Institute of Experimental clinical Research at Aarhus University (Institut For Eksperimentel klinisk Forskning ved Århus Universitet).
Dansk resumé:
PET centret på Århus Universitetshospital fik i febuar 1996 tilladelse fra Sundhedsstyrelsen til anvendelse af traceren 18F-DOPA til humant brug og kunne derefter indlede undersøgelser af dopaminmetabolismen ved anvendelse af positron-emissionstomografi. Studierne er fokuseret på regulationen af dopamindannelsen hos raske og hos patienter med Parkinsons sygdom. Ved de humane projekter undersøges i alt 3 grupper af personer, raske, personer i behandling for Parkinsons sygdom gennem nogle år, og personer med nyopdaget Parkinsons sygdom. Hos alle grupper undersøges dopamindannelsen med PET, dels før og dels efter medicinering med L-DOPA præparater. Disse undersøgelser er under fortsat afvikling. Præliminære resultater af disse undersøgelser vil blive præsenteret ved BRAIN 97, XVIII International Symposium on Cerebral Blood Flow and Metabolism, Baltimore Maryland USA juni 97.
På PET centret er desuden udviklet en dyreeksperimentel model med anvendelse af helt unge Yorkshire-grise samt voksne minigrise til undersøgelse af dopamindannelsen i hjernen. Resultater fra PET centret har vist, at dyremodellen er anvendelig til bestemmelse af 18 F-DOPAs optagelse og omsætning i hjernen. Forsøgsmodellen er sammenlignelig med andre dyremodeller for dopamins metabolisme, som anvender nonhumane primater og gnavere. Bestemmelse af den basale dopaminmetabolisme hos ungdyr er afsluttet og vil blive sammenholdt med resultaterne fra igangværende undersøgelser på voksne dyr. Disse resultater danner grundlag for fremtidige dyreeksperimentelle undersøgelser på PET centret. Resultater fra disse undersøgelser vil også blive præsenteret ved BRAIN-PET-97, Washington, USA. Juni 1997.
Fremtidige undersøgelser vil i første omgang fokusere på hvilke faktorer der kan regulere aktiviteten af enzymet DOPA-decarboxylase og dermed dannelsen af dopamin. I et etableret samarbejde med forskningsgrupper i Odense, København, Lund, Stockholm, Upsala og Cambridge deltager PET-Centret i undersøgelse af metaboliske og immumologiske mekanismer ved transplantation af føtalt CNS væv til behandling af Parkinsons sygdom. Disse undersøgelser vil blive afviklet inden for de næste år.
Referencer:
E.H.Danielsen, D.F.Smith, A.D.Gee, T.K. Venkatachalam, S.B.Hansen, A.Gjedde.1997” The metabolism of 18 F]-DOPA in pigs estimated by PET.” abstrakt til BRAIN-PET-97, Washington, USA. Juni 1997.
E.H. Danielsen, D.F. Smith, D. Bender, A.D. Gee, S.B. Hansen, A. Gjedde.1997 ”No up or down regulation of DOPA decarboxylase act. in short term DOPA treatment of normal volunteers and DOPA holiday in Parkinson's Disease.”Abstrakt til BRAIN 97 XVIIIth International symposium on Cerebral Blood Flow and Metabolism, Baltimore Mayland USA juni 1997.
This project is carried out in collaboration between the Montreal Neurological Institute at McGill University and the PET and MR Centers at Aarhus University Hospitals. The purpose is to investigate the dynamic regulation of regional cerebral blood flow (rCBF) and cerebral metabolic rate for oxygen (CMRO2) during physiological stimulation, using PET and novel combinations of fMRI measures to produce quantitative maps of CMRO2. We intend to investigate the dynamic regulation of rCBF by infusion of agents that interact with nitricoxide (NO) release, or which cause a vascular affect independent of NO.
We will implement the novel fMRI based Oxidative Index (OI) (CMRO2/rCBF). We will use OI to measure the effect of visual stimulus frequency and duration on the balance between oxidative and glycolytic energy metabolism. To do so, we will implement and validate the relation between changes of OI and the change of the tissue content of deoxyhemoglobin. The latter is the basis for the blood oxygenation-level dependent (BOLD) fMRI signal, R delta OI= 1-[(1-R delta BOLD)/(1-Rdelta PERF) a] where R is the relative change of the measure, and a the power of the function that relates venous and capillary volume changes to changes of rCBF. Once validated against measurements with PET, this relation will allow CMRO2 changes which occur during steady-state visual and vibrotactile stimulation to be measured by fMRI. We will test the hypothesis, that OI is increased by sensory stimulation only when the concentration of lactate (which increases with stimulus load) exceeds a threshold. We will manipulate the stimulus (e.g., frequency of presentation) to modulate the relative activities of oxidative and glycolytic pathways. We predict, that sudden increases in stimulus load will raise the proportion of glycolytic ATP generation, while more gradual increases will raise oxidative ATP production, resulting in changes of the OI.
In an allied project, we intend to test the hypothesis, that the effect of NO derived from endothelium or neurons is strongly modulated by NO-synthase independent agents, acting on the vascular smooth muscle or on the guanylate cyclase dependent second messenger system. We will test this hypothesis by comparing changes of basal and activated rCBF induced by infusion of the endothelial NO precursor L-arginine with those induced by a vasodilator which acts independently of endothelial NO synthesis (nitroglycerin).
L-Arginine infusion raises basal but not activated rCBF, and the effect wears off within 15 minutes, despite persistent elevation of plasma L-arginine. We will use nitroglycerin to identify vascular (endothelial or smooth muscle) NO-synthase independent mechanisms responsible for this wearing-off. At least in part, neuronally derived NO mediates the blood flow increase associated with neuronal activation. Recent studies show, that neuronal activation may proceed without a coupled blood flow increase. This suggests, that the effect of neuronally derived NO may be abolished by NO synthase independent regulatory mechanisms. To investigate this possibility we will compare the effects of the NO-independent vasoconstrictor indomethacin on basal and activated rCBF and regional glucose consumption (CMRglc). In states of alleged supersensitivity (migraine) or subsensitivity (smokers) to NO, we will use L-arginine and nitroglycerin infusion to identify alterations in NO synthesis and sensitivity.
Physiological Variables. During the past phase of this grant, we produced the only existing statistical-parametric maps of transient changes of FDG-metabolism and CMRO2 in the living human brain. To do so, we refined the 18FDG double injection method for the measurement of FDG-metabolism in two physiological states within 50 min (Murase et al. 1996). We developed and tested `two-compartment’ methods for rapid (3 min) estimation of cerebral water clearance (an index of CBF) and CMRO2 by PET, introducing correction for non-extracted intravascular radioactivity (Ohta et al. 1992, Ohta et al. 1996). With this, we demonstrated spatially non-coincident rCBF and vascular (Vo) activation foci during vibrotactile stimulation (Fujita et al. 1997).
Oxidative Decoupling and Recoupling. We confirmed and extended the observations that, during acute vibrotactile or simple photic stimulation, there is decoupling of the changes of CBF and CMRglc from those of CMRO2. We found, that this decoupling persists for as long as 30 min (Gjedde 1993, Ribeiro et al. 1993). We also found that under different but very specific conditions of visual stimulation (monocular presentation of hemiannular reversing [4 and 8 Hz] checkerboard to left visual hemifield), there is a parallel (i.e. coupled) increase of CBF and CMRO2 but while the CBF increase is maximal at 8 Hz, the CMRO2 increase is maximal at 4 Hz. Marrett et al. (1993). We believe, that this stimulus selectively activates neurons with a high content of the enzyme cytochrome oxidase (so-called "blobs"’). This observation was confirmed using different stimulus presentations (circular yellow-blue checkerboard to both eyes). In general, we found that CBF changes exceeded CMRO2 changes, as also shown by their non-linear correlation found (Figure 1).
According to the present hypothesis, there may exist a gradual (coupled) increase of the Oxidative Index (OI) over time, due to accumulation of metabolites which raise oxidative metabolism, and that with further increases in stimulus load (e.g. reversal frequency) there is gradual decline in the ratio of oxidative to glycolytic ATP generation (decoupling).
Cerebral Blood Flow Deregulation. We showed that intravenous infusion of L-arginine, the precursor of nitric oxide (NO), increased basal but not activated regional CBF. The initial effect of L-arginine infusion on mean whole brain rCBF is likely to result from increased NO production by endothelial no synthase, due to reversal of tonic glutaminergic inhibition.
Activation-induced blood flow increases may therefore depend on the level of tonic glutamine inhibition of NO.
Acknowledgements:
Supported by MRC Canada (SP-30) and MRC Denmark (12-1633, 12-1634).
Dansk resumé:
Hjernen er afhængig af en uafbrudt forsyning med ilt. Uden ilt indtræder bevidstløshed på få sekunder. Alligevel kan hjernen tilsyneladende udføre arbejde uden at behøve mere ilt, trods betydelige stigninger af hjernens blodforsyning. Forklaringen på dette misforhold kan være, at energiforbruget ved udførelsen af hjernens arbejde varierer meget mellem de forskellige opgaver, så visse opgaver kan udføres uden tilførsel af mere ilt, mens andre opgaver kræver mere ilt. Blodforsyningen styres efter denne opfattelse af andre mekanismer end energiforbruget. Det nærværende studium har til formål at afdække de principper, hvorefter hjernens iltforbrug styres.
Referencer:
Ohta S, Meyer E, Thompson CJ and Gjedde A (1992) Oxygen consumption of the living human brain measured after a single inhalation of positron emitting oxygen. J Cereb Blood Flow Metab 12: 179-192.
Gjedde A, Kuwabara H (1993) Absent recruitment of capillaries in brain tissue recovering from stroke. Acta Neurochir 57: 35-40.
Gjedde A (1993) The energy cost of neuronal depolarization. In: Functional Organization of the Human Visual Cortex, eds. Gulyas B, Ottoson D, Roland PE, Pergamon Press, Oxford, pp. 291-306 [P432]
Marrett S, Fujita H, Meyer E, Ribeiro L, Evans AC, Kuwabara H, Gjedde A (1993) Stimulus specific increase of oxidative metabolism in human visual cortex. In: Quantification of Brain Function. Tracer Kinetics and Image Analysis in Brain PET (eds Uemura K, Lassen NA, Jones T, Kanno I), Excerpta Medica, Amsterdam, pp. 217-228.
Ribeiro L, Kuwabara H, Meyer E, Fujita H, Marrett S, Evans AC, Gjedde A (1993) Cerebral blood flow and metabolism during nonspecific bilateral visual stimulation in normal subjects. In: Quantification of Brain Function. Tracer Kinetics and Image Analysis in Brain PET (eds Uemura K, Lassen NA, Jones T, Kanno I), Excerpta Medica, Amsterdam, pp. 229-236.
Ohta S. Meyer E. Fujita H. Reutens DC. Evans A. Gjedde A. (1996) Cerebral [15O]water clearance in humans determined by PET. 1. Theory and normal values. J Cereb Blood Flow Metab 16: 765-780.
Gjedde A (1996). The relation between brain function and cerebral blood flow and metabolism. Chapter 2, Cerebrovascular Disease (ed Batjer HH). LippincottRaven, Philadelphia, pp. 23-40.
Fujita H, Meyer E, Reutens DC, Kuwabara H, Evans AC, Gjedde A (1997) Cerebral [15O]water clearance in humans determined by positron emission tomography. 2. Vascular responses to vibrotactile stimulation. J Cereb Blood Flow Metab, 17: 73-79.
Recent results indicate that it may be possible to measure cerebral blood flow (CBF) by dynamic magnetic resonance imaging (MRI) of susceptibility contrast changes (1) during paramagnetic contrast agent bolus passage (2). The technique has gained much attention, since it seems capable of demonstrating viable tissue after acute stroke, and hence provides a powerful tool in predicting outcome. In the past year, we have compared the NMR bolus technique with [15O]butanol PET CBF (3,4) in a pig hypercapnia model, as well with [15O]water PET CBF in humans. In the following is a brief summary of approach and findings. The work was performed in a collaboration between Dept. of Neuroradiology and PET Center, Århus University Hospitals.
The first study was designed to test whether CBF can be determined on a pixel-by-pixel basis using MR in states of high flow and blood volume. These physiological conditions are known to represent a challenge to the MR technique(2). Also, the studies should provide first insight into whether MR flow rates can be compared among subjects. (See human studies below).
Six female Danish country bred Yorkshire pigs weighing 40 kg were used in the experiments. The project was approved by the Danish National Committee for Ethics in Animal Research. Hypercapnia was induced by decreasing respiratory rate and tidal volume. MR Imaging was performed using a GE Signa 1.0 Tesla imager. Spin echo EPI was performed during bolus injection of Gadodiamide (OMNISCAN®) at a rate of 10-15 ml/sec. To determine tissue and arterial tracer time concentration curves, a linear relationship between paramagnetic contrast agent concentration and the change in transverse relaxation rate (R2 ) was assumed. The arterial input function was determined from pixels around a large vessel (3).
Figure 1: Regional CBF values determined with NMR (top) and PET (bottom) at normo- (left) and hypercapnia (right). Notice the good agreement between regional values and responses to PaCO2 (indicated for each image). The distinction between grey and white matter is superior in the NMR CBF maps, due to higher intrinsic resolution (2.2 mm vs. 4.5 mm in the PET acqusitions, same slice thickness). NMR CBF values were normalized to the injected dose.
Pixel concentration time curves were deconvolved with the arterial input using singular value decomposition (2), and the maximum height of the resulting tissue residue curve was assumed to be proportional to CBF. PET studies were performed in a Siemens ECAT EXACT HR PET scanner. PET data were co-registered with MR data, and then transformed and resampled to the spatial location and resolution of the MR data to allow direct comparison of the two techniques. Raw PET image data were then fitted to a novel 2-compartment model (5) using nonlinear, least squared regression analysis on a pixel-by-pixel basis. Figure 1 shows typical maps of CBF using MR and PET, respectively. There was in general good agreement between the two modalities. Also, measurements provided a model constant that can convert MR flow units into true flow rates (ml/100ml/min).
In the second study, we compared the NMR bolus technique with [15O]water PET CBF in humans. Given the model parameter determined above, we wished to compare absolute flow rates in various anatomical structures within and among subjects. Six volunteers (3M, 3F, aged 22-32 years) were examined with the PET and NMR protocol described above. Model constants are now fixed from animal experiments (See animal section above) to yield NMR CBF values in ml/100ml/min. Table 1 summarizes the mean CBF (ml/100ml/min) values for 6 regions of interest (ROI) in 3 of 6 subjects, with standard deviations derived from the population of pixels within the ROI.
Table 1
Subj. | Frontal PET MR | Temporal PET MR | Occip. PET MR | Basal Ganglia PET MR | Frontal White PET MR | Posterior White PET MR |
| # 1 | 39±9 42±4 | 37±7 44±6 | 36±7 38±5 | 48±10 43±3 | 32±6 34±3 | 17±6 19±2 |
| # 2 | 44±9 38±9 | 47±12 37±11 | 47±10 36±6 | 48±8 48±6 | 38±9 26±5 | 25±8 21±5 |
| # 3 | 32±4 43±9 | 30±6 29±6 | 28±5 23±6 | 27±5 29±7 | 17±4 16±5 | 16±5 12±2 |
There was generally good agreement of CBF determined with the two methods. The preliminary data thus indicate that absolute CBF values may be obtainable with NMR susceptibility contrast imaging of paramagnetic tracer bolus passages by normalizing the arterial input to the injected dose and using an universally determined constant of proportionality.
Acknowledgements:
This study was supported by Århus University Research Foundation, The Danish Medical Association Research Foundation (Højmosegårdlegatet), The Institute of Experimental Clinical Research, The Lippert Foundation, and The Michaelsen Foundation.
Dansk resumé:
En ny teknik til bestemmelse af hjernens gennemblødning v.h.a. MR-skanning har på det seneste vakt opsigt, idet metoden tilsyneladende kan demonstrere væv, som er "truet" ’hos patienter med blodprop i hjernen. Denne viden er af stor betydning for valg og senere monitorering af behandling hos denne store patientgruppe. Metoden er stadig under udvikling, og det seneste år er teknikken sammenlignet med PET, som er "reference-metoden"’ til bestemmelse af hjernens blodgennemstrømning. Resultaterne har vist god overensstemmelse mellem PET og MR målinger i både dyrestudier og de første forsøg på mennesker, og arbejdet fortsætter nu hen mod rutinemæssig anvendelse af teknikken hos blodprop-patienter.
Referencer:
1.•Villringer et al. Magn. Res. Med 6, 164-174, 1988.
2.•Østergaard, Weiskoff, Chesler, et al, Magn Res. Med 36, 715-726, 1996.
3.•Østergaard, Sorensen, et al, Magn. Res. Med. 36, 726-736, 1996.
4.•Sorensen, Tievsky, Østergaard, et al, JMRI, 7, 47-55, 1997.
5.•Ohta S et al. J Cereb Blood Flow Metab 16, 765-780 1996.
Stable Xenon is widely used in measurements of cerebral blood flow. However, several studies have indicated that inhalation of stable Xenon may alter CBF and thus affect regional CBF estimates determined by Xe CT-CBF. In this study, we examined the regional effects of 30% stable xenon inhalation on regional CBF by 15O water PET in normal volunteers.
Eight young, healthy volunteers were studied using a Siemens ECAT EXACT HR PET scanner, operated in 3D mode. Xenon was administered using a Xetron III (ANZAI) xenon inhalator, starting gas administration 2 minutes prior to the PET CBF measurement. To measure CBF, i.v. injection of 500 MBq 15O labeled water was performed, followed by nineteen arterial blood samples and 12 PET brain images over 3 minutes. PET image data were then fitted to a 2-compartment model using nonlinear, least squared regression analysis on a pixel-by-pixel basis. Mean absolute CBF values in 3 cortical regions (frontal, occipital and temporal), basal ganglia, cerebellum and white matter were then determined and compared.
None of the subjects showed changes in white matter CBF. Two subjects showed little or no change (<2.5%) in average gray matter rCBF. The remaining subjects showed average changes in gray matter CBF ranging from 10 to 50% (mean 25% for all 8 subjects). Regionally, basal ganglia showed the largest increase in rCBF (mean 40%) while cortical gray matter showed a mean increase of 25% for temporal gray matter and 19% for both frontal and occipital gray matter. Cerebellum showed an average increase in CBF of 13%.
In the two subjects not showing rCBF changes, one hyperventilated slightly during xenon inhalation while the other inhaled only 15% Xenon. Our results are in agreement with previous findings using less localized techniques.
Thus, regional Xenon CT CBF estimates may be affected by the variability in the Xenon effect among brain regions. Furthermore, caution should be exercised when comparing regional gray matter CBF values among subjects due to the high variability of overall Xenon reactivity.
Acknowledgements:
This work was supported by The Neurosurgical Research Foundation, The Neuroradiological Research Foundation, The Michaelsen Foundation, and”"Fonden til Neurologiens Fremme".
Dansk resumé:
Inhalation af ædelgassen Xenon anvendes sammen med CT-skanning til bestemmelse af hjernens blodgennemstrømning. Det har imidlertid vist sig, at gassen ændrer hjernens gennemblødning. Dette studie har med PET kortlagt disse ændringer. Det viste sig, at gennemblødningen øges i hjernens grå substans med 25%, i visse regioner op til 40%. Effekten varierer fra person til person. Denne viden vil nu indgå i vurderingen af data opnået med Xenon CT metoden.
Referencer:
Giller et al. AJNR 11 177182(1990). Hartmann et al. Stroke 222 182-189 (1991).
Bergholt et al. Acta Neurol. Scan. Suppl. 166 45-49 (1996).
The purpose of this project is to study cerebral ischemia in the acute phase of severe brain trauma, and changes in cerebral monoaminergic tone as a consequence of the trauma. The studies include »Positron Emission Tomography Investigation of Cerebral Blood Flow and metabolism During the first Week After Severe Brain Trauma«, a clinical study on neurotraumatized patients from the Department of Neurosurgery, Aarhus University Hospitals, and »Effect of Cerebral Cortical Trauma on Monoaminergic Neurotransmission and Its Consequences for Cerebral Blood Flow, Oxygen Metabolism and Glucose Metabolism in Pigs«, an experimental study on pigs, which are subjected to a standardized cerebral cortical lesion. In the first study, we used methods which we have previously evaluated, to measure regional cerebral blood flow, oxygen consumption, and glucose metabolism in comatose neurotraumatized patients. The purpose of the study is to evaluate these regional variables in correlation to the intracranial pressure and treatment by hyperventilation or indomethacin infusion. This »physiological« degree of brain damage found by PET will be compared to the »anatomical« brain damage found on CT-scan. The studies have revealed discrepancies between flow and metabolism (uncoupling), indicating that evaluation of cerebral ischemia in brain trauma must include metabolic variables. In the second project, we studied a series of experimental animals to evaluate the effect of cerebral cortical trauma on seretonergic (5-HTergic) and dopaminergic neurotransmission. The purpose of this study is to test the hypothesis that focal brain trauma reduces monoaminergic tone in the brain, explaining changes in cerebral blood flow and metabolism, and long term psychological changes after head injuries. The experiments have shown clear side differences in monoaninergic activity in the basal ganglia corresponding to the trauma. A normal material of cerebral blood flow, oxygen consumption and glucose metabolism in pigs has been established (1), and a series of studies to establish normal values of cerebral oxygen consumption in humans is coming to the end. In parallel with these, a study of coupling between blood flow and oxygen consumption is on going. On the basis of PET data, we propose an explanation for the BOLD-MR signal (2).
Acknowledgements:
MRC Denmark (12-1633 and 12-1634). Institute of clinical exp.science.
Dansk resumè:
Kranietraumers akutte effekt på hjernens blodgennemstrømning, metabolisme og mono-aminerge neurotransmission undersøges, dels ved studier af komatøse patienter med kranietraumer, og dels ved experimentelle undersøgelser på grise, som udsættes for et standardiseret cerebralt cortikalt traume. Undersøgelser af hjernens oxygenforbrug er etableret og evalueret, og der er opnået normalmaterialer dels hos grise og dels hos raske forsøgspersoner.
Referencer:
(1) P.H. Poulsen, D.F. Smith, L. Østergaard, E.Danielsen, A. Gee, S.B. Hansen, J. Astrup and A.Gjedde: In Vivo Estimation of Cerebral Oxygen Consumption and Glucose Metabolism in the Pig by [15O] Oxygen Inhalation and Dual Injections of [18F]Fluorodeoxyglucose. (Submitted).
(2) Poulsen, P.H., Østergaard,L., Hansen, S.B. and Gjedde: A.Physiological Basis of BOLD: Cerebral Oxygen Delivery Constraints Revealed by [15O]Oxygen and [15O]Water PET. (Abstract, Human Brain Mapping `97).
Determination of myocardial glucose uptake (MGU) with 2-[18F]fluoro-2-deoxy-D-glucose (FDG) and Positron Emission Tomography (PET) is based on the tracer model developed for 2-deoxy-D-[14C]glucose and quantitative autoradiography in the brain. The method was adapted to the heart, recognizing that myocardial uptake of FDG and glucose occurs through a series of similar steps. These steps include facilitated transport of hexoses into the myocyte by transporter proteins and phosphorylation by the enzyme hexokinase. Since the phosphorylated tracer analog is neither dephosphorylated nor a substrate for the glycolytic pathway, the accumulation of FDG represents a traces of the glucose uptake.
The FDG method has provided valuable qualitative information about reversibly ischemic myocardium in patients with left ventricular dysfunction. However, the quantitative assessment of MGU by FDG is difficult because the correction factor that equates net myocardial FDG uptake to net MGU, the »lumped constant« (LC), is not a true constant.
By modulating circulating levels of insulin and/or free fatty acids, with hyperinsulinemic euglycemic clamp, somatostatin infusion, or heparin administration in healthy volunteers, we have provided evidence suggesting that the LC varies in accordance with its physiologic dependence on membrane transport rates and hexokinase activity in the normal human heart. To further study the mechanisms for the variability of the LC, we established a collaboration with scientists from The University of Texas Medical School, Houston, Texas. In the isolated working rat heart, we measured rates of myocardial glucose uptake by tracer ([2-3H]glucose) and tracer analog methods (FDG) under a variety of metabolic conditions. We also quantified the relations between rates of glucose and FDG membrane transport and phosphorylation. Compared to glucose alone, the LC decreased in the presence of insulin, or beta-hydroxybutyrate, or both. However, the LC could always be predicted from unidirectional and steady-state FDG retention of the individual experiments and independently determined ratios between glucose and FDG transport and phosphorylation rates. True rates of MGU can therefore be measured individually with FDG alone and the model is applicable to the normal human heart. At this point we cannot be certain whether the model is directly applicable to metabolically deranged heart muscle. Future studies will focus on reversibly ischemic dysfunctional heart muscle (»hibernating myocardium«) and disorders characterized by insulin resistance. We propose that the term »lumped constant« is a misnomer and should be replaced by the term lumped quotient.
Acknowledgements:
The Heart Society, Novo Nordic Foundation and The Institute of Experimental Clinical Research.
Dansk resumé:
Undersøgelser ved PET-centret har vist at samlekonstanten, som anvendes ved beregning af hjertets glukoseoptagelse ved hjælp af 18F-Fluoro-deoxy-glukose ikke er en ægte konstant. Samlekonstanten varierer afhængigt af den relative indflydelse af membrantransport og fosforylering på den totale myokardiale hexose optagelse. Vi har i såvel dyreeksperimentelle som humane studier påvist, at individuelle samlekonstanter kan bestemmes ved hjælp af den unidirektionale optagelseshastighed for FDG og netto FDG optagelseshastigheden. Samlekonstanten er en misvisende betegnelse. Vi foreslår istedet betegnelsen samlekvotient.
Cigarette smoking is known to be an important risk factor for development of coronary artery disease and sudden cardiac death. However, the mechanism of smoking-induced coronary artery disease is not completely understood. It may involve both endothelium-dependent and -independent factors and alterations might be located in both the epicardial arteries and in the coronary microcirculation.
Previous studies have demonstrated that smokers over a wide age-range show a decrease in endothelial function of their peripheral arteries. However, it has not been possible to demonstrate any changes in the coronary vasomotor response of epicardial vessels to acetylcholine in smokers.
Furthermore it has been shown that acute smoking markedly lowers the myocardial flow reserve in young smokers. This reduction in flow reserve is caused by smoking induced increases in resting myocardial blood flow and decreases in hyperemic blood flow. However, the myocardial blood flow response to cold as an indirect marker of endothelial function has not been studied in humans. Therefore, the aim of this study was to investigate, in a group of young smokers, the myocardial blood flow at rest, during cold pressor testing as an index of endothelium-dependent vasomotion, and during dipyridamole-induced hyperemia. (MBF) was measured in 15 short-term smokers (>10 cigarettes daily for at least 3 years, age 24 ± 2 years) and 15 non-smokers (24 ± 3 years). MBF was measured at rest, during a 90 sec. cold pressor test (CPT) for evaluation of endothelium-dependent vasodilatation, and during dipyridamole infusion 0.56 mg/kg over 4 min. for evaluation of endothelium-independent vasodilatation. The rate-pressure product (RPP) was similar in the smoking and nonsmoking groups both at rest (7554±1428 vs. 7144±2003, P=NS), during CPT (9351±2189 vs. 8215±2930, P=NS) and during dipyridamole (11199±2214 vs. 10614±2611, P=NS). Resting MBF was similar in the two groups (0.82±0.14 vs. 0.79±0.29). CPT did not alter MBF significantly in the two groups (0.85±0.17 vs. 0.82±0.14 and 0.86±0.27 vs. 0.79±0.29, respectively, P=NS). However, when corrected for changes in RPP, the smoking group showed a 20% reduction in MBF (1.12±0.29 vs. 0.93±0.20, P=0.014) while the non-smoking group showed an unchanged RPP corrected MBF (1.12±0.26 vs. 1.09±0.32, P=NS). Dipyridamole MBF was similar in the two groups (2.23±0.81 vs. 2.44±0.68, P=NS).
Conclusions: Young smokers and non-smokers have similar resting myocardial perfusion. Endothelium-independent hyperemic myocardial perfusion also seems to be preserved. However, in young smokers a paradoxical 15% reduction in MBF was observed during CPT (endotheliumdependent vasodilatation) when MBF was corrected for the increase in RPP. These findings suggest an abnormality in coronary vasomotion possibly related to endothelial dysfunction in short-term smokers.
Acknowledgements:
The Heart Society and Novo Nordic Foundation.
Dansk resumé:
Rygning er en kendt risikofaktor for udviklingen af præmatur hjertesygdom. Den præcise virkningsmekanisme for udviklingen af hjertesygdom er ikke kendt. Vi har undersøgt 15 unge rygere som blev sammenlignet med 15 ikke-rygere. Undersøgelsen viste, at der hos rygere allerede efter få års rygning ses et ændret reaktionsmønster på endothelstimulation, idet der sker en paradoks relativ reduktion af perfusionen hos rygere modsat ikke-rygere. Der kunne ikke påvises nogen endotheluafhængig reduktion i myokardiegennemblødningen.
Approximately 20 % of all patients undergoing coronary arteriography because of chest pain, reveals no significant stenosis. About 50 % of these patients have objective signs of myocardial ishchemia as evidenced by a positive tread mill test or increased lactate production after pacing [Kemp HG, JACC 1986]. A defect in the enzymes catalyzing production of Nitric Oxide (NO) has been proposed as a possible pathological mechanism. NO is believed to play a central role in the regulation of coronary tone. NO is produced primarily in the endothelial cells, and release of NO is stimulated by a number of substances and shear stress [Furchgott, Nature 1980;228:373-6, Palmer, Nature 1987;327:524-26]. The substrate for NO production is L-arginine and the production is controlled primarily by two Nitric Oxide Synthetase Enzymes INOS and E-NOS.
Several recent publications [Quyyumi Circulation 1996;94:130-134,] have investigated the effect of substrate supplementation on NO release. These studies have found that L-arginine enhances the vasodilatory capacity of large conductance vessels when stimulated with acetylcholine. It is however not clear whether L-arginine affects the microcirculation and thereby perfusion. Several investigations have suggested that endothelium independent vasodilatation is impaired in patients with syndrome X (HEB am J Cardiol 1996). Whether this causes the myocardial perfusion to decrease is not clear.
The aim of the present study was therefore to investigate whether endothelium dependent and independent vasodilatation is impaired in patients with syndrome X and whether L-arginine supplementation enhances endothelium dependent vasodilatation at the level of microcirculation in patients with angina pectoris and normal coronary arteries.
Myocardial blood flow (MBF) was quantified under baseline conditions with 13N-Ammonia PET (2 Compartment model) at rest, during cold pressor testing (MBF-CPT) and during iv dipyridamole in 15 female patients (53±7 years) with syndrome X (positive treadmill test, negative hyperventilation test an normal arteriogram). Twenty-two healthy volunteers served as controls. One group consisted of 10 age and sex matched volunteers (55±10 years). The other control group consisted of 12 young females (24±5 years). In the patients MBF-rest and MBF-CPT were also measured after infusion of L-arginine (6.7 mg/min over 45 minutes). The rate pressure product (RPP) was unaltered after L-arginine at rest (7892±850 vs. 8276±1400, group A controls) and after Cold pressor testing (11021±2160 vs. 11553±2210). Similarly L-arginine did not affect MBF at rest (0.85±0.16 vs. 0.90±0.14 ml/g/min) or during Cold pressor testing (0.94±0.10 vs. 1.04±0.18 ml/g/min). Moreover CPT increased MBF to a similar degree in controls (23%) in patients under baseline conditions (16%) and after L-arginine infusion (18%) respectively (p=NS). In contrast the hyperemic response to Dipyridamole was blunted (1.72±0.57 vs. 2.30±0.32 ml/g/min, p<0.05) resulting in a significant reduction in the coronary flow reserve in patients relative to controls (1.89±0.62 vs. 3.45±1.03 ml/g/min, p<0.01). Thus, coronary vasomotion in response to cold is normal and unaltered by iv L-arginine suggesting preserved endothelial function in patients with syndrome X. However, the attenuation in coronary vasodilatory capacity and flow reserve suggest a dysfunction in the endothelium independent coronary smooth muscle cell relaxation in these patients.
Acknowledgements:
The Heart Society.
Dansk resumé:
Omkring 20 % af alle patienter som får udført koronarangiografi har ikke betydende stenoser. Omkring 50 % af disse patienter har objektiviserbar tegn på myocardieiskæmi. En defekt NO syntese har været foreslået som basis for iskæmisymptomer hos disse patienter. Vi har i dette projekt undersøgt effekten af stimulation af NO syntesen v.h.a. substrat tilførsel i form af L-arginin indgift. Vi har ikke kunnet påvise nogen effekt på myocardiegennemblødningen hverken under basale omstændigheder eller under endothelstimulation. Derimod fandtes en betydelig nedsættelse af den endotheluafhængige vasodilatatoriske reserve hos denne patientgruppe.
Figur 1: Patient på 54 år med klassisk mis-match mønster med nedsat perfusion og øget glucoseoptagelse sv. t. forreste koronararteries forsyningsområde. Øverst: Perfusionsundersøgelsen, nederst: Glucoseoptagelsesbilledet.
Referencer:
1. Effect of Cold Pressor Testing and iv L-arginine on Coronay vasomotion in patients with syndrome X. J. Czernin, H. Sonne M. Bøttcher HR. Schelbert. American College of Cardiology annual meeting Orlando Florida March 1996. Journal of the American College of Cardiology 1996; March issue
Patients with normal coronary arteries and chest pain (syndrome X, SX) are suspected of having a generalized microvascular abnormality. The purpose of this study was to measure both myocardial perfusion and peripheral flow, minimal vascular resistance (MVR) and the morphology of subcutaneous resistance arteries.
Methods: 16 patients with SX (56±5 years, 3 males) and 15 healthy controls matched for age, sex and blood pressure were compared. Absolute myocardial perfusion at rest and after coronary vasodilation with dipyridamole was measured with 13N-ammonia dynamic positron emission tomography. Forearm blood flow at rest and following reactive hyperaemia was measured with external Doppler ultrasound in the brachial artery. The morphology of relaxed gluteal subcutaneous resistance arteries was measured in vitro using a small vessel myograph.
Results: Mean±S.D. (number of patients or controls studied).
| Patients | Controls | ||
| Myocardial perfusion and resistance Rest | Rest (ml/g/min) Dipyridamole (ml/g/min) MVR (mmHg min g/ml) | 0.82±0.16 (15) 1.67±0.49* 55±15* | 0.82±0.11 (10) 2.11±0.61 43±14 |
| Forearm flow and resistance | Rest (ml/min) Hyperaemia (ml/min) MVR (mmHg min/ml) | 48±29 (15) 195±83** 0.60±0.35** | 61±34 (12) 300±116 0.37±0.17 |
| Artery morphology | Lumen diameter (µm) Media thickness (µm) Media/lumen ratio (%) | 214±58 (16) 11.8±2.1 5.9±1.5 | 241±57 (15) 12.9±2.5 5.6±1.3 |
Conclusion: These findings confirm that SX could be a generalized vascular disorder. Coronary maximal flow tends to be reduced and coronary MVR to be increased. Forearm maximal flow is reduced and peripheral MVR is increased in SX. This is not reflected by large morphological changes in the subcutaneous resistance arteries, although the tendency for a decreased lumen diameter in SX is consistent with an increase in MVR.
Acknowledgements:
Servié International Research Foundation.
Dansk resumé:
Syndrom X er kombinationen af angina pectoris, normal koronarangiografisk undersøgelse og positivt arbejds-EKG. Den patofysiologiske baggrund for syndromet er uafklaret, men måske skyldes symptomerne i en stor del af tilfældene abnorm mikrocirkulation. Flere studier har afsløret nedsat koronar-flow reserve. Nye undersøgelser viser en reduceret hyperæmisk respons tydende på en øget perifer karmodstand hos disse patienter. Hvorvidt det drejer sig om en funktionel eller strukturel abnormitet i den perifere mikrocirkulation er endnu uafklaret. Formålet med projektet var at undersøge om patienter med syndrom X har abnorm struktur af små perifere subkutane arterier og sammenholde disse data med det perifere blood-flow bestemt med ultralyd og koronar flow reserve bestemt med PET-skanning. Undersøgelsen viste, at strukturen af små modstandskar var uændret hos patienter i forhold til normale kontrolpersoner. Det hyperæmiske perifere blood flow og dipyridamol induceret myocardial perfusion var reduceret hos patienter. Resultaterne viser, at syndrom X er en mikrovasculær generaliseret karsygdom, men uden signifikante morfologiske ændringer i de subkutane modstandskar.
PET has been used widely to study in vivo metabolism and blood perfusion of the brain and heart, but seldom of the liver despite its importance in the regulation of metabolism. It may, however, be difficult to interpret PET studies of the liver because of its anatomical location and dual blood supply from the hepatic artery and the portal vein. PET studies of organ metabolism and perfusion include iv injection of the positron labelled tracer, dynamic scanning of the organ in subsequent time intervals (of, for example, 5 sec to 10 min's duration) for 15-90 min, and simultaneous blood sampling from a peripheral artery for radioactivity measurements. In brain and heart studies, PET data are usually evaluated by means of various one-inlet compartment models with the time course of the brain and heart radioactivity evaluated in relation to the input radioactivity concentration as measured in the arterial blood. If oneinlet models are applied to liver PET measurements, the fit of the model to the measured data often show systematic deviations. This is in accordance with the dual inlet to the liver via the hepatic artery (around 0.3 L blood/min) and the portal vein (0.9 L/min).
We wish to develop removal kinetic models with accounts for the dual inlet and applicable to dynamic PET scanning data. We have performed a number of studies in anaesthesized pigs with H2O and CO as diffusible and vascular PET-tracers, respectively. The studies consist of dynamic PET scanning, blood sampling in a peripheral artery, the portal vein, and the hepatic vein as well as measurements of the blood flow rates in the hepatic artery and the portal vein by means of ultrasound transit-time flowmeters. The experimental study is not yet completed, but preliminary analysis shows good fit of the dual inlet models to the experimental measurements. Models will be developed to allow for the determination of the metabolic parameters and the hepatic blood flow rate, also by simple non-invasive PET studies in humans.
Acknowledgements:
The study is supported by the Novo-Nordic Foundation and the Aarhus University Research Foundation.
Dansk resumé:
Leveren har stor betydning for organismens metaboliske regulation. Med PET kan man kvantitere metaboliske processer og blodgennemstrømningen in vivo, men PET har kun i ringe omfang været anvendt til leverundersøgelser. Leveren er vanskeligt tilgængelig for undersøgelser in vivo p.g.a. dens anatomiske lokalisation og dobbelte blodforsyning, fra en pulsåre og portåren (fra tarm til lever). Formålet med projektet er at udvikle en PET metode for leveren. Bedøvede grise gives iv injektion af PET tracerne H2O og CO som henholdsvis diffusibel og vaskulær tracer. Samtidig med PET skanningen tages blodprøver fra leverens pulsåre, portåren og levervenen; gennemblødningen af leverens pulsåre og af portåren måles med operativt anbragte ultralydsflowmetre, og leverens totale gennemblødning måles med infusion af ICG. Fra disse data er vi ved at udvikle matematisk-fysiologiske modeller, dels til beskrivelse af betydningen af den dobbelte blodforsynings betydning for vurdering af PET data af leveren, dels til simpel non-invasiv PET skanning til måling af leverens metabolisme og blodforsyning hos mennesker.
The normal liver is profusely innervated but the functions of the liver nerves are not well described. The effects of the central nervous system and of the liver nerves on the regulation of the hepatic glucose metabolism has gained more interest during the last three decades because of the increasing number of liver transplantations. The liver donors are brain-dead which may affect the metabolism. The liver and the transplanted livers are denervated. Denervation of the liver does not seem to affect the hepatic glucose production at rest or during work. Based on these observations we hypothesize that central nervous system and the liver nerves become important during severe metabolic stress such as induction of hypoglycemia and induction of brain death.
We studied the hepatic glucose production (HGP) during normoglycemia and during an insulin induced hypoglycemic clamp in liver denervated (D) and sham operated control minipigs (C). During hypoglycemia, HGP and arterial plasma glucagon was higher in D than in C (P<0.05). Initial hepatic glycogen was higher in D than in C (P<0.05) but it decreased similarly in both groups during hypoglycemia.
We also studied the effects of intravenous glucose/insulin infusion in brain-dead pigs on the hepatic glycogen content. Four groups of 40 kg pigs were studied: brain dead and not brain-dead pigs given intravenous glucose/insulin or saline infusion. In brain-dead pigs given saline liver glycogen decreased during the first six hours after induction of brain death (P<0.05) and increased during the next six hours (P=0.05). These changes were accompanied by transient increases in plasma adrenaline, glucose, free fatty acids, and glucagon. Following glucose/insulin infusion, hepatic glycogen increased steadily in both brain-dead and in not-brain-dead pigs. In brain-dead pigs the increases in the blood measurements just mentioned were smaller following glucose infusion than following saline infusion. In this model, we also studied the effects of intravenous glucose/insulin infusion and induction of brain death on hepatic phospholipid metabolism, pH and ATP concentration as measured by 31P NMR spectroscopy. In control pigs given glucose/insulin and in both groups of brain-dead pigs breakdown of phospholipids seemed to decrease. There was an association between liver concentrations phospholipid breakdown products and glycogen in control animals (r2=0.54) which leads to the hypothesis that the phospholipid breakdown products act as osmotic buffers for changes in the glycogen concentration. During glucose/insulin infusion hepatic intracellular pH decreased (P<0.002). Liver ATP did not change.
In conclusion, hepatic denervation resulted in increased hepatic glucose production during hypoglycemia. This might be caused by increased glucagon or by lack of neural input to the liver. The decrease in hepatic glycogen content after brain death could be prevented by intravenous glucose/insulin infusion probably because of a reduction of the adrenaline response to the induction of brain death. Glucose/insulin infusion and induction of brain death seemed to reduce the breakdown of phospholipids.
Acknowledgements:
This work was supported by Aarhus University, Danish Diabetes Association, Novo-Nordic Foundation, Institute for Experimental Clinical Research (ÅUH) and "Knud Højgaards Fond".
Dansk resumé:
Den normale lever er rigt innerveret, men levernervernes funktioner er kun sparsomt beskrevet. Vi fremsatte den hypotese, at centralnervesystemet og levernerverne er af væsentlig betydning for leverens glukoseomsætning under svær metabolisk stress. Vi undersøgte den hepatiske glukoseproduktion (HGP) under normoglykæmi og under insulininduceret hypoglykæmisk clamp hos leverdenerverede (D) og skamopererede minigrise. Under hypoglykæmi var HGP og plasma glukagon størst hos D. Leverglykogen var under hele forsøget størst hos D. Vi undersøgte effekten af intravenøs glukose/insulininfusion og saltvandsinfusion til hjernedøde og ikke-hjernedøde grise på leverens glykogenindhold målt biokemisk, og på leverens fosfolipidmetabolisme, pH og ATP-indhold blev undersøgt målt ved hjælp af 31P NMR spektroskopi. Hos hjernedøde grise, som fik saltvandsinfusion, faldt leverens glykogenkoncentration i løbet af de første seks timer efter induktion af hjernedød. Derefter steg den. Sammen med dette temporære fald i leverglykogen sås temporære stigninger i plasma adrenalin, glukagon og blod glukose. Hos både hjernedøde og ikke-hjernedøde grise medførte glukose/insulininfusion, at leverglykogen steg jævnt mod en fordobling efter tolv timer. Resultaterne af 31P NMR spektroskopien tyder på at nedbrydningen af fosfolipider i leveren faldt under glukose/insulin infusion og efter induktion af hjernedød. Leverens pH faldt under glukose/insulininfusion (P<0,002), mens leverens ATP koncentration holdt sig uændret. Det konkluderes, at faldet i leverens glykogenindhold efter induktion af hjernedød kunne forhindres ved hjælp af intravenøs glukose/insulininfusion. Måske skyldes dette en hæmning af adrenalinresponset. Glukose/insulininfusion og induktion af hjernedød syntes at reducere nedbrydningen af fosfolipider. Det konkluderes, at leverdenervation medførte en øget hepatisk glukoseproduktion under hypoglykæmi. Dette kan skyldes øget glukagon eller manglende nervøs stimulation af leveren.
Referencer:
Klaus Roelsgaard, Hans Erik Bøtker, Hans Stødkilde-Jørgensen, Frederik Andreasen, Steen Lindkær Jensen, Susanne Keiding. Effects of brain death and glucose infusion on hepatic glycogen and blood hormones in the pig. Hepatology 1996; 24: 871-875.
Klaus Roelsgaard, Hans Stødkilde-Jørgensen, Steen Lindkær Jensen, Susanne Keiding. Effects of glucose infusion and brain death on hepatic phosphorous metabolism in the pig. Submitted.
Klaus Roelsgaard, Susanne Keiding, Jesper Mortensen, Hans Erik Bøtker, Anette Mengel, Frederik Andreasen, Mogens Vyberg, Steen Lindkær Jensen, Niels Møller. Liver denervation in pigs: Effect on hepatic glucose production during normo- and hypo-glycemia. Submitted.
Klaus Roelsgaard, Steen Lindkær Jensen, Susanne Keiding: Effects of brain death and glucose infusion on hepatic glycogen and blood hormones in the pig. International Association for the Study of the Liver (IASL) biennial scientific meeting, Cape Town, Sydafrika 1996, Hepatology 1996; 23; I-72.
Klaus Roelsgaard, Niels Møller, Jesper Mortensen, Steen Lindkær Jensen, Susanne Keiding. Hepatic glucose production during normo- and hypo-glycemia in pigs -Effect of hepatic innervation. European Society for Clinical Investigation (ESCI) 30th Annual Meeting, Interlaken, Schweitz 1996, Eur. J. Clin. Invest. 1996; 26 Suppl. 1: A-47.
Primary sclerosing cholangitis (PSC) is a chronic liver disease in patients with ulcerative colitis or Crohn's disease. Most patients have no, or only minor symptoms from the liver disease, but may develop cirrhosis or cholangiocarcinoma (CC) (5-15%). At the time of diagnosis, the tumour nearly always has spread so widely into the liver that the disease is fatal within a short period of time, even after resection or liver transplantation. However, if a small tumour is diagnosed coincidentally in connection with liver transplantation for cirrhosis, the survival prognosis is good.
We therefore designed a PET method to test its value of early detection of cholangiocarcinoma in patients with PSC. PET images yield quantitative measures of metabolic processes in vivo. [18F]fluoro-deoxyglucose (FDG) is a positron-labelled glucose analog which is widely used in tumour diagnostics. Tumours accumulate FDG and FDG-6-phosphate due to increased activity of hexokinase and cell membrane glucose transporters. We hypothesized that FDG would be especially suitable for the detection of cholangiocarcinoma because the hepatocytes have a high content of glucose-phosphatase in contrast to the bile duct cells. Accordingly, after uptake in the cells, FDG rapidly returns to the blood from the hepatocytes (probably also in livers with PSC) whereas FDG-6-phosphate accumulates in the cholangiocarcinoma cells. Sites of cholangiocarcinoma therefore appear as »hot spots«.
In the first part of the study, patients with PSC, some with, some without cholangiocarcinoma, as well as patients with cholangiocarcinoma in otherwise normal livers were investigated. Until now, 8 patients with PSC, 3 patients with PSC +cholangiocarcinoma, and 2 patients with cholangiocarcinoma have been investigated. FDG was given intravenously followed by dynamic scanning of the liver for 90 min with simultaneous blood sampling from an artery for determination of plasma radioactivity concentrations.
The cholangiocarcinoma tumours were clearly seen as ”hot spots” (Fig.1). The time-activity curves in Fig. 2 illustrate the accumulation of radioactivity in cholangiocarcinoma versus the rapid turnover of radioactivity in non-malignant liver tissue. The preliminary results (Fig. 3) showed a marked difference between the calculated "metabolic clearance" values (K = (K1•· k3)/(k2+ k3)) (1) in the cholangiocarcinoma tissue and the PSC (and normal) liver tissue.
We are presently recruiting more patients with cholangiocarcinoma and analyzing the data in order to optimize the detection of very small tumours. In a subsequent project, a group of patients with PSC will be studied at regular intervals to evaluate the prognostic value of the FDG investigation.
Figure 1: PET image of the liver after iv inj. of FDG in a patient with PSC and cholangiocarcinoma.
Figure 2: Time course of tissue radioactivity.
Figure 3: Metabolic FDG clearance.
Acknowledgements:
The study is supported by the Danish Cancer Society.
Dansk resumé:
Primær skleroserende kolangitis (PSC) er en kronisk kolestatisk leverlidelse, som især ses hos patienter med kronisk inflammatorisk tarmlidelse. De fleste patienter har kun få symptomer fra leversygdommen, men sygdommen kan udvikle sig til skrumpelever eller kolangiokarcinom (CC, galdegangskræft) (5-15%). Når diagnosen kolangiokarcinon stilles, er kræften næsten altid så udbredt i leveren, at det er udsigtsløst at operere. Hvis man imidlertid »tilfældigt« finder kolangiokarcinom ved f.eks. levertransplantation for skrumpelever, er overlevelsesprognosen god.
Vi vil derfor udvikle en PET undersøgelse til diagnosticering af små kolangiokarcinom tumorer hos patienter med PSC. [18F]fluoro-deoxyglucose (FDG) er formentlig specielt velegnet til detektion af denne tumor, idet selve levercellerne indeholder store mængder af et enzym (glukosefosfatase), som nedbryder FDG produkterne i modsætning til galdevejscellerne (og dermed kolangiokarcinomet). Vi har undersøgt et mindre antal patienter med PSC eller CC (±PSC), og som forventet ophobes der radioaktivitet i kræftknuderne, men ikke i PSC levervæv (Fig. 1 og 2). Fig. 3 viser at der er betydelig forskel på de beregnede værdier af »FDG metabolisk clearance« for PSC og kolangiokarcinom. Vi er ved at undersøge hvor små tumorer vi med rimelig sikkerhed kan detektere. Efterfølgende vil metoden blive evalueret i et prospektivt studie i en udvalgt gruppe patienter.
Referencer:
1.Gjedde A, Calculation of cerebral glucose phosphorylation from brain uptake of glucose analogs in vivo. Brain Res Rev 1982; 4: 237-74.
2. S. Keiding, H. Højgaard Rasmussen, A. Gee, S.B. Hansen, J. Møller-Petersen. Detection of cholangiocarcinoma in patients with primary sclerosing cholangitis by positron emission tomography (PET). Poster at the 31thScientific Meeting of the European Society for Clinical Investigation, Kiel, March 1997.
Breast cancer is a common malignant disease in women. At the operation, the lymph glands in the axilla are routinely removed due to the risk of spread of malignant cells. This part of the operation is often followed by lymph oedema, pain, and problems with movements of the arm. In half of the cases, no malignancy is found in the axilla glands by subsequent histological examination, and the operation thus was »unnecessary«.
There are no methods currently available to determine, before the operation, whether there are malignant glands in the axilla, neither by clinical examination, X-ray, ultrasound, MR, or CT, all being »morphological« examinations. PET provides images of metabolic processes in vivo, and since malignant tumours often have increased metabolic activity PET often is useful in the detection of malignant tumours. [18F]fluoro-deoxyglucose (FDG) has been used to demonstrate breast cancer and malignant glands in the axilla. The method has, however, not yet been developed with emphasis on the detection of small malignant glands in unselected patients. We also want to test the hypothesis that [11C-methyl]methionine is more sensitive and specific than FDG.
We therefore initiated a study in which patients with primary breast cancer are examined. The figures give examples of the images obtained after FDG injection in one patient. Fig.1 illustrates the accumulation of FDG in a »hot spot« corresponding to the breast cancer (as well as accumulation in the heart region). Fig. 2 demonstrates the use of parametric imageing technique in a picture of the calculated pixel values of the »metabolic clearance« of FDG. We are presently recruiting more patients. In a subsequent study, the prognostic values of the method will be evaluated.
Figure 1: PET image of a breast cancer. Sum of data from 30-90 min after iv injection of FDG.
Figure 2: PET image of a breast cancer. Parametric image of the "metabolic clearance, K" of FDG from the Patlak-Gjedde plot.
Dansk resumé:
Ved operation for brystkræft fjernes i dag lymfeglandlerne i aksillen, hvilket hyppigt forårsager postoperativt armbesvær. Formålet med projektet er at udvikle en PET metode som kan påvise aksilmetastaser med så stor sikkerhed, at man kan undlade aksiloperation hos den halvdel af kvinderne, som ikke har maligne forandringer i aksilglandlerne.
Med PET kan man opnå kvantitativ billedmæssig fremstilling af stofskifteprocesser in vivo. Pga øget stofskifte ophober mange kræftknuder injiceret [18F]fluoro-deoxyglukose (FDG), men de nuværende metoder er ikke sensitive nok. Vi vil derfor også undersøge anvendeligheden af [11C-methyl]methionin samt udvikle en mere præcis billedanalyse. I første delprojekt optimeres metoden med henblik på at afsløre små maligne knuder og samtidigt være klinisk anvendelig. Herefter vil vi en et nyt delprojekt undersøge sensivitet og specificitet af metoden i et større konsekutivt patientmateriale.
Fig.1 viser akkumulation af radioaktivitet i brystcancer efter injektion af FDG hos en af vore første patienter. Fig. 2 giver et eksempel på billedmæssig fremstilling af beregnede parametre for FDG metabolismen, såkaldt "metabolic clearance".
Referencer:
Adler LP, Crowe JP, Al-Kaisi NK, Sunshine JL. Evaluation of breast masses and axillary lymph nodes with [F-18]2-deoxy-2-fluoro-D-glucose PET. Radiology 1993; 187: 743-50.
Leskinen-Kallio S, Någren K, Lehikoinen P, Ruotsalainen U, Joensuu H. Uptake of 11C-methionine in breast cancer studied by PET. An association with the size of S-phase fraction. Br J Cancer 1991; 64: 1121-24.
Osteogenic sarcoma is treated by preoperative chemotherapy and operative resection. The purpose of the chemotherapy is to minimize the tumour size and risk of metastases during the operation. In the planning of the operation it is important to evaluate the viability of the tumour tissue following the chemotherapy treatment. We routinely use X-ray examination, conventional MR scanning as well as dynamic MR scanning with gadolinium contrast to give a measure of the blood perfusion. However, these procedures cannot always be used to distinguish between tumour tissue and inflammatory tissue.
The purpose of the study is to test the hypothesis that the amount of osteoid producing tissue can be measured quantitatively by means of accumulation of [18F]fluoride and be distinguished from inflammatory tissue as measured by the accumulation of [18F]fluoro-deoxyglucose (FDG). This will be compared with the results of the histological examination of tissue removed at the operation. The result of one of the first studies is shown in Fig.1.
Acknowledgements:
The study is supported by the Danish Cancer Society.
Dansk resumé:
Kræft udgående fra knoglecellerne (osteogene sarkomer) er sjældne. De behandles med operativ fjernelse af tumoren, forudgået af kemoterapi. Vurdering af udbredningen af aktiv tumor efter kemoterapi behandlingen er vigtig i planlægning af operationen. Hverken med konventionel eller dynamisk MR skanning kan man med sikkerhed vurdere mængden af aktivet tumorvæv, idet ikke kun tumorvæv, men også reaktive forandringer lader op. Formålet med projektet er at undersøge om man med PET skanning med [18F]fluorid og [18F]fluoro-deoxyglucose (FDG) kan skelne mellem aktivt tumorvæv og reaktive forandringer. De histopatologiske fund ved undersøgelse af operationspræparatet anvendes som facitliste. Fig.1 viser PET billeder fra en af vore første patienter.
Figure 1: Patient with osteogenic sarcoma: PET scanning with 18F-fluorid.
Figure 2: Patient with osteogenic sarcoma: PET scanning with 18F-FDG.
Endogenous and exogenous amino acids labelled with 11C and 18F have been used in PET, for investigating processes such as protein synthesis rates, amino acid transport and neurotransmitter synthesis in both animals and living man. As the synthesis and synthetic application of chiral aziridines has become of great interest in recent years (1), the application of aziridine-2-carboxylates to the synthesis of 11C and 18F labelled amino acids was investigated (Scheme 1).
Initial work on the labelling of ß-18F-fluorophenylalanine (2) via the nucleophilic fluorination of aziridine precursor 1a lead to a fully automated synthesis with a 5% overall radiochemical yield (decay corrected to EOB) and a synthesis time of 60 minutes including HPLC purification. Work is in progress to synthesise ß-18F-fluorodopa in a similar manner. This investigation has been expanded to the use of 11C cyanide in aziridine ring-opening. The labelled precursor ß-11C cyano alanine, isopropyl ester was produced in 4 minutes by reaction of 11C cyanide with N-tert-butoxycarbonyl-2-aziridine isopropyl carboxylate 1 (R' = H, R = iPr) in 46% radiochemical yield (decay corrected to EOB). After purification by preparative HPLC, the reduction and/or hydrolysis of this intermediate gave the three potentially useful amino acids 4-11C asparagine 4, 4-11C aspartate 5 or 4-11C-2,4-diaminobutyric acid 6 (Scheme 2). The racemic aziridine precursor was resolved by preparative HPLC using a Chiracel OD-H cellulose carbamate column and a single enantiomer was labelled and hydrolysed to give 4-11C aspartate. Chiral HPLC analysis of the product showed complete racemisation. We are currently attempting to establish whether racemisation occurs during the labelling reaction or during acidcatalysed hydrolysis.
2-Aziridine isopropyl carboxylate was prepared by reaction of 2,3-dibromo isopropyl propionate with liquid ammonia (3). Treatment with di-tert-butoxycarbonate gave N-tert-butoxycarbonyl-2-aziridine isopropyl carboxylate 1b. This precursor (5 mg) was dissolved in a solution of tetrabutyl ammonium hydrogen carbonate (0.1 M) in anhydrous DMF (300 µl). Subsequent to the trapping of 11C cyanide, the solution was heated at 120 °C for 4 minutes. Following dilution with 0.05M phosphoric acid (500 µl), crude 3 was injected directly on to a Nucleosil 5 C18 HPLC column (10 x 250 mm) eluted with 15/85 acetonitrile/water at a flow rate of 8 cm3 min-1. ß-11C cyano alanine, isopropyl ester eluted at 10-12 minutes and the collected fraction was evaporated to dryness prior to hydrolysis (10 min., 0.1 M HCl, 110 °C for 4; 5 min., 5 M HCl, 110 °C for 5) or reduction (5 min. with 10 mg COCl, 30 mg NaBH4 in methanol, 90 °C, followed by acidification and sep-pak purification for 6). The radioactive products were neutralised by passage through a column of Biorad AG11 A8 ion exchange resin prior to analysis on a Crownpak Cr+ chiral HPLC column (4 x 150 mm) eluted with perchloric acid (pH 1.0) at 0 °C.
It has been show that aziridine-2-carboxylates are promising as precursors for ring opening reactions with 11C and 18F-labelled nucleophiles. There is potential for further development of this work, for example by the use of 11C methyl lithium as a nucleophile for ring-opening to produce 4-11C valine or isoleucine. It is expected that the use of milder labelling conditions may reduce racemisation. This general labelling strategy is, of course, not limited to amino acids, and should also be applicable to the synthesis of labelled amines.
Dansk resumé:
Endogene og eksogene aminosyrer mærket med 11C og 18F er blevet brugt i PET til at undersøge protein syntese hastigheder, aminosyre transport og neurotransmitter syntese. Vi har undersøgt azirdin-2-carboxylater som reaktanter i nukleofile ring-åbnende reaktioner med 18F -fluorid og 11C-cyanid. Syntese af specielle aziridiner og optimering af mærkningsbetingelserne har ført til syntese af nogle brugbare mærkede aminosyrer.
Referencer:
1.•D. Tanner, Angew. Chem. Int. Ed. Engl., 33, 599-619 (1994).
2.•Gillings, N. M., Venkatachalam T. K. and Gee A. D. J. Label. Compound. Radiopharm, 37, 133, (1995).
3.•Von Kyburz, E.; Els, H.; Majnoni, St.; Englert, G.; von Planta, C.; Fürst, A. and Plattner, Pl. A. Helv. Chim. Acta 49, No. 41, 359. (1966).
2-18F Fluoro-2-deoxy-D-glucose (FDG) is a widely used radiopharmaceutical for the measurement of glucose utilisation by PET. Despite the widespread use of the tracer, it should be remembered that FDG is a glucose analogue. The estimation of endogenous glucose transport and phosphorylation based on FDG data usually relies upon the use of a conversion factor, the so called "Lumped Constant" (LC). This constant has been experimentally determined in healthy tissue, in animals and man. However, there is little indication that the LC has the same value in diseaed tissue. Indeed, recent reports show that the LC can significantly vary in magnitude even in healthy tissue (1). The Lumped Constant is therefore not a "constant"’but a variable. To enable the calculation of the LC in various normal and diseased tissues a simplified synthesis of [1-11C]Glucose was developed. The synthesis utilises the Kiliani-Fischer synthesis [2,3,4], modified with a solid phase supported reaction of n.c.a. 11C-CN with arabinose to shorten reaction times, simplify work-up procedures and automation, and increase the amount of tracer available for injection.
15 mg (1.15 x 10-4 mol) of D(-)arabinose were dissolved in 0,1 ml of 0,033 M borate buffer 4 and loaded on an Alumina N Sep-PakTM preconditioned with 10 ml of 0,033 M borate buffer. NH411CN was passed over the Sep-PakTM, after which the radioactivity was eluted using 2,5 ml of 30% HCOOH directly into a reduction vessel containing ca. 0,5 g of Raney© nickel slurry. After 7 min at 383 K the solution was transferred via a column filled with the 0,8 g BIO-RAD AG© 50W-X8 (100-200 mesh) cation exchange resin, 1,5 g BIO-RAD AG© 11A8 (50 -100 mesh) ion retardation resin and a vented Millipore filter directly into a 2 ml HPLC loop. The 1-11C-D-glucose fraction was collected and the total volume adjusted to 10 ml, using isotonic saline.
| Reaction parameters: | solid phase synthesis | previous report 4 |
| Syntheses time: | 38 min (EOB) | 50 -55 min |
| Trapping efficiency: | >99% on Sep-PakTM | >99% in buffer |
| Elution efficiency from Sep-PakTM: | >95% | --- |
| AldonitrileYield: | >90% | >95% |
| RCY of 1-11C glucose and 1-11C mannose after exchange resin work up: | 10 -30% | approx. 20 -30% |
| Yield of 1-11C glucose (noncorrected): | 2.5 -8.5% | 1.5- 3.5% |
| Yield of 1-11C D-glucose (decaycorrected): | 5-15% (EOB) | 10 -20% (EOB) |
| Ratio glucose/mannose: | 1.2 -1.7 | 1.5-2.1 |
| Radiochemical purity: | >95% | >95% |
Discussion
The solid phase supported synthesis simplifies the previously reported procedures for the preparation of 1-11C-D-glucose as a pH control and rotary evaporation step before HPLC is avoided. Due to the instantaneous reaction of the cyanide with the arabinose/borate complex on the Al-N Sep-PakTM, this simplified synthesis procedure reduces the overall reaction time by 12-17 min (4) . Nevertheless the crucial and yield determining step of the synthesis of labelled carbohydrates using the Kiliani-Fischer approach is the reduction of the aldonitriles to the corresponding sugars. The use of complex hydrides (NaBH4, NaB(OCH3)3H, LiAlH4, LiAl(N(C2H5)2)3H5, LiAl(N(C4H9)2)3H5)) resulted either in an overreduction or in a nonreduction of the aldonitriles. Of the hydrides tested, NaBH4 was the only reagent able to produce radiolabelled sugars (radiochemical yields of about 10%). However, the previously reported Raney© nickel and formic acid reduction gave higher yields of labelled sugars, so this procedure was adopted for the reduction step.
Figure 1: Experimental Setup:
Dansk resumé:
2-18F fluoro-2-deoxy-D-glukose (FDG) er et radioaktivt lægemiddel, som ofte bruges til måling af glukose forbrug. Dette sporstof er en glukose analog, og når det bruges til måling af glukose transport og fosforylering, er det nødvendigt at bruge en faktor kaldes“"the lumped constant". Desværre er denne konstant ikke den samme i raskt og sygt væv. Derfor er 11C glukose bedre end FDG til kvantitativ måling af disse processer. Vi har derfor prøvet at udviklet en simplificeret syntese af 11C glukose. Vi har brugt en "solid phase supported"”reaktion i en automatiseret proces. Denne er bedre end tidligere beskrevne synteser, og giver et radiokemiske udbytte på 5-15% 11C glukose efter 38 minutter.
Referencer:
1. Hawkins R. A., Mans A. M., Davis D. W., Vina J. R. and Hibbard L. S. -Am. J. Physiol. 248: 170 (1985)
2. Shiue C. Y. and A. P. Wolf -J. Label. Compds. Radiopharm. 22: 171 (1985)
3. StoneElander S., Halldin C., Långstrom B., Blomqvist G. and Widen L. -Appl. Rad. Isot. 43: 721 (1992)
4. Dence C. S., Powers W. J., and Welch M. J. Appl. Rad. Isot. 44: 971 (1993)
5. Cha J. S. -Bull. Korean Chem. Soc. 13: 670 (1992)
The choice of synthetic strategy for labelling tracers with short-lived radionuclides is influenced by a number of factors including the availability of labelling precursors which can be produced in high radiochemical yields in the minimum of time. Of the 11C-labelling precursors that are readily available, 11CO2 , 11C-Cyanide and 11C-methyl iodide/triflate are the most commonly used. Other labelling agents such as 11C-benzyl iodides (Scheme1), have been used in labelling syntheses, but have received limited application in‘"routine" labelling applications, perhaps due to their relatively long synthesis times (20 -25 min) (1-3) and complexity of automation (which includes a liquid-liquid extraction procedure). In order to develop a rapid and conveniently automated synthesis of benzyl iodides, the potential of solid phase-supported Grignard reactions with 11CO2 was investigated.
Phenyl magnesium bromide (100 µl, 1M) was added to a C-18 Sep-Pak under an inert atmosphere and flushed with nitrogen. After connection to the target delivery line, 11CO2 was passed directly through the Sep-Pak in a stream of nitrogen gas at a flow rate of 1000 cm3/min. Immediately after trapping of 11C radioactivity, 200 ml of lithium aluminium hydride (LAH, 1 M in THF) was added. After reacting for ca. 15 s, the Sep-Pak was flushed with a stream of nitrogen for ca. 15 s and 500 µl 57 % hydriodic acid (HI) was slowly (30 s) added. The Sep-Pak was then washed with 20 cm3 H2O and again flushed with nitrogen for 30 s. The resulting product was eluted with ether, via a silica Sep-Pak and drying column (containing sodium sulphate, magnesium sulphate and calcium sulphate) to a septum-equipped vial. In a number of experiments, the ether was evaporated and the crude product reacted with sodium ethoxide (ca. 1 mg sodium in 2 cm3 ethanol, 5 min 40 °C). In one experiment, 4-Cl-phenyl Mg Br was used as the Grignard reagent in an analogous manner. The reactions were followed by reverse phase HPLC. Automation was performed using pneumatically operated Rheodyne- 3-way slider valves actuated by computer-controlled relays (Scheme 1).
The trapping efficiency of 11CO2on the Grignard impregnated Sep-Pak was >99 % even at the high flow rate of 1000 cm3 /min. Addition of LAH and HI were carefully added to the Sep-Pak (over 15 and 30 s respectively) in order to prevent too vigorous a reaction. The crude product was purified from hydrophilic by-products by eluting the C-18 matrix with water. Benzyl iodides were subsequently eluted with ether via a Si Sep-Pak (to decolourise the solution) and a drying column. Evaporation of the ether afforded 11 C-benzyl iodides in > 95 % radiochemical purities, a synthesis time of 8 min (after EOB) and decay corrected radiochemical yield of 40 -45 %. In a typical production starting with 17 GBq (460 mCi) 11CO2, 4.5 GBq (122 mCi) of 11C-benzyl iodide was produced at EOS. Reaction of the 11C-benzyl iodide with sodium ethoxide at 40 °C for 5 min produced the corresponding 11C-labelled ethyl ether. The time taken for the successive reaction steps is outlined below:
| Time after EOB (min) | Description |
| 0 | Trap 11CO2on C-18 Sep-Pak |
| 1.5 | Add LAH™ |
| 2 | Add HI |
| 4 | Wash Sep-Pak with H2O |
| 5.5 | Elute 11C-benzyl iodides with ether |
| 6.5 | Ether evaporation |
| 8 | EOS |
Radioactivity losses (ca. 50%) were mainly located on the C-18 Sep-Pak and in the water used for flushing the C-18 Sep-Pak, the former indicating irreversible bonding of Grignard derived compounds to the C-18 matrix itself and the latter indicating either incomplete reduction of the Grignard or iodination of the alcohol. The radiochemical yield of the procedure may be further improved by the optimisation of Grignard, LAH and HI solution concentrations and reaction times/temperatures of the critical reaction steps.
Conclusions:
It has been shown that 11C-benzyl iodides can be rapidly synthesised by the reaction of Grignard reagents immobilised on a C-18 matrix. The chromatographic properties of the C-18 solid phase support can be subsequently utilised to purify the labelled benzyl iodides. Furthermore, the procedure is easy to automate, and therefore may provide a stimulus for its more widespread use as a labelling agent.
Scheme 1. Automation Setup for the Synthesis of Benzyl Iodides.
Dansk resumé:
Valg af metode til mærkering af sporstoffer afhænge af tilgængeligheden af radioaktiv reaktans. De mest brugte, som også er de mest enkle at lave, er 11C carbon dioxid, 11C cyanid og 11C methyl iodid. 11C benzyl iodider er blevet brugt, men de beskrevne synteser tager 20-25 minutter og er svære at automatisere. En metode til fremstilling af 11C benzyl iodider ved hjælp af en "solid phase supported synthesis"”er blevet udviklet. Denne giver det ønskede produkt efter kun 8 minutter og med et radiokemiske udbytte på 40-45%. Denne syntese er blevet automatiseret ved brug af pneumatiske ventiler.
Referencer:
1. Antoni, G and Langstrom B.: J. Labelled Compd. Radiopharm. 24:125 (1987)
2. Fasth K.-J., Antoni G. and Langstrom B.: J.Chem.Soc., Perkin Trans. I: 2081 (1988)
3. Fasth K.-J., Antoni G. and Langstrom B.: Appl. Radiat.Isot. 41:611 (1990)
The terminal sterilisation of formulated radiopharmaceuticals is often achieved using a 0.22 mm membrane filter which is designed to remove microorganisms and particles before passage into a sealed sterile vial. In automated syntheses using this method of sterilisation, the presence of airbubbles in the pre-sterilised solution may cause high back pressures and resulting in a‘"blocked"’filter due to the formation of an airlock above the filter membrane. This problem can be overcome using a so-called "vented" filter. These filters posses a 0.02 µm hydrophobic filter (the so-called vent) which allows air, but not liquid, to be expelled. This effectively removes airlocks, allowing the formulation to pass unhindered over the 0.22 mm membrane (Scheme 1).
During the process of automating the syntheses of 6-[18F]-fluoro-DOPA and [1-11C]-glucose, the properties of vented sterile filters were investigated with a view to simplifying the automation setup of semi-preparative HPLC loop filling.
A pneumatically actuated Rheodyne injector was fitted with a 5 ml loop (i.d. 2 mm). The loop loading port was connected to PEEK tubing (i.d. 0.51 mm, 5 cm long), a low dead volume PEEK union and a female luer fitting. A closed conical reaction vial was equipped with a helium inlet and a Teflon transfer tube (i.d. 0.7 mm) connected to a male luer fitting. A Vented Millex-GS sterile filter (SLGS V25 5F) was connected between the Teflon transfer tube and the PEEK HPLC loop filling line (Scheme 2). Crude 6-[18F]-fluoro-DOPA (in 1.6 ml 57 % HI + 3.5 ml 3 M NaOH) or [1-11C]-glucose (in 2.5 ml 5 % formic acid) were transferred from the reaction vial via the vented filter to the HPLC injection loop using an over pressure (15 psi, 1 bar) of helium. Upon complete transfer of the crude product the autoinjector was activated and the solution chromatographed using an appropriate column.
Crude solutions of 6-[18F]-fluoro-DOPA or [1-11C]-glucose were transferred via the vented filter to the HPLC loop (i.d. 2 mm) and injected onto the column within 30- 60 seconds after application of a helium overpressure. Initial trials with a smaller bore injection loop caused too slow transfer of the product over the filter. Use of nitrogen over pressure caused the stopper of the reaction vial to open, presumably due to the decreased compressibility of nitrogen compared with helium gas. Losses of radioactivity were in the order of 5 % of the radioactivity contained within the reaction vessel. This is attributable to the post membrane dead volume of the filter (ca. 50 ml). An advantage of the described setup is the simplification of automation procedures, which otherwise may require the use of syringe drives and extra valves. The filters are tolerant of a wide range of solvents including alcohols and alkanes and buffer solutions which allows considerable scope in the applications of both straight and reverse phase chromatography.
This method has been applied in over 50 syntheses of 6-[18F]-fluoro-DOPA or 1-[11C]-glucose with excellent reproducibility and reliability and lends itself to the automation of routine tracer purifications.
Scheme 1. Diagram of a Vented Sterile Filter.
Scheme 2. Automation of HPLC Purifications Using a Vented Sterile Filter.
Dansk resumé:
Endelig sterilisering af radioaktive sporstoffer bliver ofte lavet med et 0.22 µm membran filter, hvilket fjerner mikroorganismer og partikler før leveringen i et sterilt hætteglas. I automatiserede produktioner bliver et ventileret filter brugt, fordi et normalt filter vil blive blokeret, hvis der er luft bobler i opløsningen. Efter levering af hele opløsningen ved hjælp af helium gas, vil filtret blive blokeret ved brug af non ventileret filtere. Dette fænomen er blevet brugt til automatisk levering af radioaktive sporstoffer til et "sample loop"” før injektion på en kromatografi kolonne. Denne proces har forenklet vores automatiske produktion af 18F fluoro-DOPA og 11C glukose.
Introduction:
Recommended procedures for the analysis of partially acetylated 18F-FDG derivatives have included HPLC chromatography using a LC-NH2 (amino) column in conjunction with a refractive index (RI) and radio detector or silica gel TLC using a radiodetector. However, due to advances in chromatography technology a growing number of FDG producers are currently using a Dionex Carbopac column in conjunction with pulsed amperometry detection (PAD) as a sensitive indicator of non-radioactive sugar derivatives in formulated FDG solutions (1). This technique has been found to be many times more sensitive than other recommended chromatographic procedures. The validation of new methods however must be carefully performed before using a chromatographic system: During the validation of the Dionex carbohydrate analysis system in our laboratory some years ago, it was determined that partially acetylated 18F-FDG (Ac18F-FDG) , arising from an incomplete hydrolysis of the labelled sugar precursor) was not detectable using this chromatographic system. It was concluded that the Ac18F-FDG was either "sticking"’on the column or becoming hydrolysed to FDG under the chromatographic conditions used. In the light of recent findings (2, 3) the latter seemes more feasible. However, it has recently come to the authors attention that some important sources of literature may be advocating the Dionex HPLC analysis of FDG in an inappropriate manner (ie. for the analysis of partiallly acetylated FDG in formulated products)(4). An investigation of the chromatographic properties of acetylated sugars using three different analytical systems was therefore carried out in order to confirm or refute our previous findings.
Experimental:
FDG was synthesised by a Merrifields resin supported fluorination of tetra-O-acetyl-D-mannose triflate (5) using a GE microlab synthesis system. After EOS, samples of the formulated product and radioactivity remaining in the resin (containing Ac18F-FDG) were subjected to chromatographic analysis on the following systems:
A)•Nucleosil LC-NH2 column (250 x 4.6 mm) eluted with acetonitrile/water (60/40) at a flow rate of 1 ml/min.
B)•Dionex Carbopac PA1 column (250 x 4 mm) eluted with NaOH (0.9625 M), at a flow rate of 0.5 ml/min.
Tetra-O-acetyl-D-mannose triflate (1) was subjected to chromatographic analysis A and B in addition to TLC using silica gel (eluent: 95/5 acetonitrile/water) (System C).
Results:
System A: Chromatograms of partially acetylated 18F-FDG derivatives using method A, clearly showed the presence of labelled byproducts which were absent in the formulated FDG final product (Fig 1a,b)
System B: 18F-FDG and Ac18F-FDG applied to the Dionex system showed only peaks corresponding to 18F-FDG and 18F-fluoride (Fig 2a,b) indicating that any Ac18F-FDG in the sample was hydrolysed by the eluent. Injection of FDG precursor (1) showed the presence of two peaks corresponding to glucose/mannose and deoxyglucose/deoxymannose (Fig 3).
System C: To ensure that 1 contained no traces of sugars or deoxy sugars which may have contributed to the peaks found on system B, the sample was analysed by TLC system C. No traces of glucose, mannose, deoxyglucose or deoxymannose were found in the sample.
Discussion:
System A and C were found to be suitable for the analysis of acetylated sugars. Using system B, Ac18F-FDG was hydrolysed on column to 18F-FDG . The FDG precusor (1) was also hydrolysed under these conditions to a mixture of mannose and/or glucose and deoxyglucose and/or deoxymannose (at the NaOH concentration used in this study, mannose co-eluted with glucose and deoxyglucose with deoxymannose). System B was found to be the most sensitive system for the detection of non-radioactive sugars and was also found to be useful in the detection of 18F-fluoride.
In light of these results, the authors conclude that LC-NH2(amino) HPLC or TLC are appropriate methods for the determination of Ac18F-FDG. The Dionex system has been found to be unsuitable for the routine analysis of Ac18F-FDG, although it is probably the method of choice for the determination of 18F-FDG non-acetylated sugars and 18F-fluoride. A combination of either system A or C with the Carbopac HPLC analysis (System B) provides a suitable set of chromatographic methods for the routine determination of radiochemical purity (18F-FDG, 18F-fluoride, and 18F-acetylated FDG) and non-radioactive sugars. It is stressed, however, that it is the responsibility of the quality assurance program associated with each FDG producer to ensure that the analytical techniques employed are appropriate for a particular application.
Acknowledgements:
MRC 12-1634.
Dansk resumé:
Som følge af nogle modsigende offentliggjorte anbefalinger, har vi gennemgået mulighederne for kvalitetskontrol af 18F-FDG. Vi har fundet at en LC-NH2HPLC kolonne er bedst til analyse af delvis hydrolyseret 18F-FDG. En Dionex Carbopac HPLC kolonne er ikke passende til denne analyse, men er meget god til analyse af 18F-FDG, 18F -fluorid, glukose, mannose og andre sukkere.
Referencer:
1.•Alexoff D.L., Casati R., Fowler J.S., Wolf A.P., Shea C., Schleyer D.R. and Shiue C-Y., Appl. Radiat. Isot., 43, 1313-1322 (1992).
2.•Fuchtner F., Steinbach J., Mading P. and Johannsen B., App. Radiat. Isot.: 47, 61-66 (1996).
3.•Varelis P. and Barnes R.K., Appl. Radiat. Isot.: 47, 731-733 (1996).
4.•Radiopharmaceuticals for Positron Emission Tomography’, Editors: Stocklin and Pike, Kluwer Academic Publishers, 1993, p 135.
5.•Toorongian S.A., Mulholland G.K., Jewett D.M., Bachelor M.A. and Kilbourn M.R., Nucl. Med. Biol.: 17, 273-279 (1990).
Due to expiration of warranty, 1996 was the first year in which responsibility for service and preventive maintenance of the cyclotron (GE PETtrace 200) as well as the PETcamera (Siemens/-CTI ECAT 961) was fully transferred to the hospital. This arrangement has worked out very satisfactorily, and thanks to our group of highly qualified technicians it has not been necessary to request assistance from the companies on any occasion. This means that operation of cyclotron and PET-camera have been possible with a minimum of downtime and at a cost significantly less than the price for a service contract from the suppliers.
The PET-camera is equipped with 3D capability, and all neurological studies of humans are routinely performed in 3D mode. In particular, activation studies with 15O water have benefitted from the substantial lowering of the injected activity compared to earlier 2D techniques. Body scans are still performed in 2D mode; however, a planned validation of the 3D technique for heart studies will show whether it is advantageous also to do these studies in 3D. Also it is the plan to move to 3D for some of the animal studies; however, upgrade to a more powerful computer for image reconstruction may be needed.
A home-built apparatus for automatic sampling and counting of blood samples was designed and constructed in 1996. This equipment will allow more accurate determination of input curves, in particular for short lived tracers (15O). Furthermore it will significantly reduce the radiation dose to the staff working with manual blood sampling today. Test and validation of the automatic sampler is scheduled for the first half of 1997.
The computer facilities at the PET-center continue to grow so that we now have 10 Unix workstations (SUN and SGI) and about 25 PCs on the local network. The Unix stations are mainly used for image analysis which mostly takes place with high level languages such as IDL and Matlab. The PCs are used for office tasks and control of various experimental setups. Major upgrades in 1996 include upgrade of the server to a SUN Ultra-2 running Solaris 2.5. Also, a DLT tape library was installed for routine backup of all data on the local network (about 40 GB) in one operation. From any computer there is access to the internet though the firewall of the hospital, and daily use of the services on the internet are essential for the research carried out in the PET-center. It should also be mentioned that a Wold Wide Web server has been setup on a PC installed outside the firewall, allowing people from outside the hospital to access this information. An anonymous FTP service is also in operation at this machine.
Cyclotron, PET-camera, and computer network are operated in cooperation with the Department of Medical Physics, which is represented in the PET-center by a medical physicist and two electronics technicians. In 1996, the two technicians visited the Siemens/CTI factory in Knoxville, TN, for training on PET-camera hardware and software.
Dansk resumé:
På grund af udløb af garantiperioden blev 1996 det første år, hvor hospitalet fuldstændigt overtog ansvar for drift og vedligeholdelse af cyklotron og PET-kamera. Denne ordning har fungeret særdeles tilfredsstillende, idet der endnu ikke har været behov for tilkald af ekstern assistance. PET-kameraet er udrustet med den nyeste teknologi til gennemførelse af skanninger efter den meget sensitive 3D teknik, hvilket benyttes rutinemæssigt for alle humane neurologiske studier.
PET-centrets computerfaciliteter er fortsat blevet styrket, og omfatter nu 10 Unix arbejdsstationer og ca. 25 PC'er. Der er installeret en Wold Wide Web server som giver adgang til information om centrets forskningsaktiviteter.
Drift af cyklotron, PET-kamera, samt computer netværk varetages i samarbejde med Afdeling for Medicinsk Fysik, som er repræsenteret ved en hospitalsfysiker og to elektroniktekniker.
1996 has been a busy year for the chemistry group. The number of tracer productions for PET studies increased by 70% compared with 1995. This has therefore been a good year for testing the quality and reliability of our working procedures and tracer production regimen. It is gratifying to see that, despite this increase, failure rates have remained low (2% in 1996, cf 2 and 6% in 1995 and 1994 respectively) and the quality high. Our new crew of laboratory technicians have undergone intensive training in the production and quality control of our‘"workhorse" radiopharmaceuticals (FDG, H2O, NH3 and FDOPA) and are now the backbone of our routine production regime. This has enabled the Quality Control and Production Managers (A. Gee and D. Bender) to concentrate on the smooth running and maintaince of standards of the chemistry facility in light of our increased activity. The year has seen the introduction of new tracers, for both research and human use, establishment of HPLC metabolite analyses and healthy progress in the basic chemistry research program.
Table I: Tracer Production in 1996
| Total Number of Productions | 1963 |
| Productions for Patients | 939 |
| Productions for Animal Studies | 392 |
| Productions for QC, system tests and research | 632 |
| Chemistry Failures | 40 |
Table II: Current Repertoire of Tracers
| Tracer | Application |
| [18F]Fluorodeoxyglucosea,b | glucose metabolism |
| [13N]Ammoniaa,b | blood flow, glutamine synthetase |
| [15O]H2Oa,b | blood flow |
| [15O]O2a,b | oxygen consumption |
| [18F]6-FluoroDOPAa,b | dopamine synthesis |
| [18F]Fluoridea,b,c | bone metabolisms |
| [11C]Methamphetamineb | amine binding sites |
| [11C]ß,ß-Difluoromethamphetamineb* | amine binding sites |
| [11C]Venlafaxineb* | monoamine reuptake |
| [11C]Racloprideb | D2-receptor |
| [11C]Methyl Spiperoneb | D2 & 5-HT2 receptors |
| [11C]Methionineb | protein synthesis |
| [11C]NS 2214b* | dopamine reuptake |
| [11C]Glucoseb,c | glucose metabolism |
| [15O]Butanolb,c | blood flow |
| [11C]COb,c | blood volume |
| [15O]COb,c | blood volume |
| [18F]ß-Fluorophenylalaninec,d* | protein synthesis, aa transport |
| [11C]Aspartic Acidc,d* | protein synthesis, aa transport |
| [11C]2,4-Diaminobutyric acidc,d* | protein synthesis, aa transport |
| [11C]Asparginec,d* | protein synthesis, aa transport |
| [11C]Nefopamb* | monoamine reuptakee |
| [11C]Methyl FPL 1221AAb* | monoamine reuptakee |
| [11C]Methyl Nitroquipazineb,c* | 5-HT reuptakee |
Table III: Syntheses Planned for 1997
| [18F]ß-FluoroDOPA | dopamine synthesis |
| [11C]PK 11195 | periferal benzodiazepine receptors |
| [11C]ß-CIT-FE | dopamine reuptake |
| [18F]Fluoro Misonidazole | hypoxia |
Licencing:
1995 saw the approval of two new radiopharmaceuticals for human use: 18F- fluoroDOPA for the measurement of dopamine synthesis and 18F -f luoride for bone imaging. Working procedures and Quality Assurance measures have been continually revised and improved during the course of the year.
New Tracers:
In 1996 the chemistry group developed syntheses for 5 new tracers: [11C]Aspartic Acid, [11C]2,4Diaminobutyric acid, [11C]Aspargine and [11C]Methyl Nitroquipazine. Manuscripts describing the results of this work are in preparation are are expected to be published in international journals during 1997. In addition, the "known"’tracers: 18F-Fluoride, 11C-Glucose, 15O-Butanol, 11C-CO and 15O-CO; have been introduced to the repertoire of available radiopharmaceuticals at the PET Centre.
Automation:
Continuing on our earlier success in the automation the synthesis of FDOPA and research fluorinations, 1996 saw the development of automated procedures for 11C-Glucose and 15O-Butanol. The flexible computer-controlled manipulation of pneumatic valves and pistons has shown to be very reliable and robust for the remote-control of routine and research radiosyntheses.
Amphetamine Project:
Work has continued on investigating the pharmacology of amphetamine derivatives. A current research theme is an investigation into the effect of amine pKa on binding affinity of amphetamine derivatives in the brain. Our approach has been to compare the in vivo binding properties of two tracers: 11C-methamphetamine and 11C-ß,ß-difluoromethamphetamine of high (10) and low (7.5) pKa respectively. These studies showed that 11C-methamphetamine was retained in the brain whereas 11C-ß,ß-difluoromethamphetamine was rapidly removed. The validity of this approach, however, assumes that the introduction of two fluorine atoms into the beta position of methamphetamine causes no steric changes in the molecule which would influence the ‘fit’ of the molecules to amine binding sites. This question was adressed in collaboration with Karl-Anke Jørgensen at the University Chemistry Department, by performing ab intio molecular modelling of the two compounds. The calculations show that the two amphetamine analogs are almost sterically identical, and only differ significantly in their charge density on their respective amine moieties. This data, supports the validity of our experimental approach and provides additional evidence that the brain binding of amphetamine analogs is directly related to the pKa of the substrate.
Development of Labelling Reagents:
The number of 11C and 18F labelling reagents which can be readily produced in high yields for routine rediopharmaceutical syntheses is limited. The devopment of new labelling agents and techniques is an important research area which may result in the labelling new tracers for diagnostic and research studies. During 1996, a new method for the rapid synthesis and purification of benzyl iodides using solid phase technology was developed. 11C-benzyl iodides are now being produced via the trapping of 11C-carbon dioxide on a C18 matrix impregnated with an appropriate Grinard reagent. Subsequent hydrolysis, iodination and purification is performed using the same matrix, affording these labelling agents in a synthesis time of ca. 8 minutes. The reaction sequence has been automated so that these new labelling reagents can be further evaluated as precursors in the synthesis of radiotracers.
Azridine Project:
An investigation into the potential use of aziridines in rapidlabelling syntheses has enabled the labelling of amino acids with 18F in the beta position. The methodology has now been applied to ring opening reactions with 11C-labelled nucleophiles. Labelling of aziridine carboxylate esters with 11C-cyanide produces b-cyano amino acid esters which can be used as a versatile intermediates for the synthesis of a number of amino acids. Depending on the choice of reduction and/or hydrolysis conditions this intermediate has been successfully converted to [4-11C]aspatic acid, [4-11C]2,4-diaminobutyric acid or [4-11C]asparagine. In vivo evaluation of these new tracers is expected in 1997.
11C-Glucose:
[2-18F]Fluoro-2-deoxy-D-glucose (FDG) is a widely used radiopharmaceutical for the measurement of glucose utilisation by PET. Despite the widespread use of the tracer, it should be remembered, that FDG is a glucose analogue. The estimation of endogenous glucose transport and phosphorylation based on FDG data usually relies upon the use of a conversion factor, the so-called lumped constant (LC). This constant has been experimentally determined in healthy tissue, in animals and man. However, there is little indication that the LC has the same value in diseased tissue. Indeed, recent reports show, that the LC can significantly vary in magnitude even in healthy tissue. The Lumped Constant is therefore not a constant but a variable. To enable the calculation of the LC in various normal and diseased tissue a simplified synthesis of [1-11C]glucose was developped. The synthesis utilises the Kiliani-Fischer synthesis, modified with a solid phase supported reaction of n.c.a. 11C-CN with arabinose to shorten reaction times, simplify work-up procedures and automation and increase the amount of tracer avaible for injection. In vivo evaluation of [1-11C]glucose is expected in 1997.
European Cooperation in the Field of Scientific and Technical Research (COST):
In 1996, A. Gee became the Danish representative for the COST B3 action on "New Radiotracers and Methods of Quality Assurance for Nuclear Medicine Application". The purpose of this action is to encourage and develop a European network for scientific collaboration and dissemination of ideas and results within a particular interest area. Current areas of collaborations include the development of tracers for brain and heart neurotransmision, enzyme inhibitors, standardisation of labelling and QA procedures and the development of new labelling reagents and methods.
Dansk resumé:
1996 har været et aktivt år for kemisektionen. Produktionen af PET-sporstoffer var 70% større end i 1995. Vores nye team af laboranter har undergået en intensiv træning i produktion og kvalitetskontrol af de PET sporstoffer, som er rygraden i vores produktion. I 1996 fik vi godkendt to nye sporstoffer til human in vivo administration: 18F fluorodopa til måling af dopaminsyntese og 18F] fluorid til knogleskanning. Kemigruppen har udviklet 4 nye sporstoffer: 11C aspartat, 11C asparagin, 11C 2,4-diaminbutyrat og 11C metyl nitroquipazin. Allerede igangværende projekter blev fortsat i 1996, bl. a. undersøgelse af amfetaminderivaters farmakologi for at finde effekten af pKa på hjernebinding, og undersøgelse af aziridiner som reaktanter i syntesen af [18F] og [11C] aminosyrer. Også en forbedret metode til syntese af [11C] glukose med brug af "solid phase supported"” reaktion er blevet udviklet. Denne syntese er blevet fuldautomatiseret og in vivo evaluering forventes i 1997.
Referencer:
Gee A.D., Gillings N., Smith D.F., Inoue O., Kobayashi K. and Gjedde A.‘"The Effect of Amine pKa on the Transport and Binding of Amphetamine Analogs in the Pig Brain: An in Vivo Comparison of ß,ß-Difluoro[N-methyl]methamphetamine and [N-methyl-11C]Methamphetamine Uning PET’": in Quantification of Brain Function Using PET, Academic Press, 42-46 (1996).
Gee A.D. ‘The Chemistry of Neuroreceptor Ligands: From Atoms to Neurons’ J. Neurochemistry, 66, 67 (1996).
Advances in clinical science often arise from discoveries made in basic research carried out using laboratory animals. We have selected pigs as laboratory animals for use at the PET Center for several reasons. First, they have been used successfully over the years by our colleagues at the Institute of Experimental Clinical Medicine (Head: Prof. J.C. Djurhuus) at Skejby Hospital where it has been possible to obtain assistance for our work. Second, the limit of resolution of our PET scanner, namely ca. 4 mm, precluded the use of small laboratory animals such as rodents for studying pharmacokinetic processes in living brain regions. On the other hand, the brain and liver of the pig are large enough for studying regional pharmacokinetics, hemodynamics and metabolism by PET (Smith et al., 1996). Pigs are relatively inexpensive and bear few health hazards for humans (Tumbleson, 1986). Fourth, pig brain shows sufficient affinity for the types of radiotracers that we are interested in, such as antidepressant drugs with specific actions on 5-HT uptake sites (Brust et al., 1996) as well as compounds interacting with dopaminergic neuropathways (Gee et al., 1996). Fifth, current developments in organ transplantation indicate that the pig may be suitable as brain tissue donor for humans afflicted by neurodegenerative diseases, a topic of interest for the PET Center. Studies on liver and cardiac function are also routinely carried out. Minipigs have offered the opportunity of using PET for studies of the nervous system and liver function in adult animals. The findings obtained using PET imaging in laboratory animals are being analysed and applied for inventing new procedures for investigating various human disorders. The results obtained by the individual projects are presented by the principal scientists elsewhere in this annual report.
Blood Flow: The pig brain's blood flow is measured during the circulation of radioactive water after i.v. bolus injection. The blood flow is calculated as the unidirectional clearance of the radioactive water, based on an arterial input curve. The measurement lasts 3 minutes and the radioactive water decays completely in 20 minutes. We have established the average brain blood flow of the normal pig to be 45 ml hg-1 min-1.
Oxidative Metabolism: The pig brain's metabolic rate of oxygen is measured during a single inhalation of radioactive oxygen. The oxygen consumption is calculated as the product of the unidirectional clearance of the radioactive oxygen and the arterial concentration of oxygen. The measurement lasts 3 minutes and the radioactive oxygen has decayed completely in 20 minutes. The average striatal oxygen consumption of the normal pig bran is 170 µmol hg-1 min-1.
Dopamine Transporter Density: The density of dopamine transporters in pig brain is measured during the circulation of radioactive NS2214 after i.v. bolus injection. The density is measured as the rate of binding (or binding potential) of radioactive NS2214, a specific dopamine transporter ligand, calculated as the ratio between the net clearance of NS2214 and the steady-state distribution volume of NS2214 in striatum (k3). The measurement lasts 90 minutes and the radioactive NS2214 decays completely in 2.5 hours. The average volume of distribution of NS2214 in pig striatum is 19 ml g-1, compared to 12 ml g-1 in the cerebellum, corresponding to a binding potential of 0.65. The normal striatal dopamine transporter density of the pig is therefore close to 75 pmol g-1.
Dopamine D2-like Receptor Density: The density of dopamine D2 receptors is measured during the circulation of radioactive raclopride after i.v. bolus injection. The density is measured as the binding potential of radioactive raclopride, a specific dopamine D2-D3 receptor ligand, calculated as the ratio between the steady-state volumes of distribution of radioactive raclopride in a binding and nonbinding region of the brain (k3/k4). The measurement lasts 90 minutes and the radioactive raclopride decays completely in 2.5 hours. The average volume of distribution of raclopride in pig striatum is 1.2 ml g-1, compared to 0.3 ml g-1 in the cerebellum, corresponding to a binding potential of 3. The normal striatal dopamine D2 receptor density of the pig is therefore close to 20 pmol g-1.
Glycolytic Rate: The metabolic rate of glucose is measured during the circulation of radioactive fluorodexoyglucose (FDG) after i.v. bolus injection. The glucose consumption is calculated as the ratio between the net clearance of the radioactive FDG and the lumped constant. The measurement lasts 45 minutes and the radioactive FDG decays completely in 10 hours. The average rate of glucose phosphorylation is 30 µmol hg-1 min-1 in the pig brain.
Dopamine Synthetic Rate: The rate of decarboxylation of DOPA is measured during the circulation of radioactive fluorodopa (FDOPA) after i.v. bolus injection. The decarboxylation rate is calculated as the ratio between the net clearance of FDOPA and the steadystate distribution volume of FDOPA (k3). The measurement lasts 90 minutes and the radioactive FDOPA decays completely in 10 hours. The average rate of FDOPA decarboxylation in the pig striatum is 0.020 min-1, compared to 0.005 min-1 in cerebral cortex, and 0.001 min-1 in cerebellum, the latter not significantly different from nil.
These average measurements in the control state are all lowered by the so-called partial volume effect due to the small size of the pig regions in relation to the power of resolution of the PET scanner. Although the partial volume effect depends on surrounding radioactivity, it is likely to be of similar magnitude for all measurements mentioned here.
Figure 1: Silhouette showing transaxial plane through head of a pig showing (in red) the location of brain regions that bind serotonin.
Acknowledgements:
This work received financial support from Institute of Experimental Clinical Research, Aarhus University. We thank the entire”"crew"”at the Animal Colony, Påskehøjgårdcentret, for their unwavering assistance.
Dansk resumé:
Der er en tradition i Århus for at anvende grise som forsøgsdyr i eksperimentel klinisk forskning, og vores erfaring er nu at grise faktisk er meget velegnede til PET skanning. For det første er de store nok til at blive undersøgt ved hjælpe af vores PET skanner. For det andet, kan mennesker omgås grise uden fare for smitsomme sygdomme. For det tredje har hjerne og lever hos grise en opbygning der har mange egenskaber tilfælles med mennesket. Vi har anvendt grise i forskningsprojekter som er omtalt andetsteds i denne årsrapport.
Referencer:
Brust, P., Bergmann, R. and Johannsen, B. (1996) Highaffinity binding of [3H]paroxetine to caudate nucleus and microvessels from porcine brain. NeuroReport 7, 1405-1408.
Gee, A.D., Gillings, N., Smith, D.F. and Gjedde, A. (1996) The effect of pKa on the transport and binding of amphetamine analogues in pig brain: An in vivo comparison of difluoro-N-methyl-methamphetamine. In: Myers, R., Cunningham, V., Bailey, D. and Jones, T. (Eds.), Academic Press, San Diego, Calif. pp. 42-46.
Smith, D.F., Glaser, R., Gee, A., Gjedde, A. (1996) [11C]Nefopam as a potential PET tracer of serotonin reuptake sites. In: Myers, R. and Bailey, D. (Eds.), Quantification of Brain Function: PET. Academic Press, San Diego, Calif. pp. 38-41.
Tumbleson, M.E. (Ed.) (1986) Swine in Biomedical Research. Volume 1. Plenum Press, New York.
The year brought in many changes regarding the medical laboratory technicians. New staff was hired due to the increased demand for scanning time. Ilse Rasmussen joined the staff in March and Vikie Larsen joined in June. The original 2 technicians transfered to other departments in the summer. To fill the vacancies Helle Larsen joined the staff in October and Gloria Stocks-Gee returned from maternity leave in November. With the new staff intensive training was required and is ongoing, with the gearing up for the increased scanning schedule to begin in February 1997.
The technicians are responsible for many areas dealing with the performance of the PET scans. These areas include scheduling and organization, clinical and research scanning, as well as chemistry and radiopharmaceutical production. The technicians work closely with the clinical co-ordinator and investigators to schedule the weeks’ scans, ensuring that the timing and tracer productions are possible and co-ordinated with the radiochemists. Journals are then prepared and patients notified of any special instructions for their scan. The technicians also do inventory and ordering of all the radiopharmaceuticals, goods and medicines required for clinical and research scans.
Clinical and research scanning use a number of varied protocols and the technicians are responsible for the acurate and reliable performance of these protocols. Among the protocol procedures are Quality Control of the Dose calibrator, PET scanner, and well counter, protocol setup, image reconstruction, long term archiving, Patient Preparation including i.v. catheters, ECG monitoring, blood pressure monitoring, drug preparation, Blood Sampling including computer set-up, whole blood and plasma, arterial and venous, Image Analysis with further processing of images, co-registration with MRI images.
The technicians also perform chemistry and hemotology testing as well as make routine radiopharmaceuticals for scanning. Processing of the blood sampling are performed per protocol. The blood plasma, and metabolic extracts are then measured using a well-counter. Evaluation of glucose and hematocrit are also perfomed routinely.
The technicians make the routine radiopharmaceuticals, H215O, 13NH3, 18FDG and have started training on the synthesis of 18FDOPA. In addition, the quality control of these tracers are performed by technicians using HPLC, GC and pH paper prior to the release for patient use.
The training of the technicians in all these areas is nearly complete. The new staff enjoy the work and ensure that the quality of PET scans are the best possible.
Dansk resumé:
Da alle 4 laboranter (Ilse Rasmussen, Vikie Larsen, Gloria Stocks-Gee og Helle Larsen) er startet i 1996 har en del af arbejdet bestået i oplæring. Dette er gået godt og de var klar til udvidet skanningstid, som startede i februar 1997.
Laboranterne er ansvarlige for mange af de ting, der vedrører PET-skanningerne. Planlægning og fordeling af skantider, indkaldelse af patienter, fremstilling af isotoper samt lave kvalitetskontrol på disse. Derudover står de for betjening af skanner, blodtryksmåling, EKG, isætning af venflon, blodprøvetagning, analysering af blodprøverne samt vedligeholdelse og kvalitetscheck af diverse apparater, her i blandt PET skanner, dosis kalibrator og tælleapparat. Efter skanning arkiveres de data, der er fremkommet og udfra disse laves billeder til analysering og senere brug. Laboranterne står ligeledes for bestilling af mange af de ting, som benyttes på PET centret.
Isotoperne, som laboranterne fremstiller, er 15O-H2O, 13NH3,18FDG og snart mestrer alle også 18FDOPA. Til kvalitetskontrol af disse benyttes HPLC, gaskromatografi og pH-måling før de frigives til brug ved humane skanninger.
Arbejdet ved skanneren indebærer opsætning af korrekt protokol, korrekt placering af personen i skanneren, samt sørge for, at denne befinder sig godt under skanningen. Det nye team nyder arbejdet og står inde for, at kvaliteten af PET skanningerne altid er bedst mulig.
The PET Center at Aarhus University Hospitals conducts nuclear medicine examinations required by specialists in and outside Aarhus County, for the purpose of diagnosis or research. With positron emission tomography (PET), it is possible to image the function of an organ after administration of a radioactive tracer.
With PET it is possible to accurately measure in vivo organ physiology, metabolism, drug distribution, and receptor function non-invasively. This unique aspect of PET is consequentially important for examinations in certain clinical situations.
Current clinical applications of PET include the diagnosis of myocardial viability, malignant diseases, Parkinson's disease, examination for dementia, and location of epileptic foci.
Before coronary bypass surgery of the heart, PET can be used to examine the regional viability of the heart muscle. This is important in certain cardiac patients where the results may contribute to the decision whether and how a surgical intervention should be performed.
Before neurosurgery for arterial venous malformation (AVM) and brain tumors, a PET examination can accurately map the position of vital parts of the brain in relation to the AVM or tumor. This may provide the neurosurgeon with useful information about how to perform the operation without damaging properly functioning areas of the brain, which may lie in proximity to the targeted area of surgery.
PET examinations are expensive and the capacity is limited, so it is important to select the clinical problems carefully. Clinical research in the PET-centre continues to identify diseases and clinical situations where PET examinations are appropriate.
An examination often consists of repeated scans with different tracers to reveal more than one function in the healthy or diseased body. In some cases, up to 12 scans are performed. A "reimbursement item" is the hospital's unit of payment. Every examination consists of several "reimbursement items", such as i.v. injection, blood sampling, and image analysis.
In 1996, the PET Center performed 1256 emission scans, consisting of 285 diagnostic scans and 971 research scans, of the latter 635 in humans, and 336 in pigs. This activity represents 5 examinations per working day. Organs were examined as listed in Table I. Table II lists the tracers used. Table III shows the examinations performed for different departments and projects.
Dansk Resume:
PET-centret ved Århus Universitetshospital udfører undersøgelser efter henvisning fra inden- og undenamtslige specialister med henblik på diagnostik eller forskning. Med positron-emissionstomografi (PET) er det efter injektion af et radioaktivt sporstof muligt at danne billeder af et organs funktion.
Ofte gentages undersøgelserne med forskellige sporstoffer for at belyse flere funktioner i den syge eller raske organisme. I nogle tilfælde kan indtil 12 undersøgelser komme på tale. En ydelse ('reimbursement item') er hospitalets betalingsenhed. Hver undersøgelse består af flere ydelser i form af injektioner, blodprøvetagning, billedbearbejdning m. fl.
I 1996 udførte PET-centret i alt 1256 undersøgelser, fordelt på 285 diagnostiske undersøgelser og 971 forskningsundersøgelser, heraf 635 på mennesker og 336 på grise. Dette repræsenterer 5 skanninger om dagen. Fordelingen på organer fremgår af tabel I. Tabel II viser de sporstoffer, der er anvendt. Tabel III viser undersøgelserne fordelt på hospitaler, afdelinger og projekter.
De vigtigste diagnostiske undersøgelser har været undersøgelse af hjertets viabilitet før eventuel operation, kortlægning af primære hjernecentre inden neurokirurgisk operation for arteriovenøs malformation eller tumorer, demensudredning, kortlægning af epileptiske foci og undersøgelse af tumorer.
Table I: Organs examined in 1996
| Subjects | Whole-body | Brain | Heart | Liver | Bone | Total |
| Clinical Patients | 2 | 37 | 43 | 1 | 0 | 83 |
| Volunteer Patients | 0 | 48 | 31 | 9 | 2 | 90 |
| Normal Volunteers | 0 | 59 | 64 | 0 | 0 | 123 |
| Research Animals | 1 | 55 | 2 | 21 | 0 | 79 |
| Total | 3 | 199 | 140 | 31 | 2 | 375 |
Please note that cancelled examinations are included in this table.
Table II: Tracers used in 1996
| Emission scans | Clinical Patients | Volunteer Patients | Normal Volunteers | Research Animals | Total |
| 15O Butanol | 0 | 0 | 0 | 40 | 40 |
| 11C-Amphetamine analog | 0 | 0 | 0 | 19 | 19 |
| 11C-Aminoacids | 0 | 0 | 0 | 6 | 6 |
| 18F-FDOPA | 4 | 12 | 9 | 10 | 35 |
| 18F-Fluoride | 0 | 1 | 0 | 0 | 1 |
| 15O-H2O | 179 | 169 | 213 | 108 | 669 |
| 18F /11C-Hexoses | 57 | 15 | 0 | 9 | 81 |
| 11C /15O-Carbon monoxides | 0 | 0 | 0 | 46 | 46 |
| 13N-NH3 | 45 | 60 | 141 | 40 | 286 |
| 15O-O2 | 0 | 4 | 11 | 24 | 39 |
| 11C-Receptor ligands | 0 | 0 | 0 | 19 | 19 |
| 11C-Reuptake inhibitors | 0 | 0 | 0 | 15 | 15 |
| Total | 285 | 261 | 374 | 336 | 1256 |
Please note that tracers produced but not injected are not included in this table.
Table III: Departments served in 1995 and 1996
| 1995 | 1996 | |||||
| Subjects | Emission Scans | Reimbursement items | Subjects | Emission Scans | Reimbursement items | |
| Clinical patients: | ||||||
| Aarhus General Hospital | ||||||
| Oncology Dept. D | 1 | 1 | 1 | |||
| Orthopaedic Surgery Dept.. E | 1 | 1 | 1 | |||
| Neurology Dept.. F | 10 | 27 | 50 | 24 | 97 | 142 |
| Neurosurgery Dept. GS | 1 | 1 | 5 | 4 | 30 | 40 |
| Hepatology Dept. V | 10 | 10 | 48 | 1 | 1 | 5 |
| Other Departments | 17 | 17 | 41 | |||
| Aarhus General Hospital, total | 38 | 55 | 144 | 31 | 130 | 189 |
| Aarhus Amtssygehus, Haematology B70 | 1 | 1 | 2 | |||
| Skejby Hospital, Cardiology Dept.. B | 41 | 76 | 117 | 43 | 85 | 132 |
| Aalborg Hospital, Neurosurgery Dept. | 6 | 50 | 63 | 7 | 67 | 80 |
| Odense Hospital, Neurology Dept.N | 1 | 2 | 4 | |||
| Other hospitals | 2 | 3 | 5 | |||
| Clinical patients total: | 87 | 184 | 329 | 83 | 285 | 407 |
| Clinical patients inside/outside Aarhus County: | ||||||
| Patients inside Aarhus County | 45 | 96 | 170 | 42 | 120 | 184 |
| Patients outside Aarhus County | 41 | 87 | 155 | 41 | 165 | 223 |
| Self payers | 1 | 1 | 4 | |||
| Human research: | ||||||
| Aarhus General Hospital | ||||||
| Anaesthesia Dept. N, project 16 | 13 | 32 | 122 | |||
| Radiology Dept. R, project 19 | 2 | 2 | 10 | |||
| Neurosurgery Dept. GS, project 01 | 3 | 9 | 30 | |||
| Neurology Dept. F, Project 07 | 29 | 148 | 206 | 10 | 52 | 72 |
| Neurology Dept. F, Project 08 | 22 | 21 | 106 | |||
| Neurology Dept. F, Project 10 | 19 | 115 | 153 | |||
| Neurology Dept. F, Project 24 | 8 | 8 | 37 | |||
| Neurology Dept. F, Project 28 | 7 | 75 | 89 | |||
| Neurology Dept. F, total | 48 | 263 | 359 | 47 | 156 | 304 |
| ENT Dept. H, project 03 | 5 | 40 | 50 | 5 | 40 | 50 |
| Hepatology Dept. V, project 26 | 9 | 9 | 45 | |||
| PET Center, project 05 | 2 | 5 | 18 | |||
| PET Center, project 22 | 1 | 6 | 7 | 1 | 8 | 10 |
| Total, PET Center, total | 1 | 6 | 7 | 3 | 13 | 28 |
| Aarhus General Hospital, total | 54 | 309 | 416 | 82 | 261 | 589 |
| Psychiatric Hospital | ||||||
| Biological Psychiatric Inst., project 06 | 4 | 26 | 34 | 31 | 173 | 233 |
| Psychiatric Hospital, total | 4 | 26 | 34 | 31 | 173 | 233 |
| Skejby Hospital, Cardiology Dept. | ||||||
| Cardiology Dept. B, project 04 | 8 | 8 | 48 | |||
| Cardiology Dept. B, project 13 | 16 | 40 | 72 | |||
| Cardiology Dept. B, project 14 | 24 | 48 | 96 | 17 | 29 | 60 |
| Cardiology Dept. B, project 23 | 15 | 38 | 68 | 57 | 132 | 243 |
| Cardiology Dept. B, project 25 | 21 | 40 | 81 | |||
| Skejby Hospital, Cardiology Dept., total | 63 | 134 | 284 | 95 | 1 | 384 |
| Human research total: | 121 | 469 | 734 | 208 | 635 | 1206 |
| Totals: | ||||||
| Clinical patients | 87 | 184 | 329 | 83 | 285 | 407 |
| Human research | 121 | 469 | 734 | 208 | 635 | 1206 |
| Human research and clinical patients | 208 | 653 | 1063 | 291 | 920 | 1613 |
| Animal research | 50 | 131 | 491 | 78 | 336 | 1152 |
| Phantoms | 1 | 2 | 8 | |||
| Research total | 172 | 602 | 1233 | 286 | 971 | 2358 |
| Examinations total: | 259 | 786 | 1562 | 369 | 1256 | 2765 |
In 1996, Albert Gjedde gave invited lectures to the Institutskolloquium, Forschungszentrum Rossendorf, Institut für Bioanorganische und Radiopharmazeutische Chemie in Rossendorf in March, to the 11th ESN Meeting of the European Society for Neurochemistry in Groningen in June, to The Sixth Asia and Oceania Congress of Nuclear Medicine and Biology in Kyoto, to the Cost B3 Conference, Preclinical Pharmacological Studies With & For Radiopharmaceuticals, Biological Tools or Models at the Service Hospitalier Frédéric Joliot in Orsay in October, and to the 504th meeting of the Jutland Society of Medical History in Aarhus in November. He attended the opening of the Institute of Neurology, Wellcome Department of Cognitive Neurology, Leopold Muller Functional Imaging Laboratory, in London in April and co-authored an oral presentation to the 26th Annual Meeting, Society for Neuroscience in Washington in November.
Susanne Keiding gave invited lectures to the Danish Association for Clinical Pharmacology in Copenhagen in March, to the Danish Association for the Study of the Liver in Copenhagen in March and in Aarhus in December, to the Scientific Colloquiem at Aarhus County Hospital in March, to the Danish Kidney Society in Holstebro in September, to the Nordic Liver Transplantation Group in Gothenburg in October. Presented posters at the Biennial Scientific Meeting in the International Association for the Study of the Liver in Cape Town in February and at the 30th Annual Scientific Meeting of the European Society for Clinical Investigation in Interlaken in April. Susanne Keiding was Council member and Vice President in the European Society for Clinical Investigation and participated in the organization of the 30th Annual Scientific Meeting of the society in Interlaken. She participated in the European Conference on Research and Application of Positron Emission Tomography in Oncology in Groningen in May, and the Scientific Meeting of the American Association for the Study of Liver Diseases in Chicago in November.
Antony Gee gave invited lectures to the 11th Meeting of the European Society of Neurochemistry in Groningen in June, to the scientific meeting of the Danish Association for Clinical Pharmacology in Copenhagen in March. Participated as Chairman for the European Nuclear Medicine Congress in Copenhagen in September, to the Cost B3 Conference, Preclinical Pharmacological Studies With & For Radiopharmaceuticals, Biological Tools or Models, at the Service Hospitalier Frédéric Joliot in Orsay in October. Participated with a poster at The First Easter School on Radiopharmaceuticals in Liverpool in March.
Klaus Roelsgaard participated with posters at the Biennal Scientific Meeting of the International Association for the Study of the Liver in Cape Town in February and to the 30th Annual Scientific Meeting of the European Society for Clinical Investigation in Interlaken in April, Dansk Endokrinologisk Selskabs Årsmøde, Ålborg and patricipated in the annual meeting of the Danish Society for Endocrinology in Aalborg in January.
Morten Bøttcher participated with posters at the annual meeting of the American College of Cardiology in Orlando, Florida in March, to the 2nd World Congress on Heart Failure in Jerusalem, to the annual meeting of the Danish Society of Cardiology in Odense in May, to the American Heart Association Meeting in New Orleans in November.
Anders Rodell and Flemming Andersen participated at the VBC 1996 Meeting, Visualization in Biomedical Computing 1996 in Hamburg in September.
Graduate student Nicholas Gillings and Dirk Bender participated with a poster at The First Ester Shool in Radiopharmaceutics in Liverpool in March and participated at the 5th Anniversary Symposium, The Frontiers of PET and its use in drug development at Uppsala University PET Centre in Uppsala, and attended the Synthia Usermeeting at Uppsala PET Centre in October.
Flemming Hermansen participated with posters at the ECAT Technical Users Meeting in Stockholm in April and at the European Nuclear Medicine Congress in Copenhagen in September.
Donald F. Smith participated with posters at the European Nuclear Medicine Congress in Copenhagen in September and at the Cost B3 Conference, Preclinical Pharmacological Studies with and for Radiopharmaceuticals at the Service Hospitalier Frédéric Joliot in Orsay in October.
Peter Johannsen participated with posters at the annual meeting at the Danish Association for Neuroscience in Sandbjerg in May and at the annual meeting of the Danish Neurological Society in Korsør and participated in the 2nd International Congress on Mapping of the Human Brain in Boston in June and in »The Decade of the Brain: Midpoint Review Joint Meeting of IPA and NorAge« in Reykjavik in October.
Søren B. Hansen participated at the European Conference on Research and Application of Positron Emission Tomography in Oncology in Groningen in May, at the Brain Imaging Seminar of the Danish Society for Clinical Physiology and Nuklear Medicin in Copenhagen in June, at the Sixth International Radiophamaceutical Dosimetry Symposium in Gatlingburg in May and at the Quantitative Medical Imaging, Danish Society for Medical Physics in Aarhus in October.
Circulation, measurements and regulation
February
5. Maurice Ptito, Professor, PhD, Université de Montréal and Montréal Neurological Institute, Canada: Studies of Residual vision in Hemicorticectomized human subjects.
12. Maurice Ptito, Professor, PhD, Université de Montréal and Montréal Neurological Institute, Canada: Hemispherectomy in monkeys: Anatomical studies.
22. Maurice Ptito, Professor, PhD, Université de Montréal and Montréal Neurological Institute, Canada: What the frog's brain tells the monkey's brain: Collicular functions.
March
4. Ida Holm, MD. Institute for Anatomy and Biology, Aarhus University : The hippocampal region of the domestic pig.
11. Donald Smith, DSc., Ph.D., PET Centre & Psychiatric Hospital, Aarhus University Hospitals: An update on PET Antidepressant binding.
18. Klaus Roelsgaard, M.D., PET Centre & Dept. L, Aarhus University Hospitals: Effects of brain death and glucose infusion on hepatic glycogen in pigs.
25. Marcus Keep, M.D., University of Lund, Dept. of Neurology: Neural transplantation in Parkinson's disease. Observations on the Lund experience.
April
15. Klaus Roelsgaard, M.D. PET Centre & Dept. L, Aarhus University Hospitals: Hepatic glucose production during hypoglycemia in pigs -effects of hepatic innervation.
22. Erik Danielsen, M.D., PET Centre, Aarhus University Hospitals: F-DOPA uptake and metabolism in the pig.
29. Peter Ott, MD., Dept. A, Rigshospitalet, Copenhagen: Hepatic ICG removal in the pig and rat liver depends on plasma protein concentration and hematocrit: Evidence of sinosoidal binding disequilibrium and unstirred water layer effects.
May
13. Albert Gjedde, Professor, PET Centre, Aarhus University Hospitals: Indicator fractionation: The use of chemical and solid microspheres.
20. Susanne Keiding, MD., D.Sc., PET Centre & Dept. V, Aarhus University Hospitals: Liver blood flow measurements: Concepts and PET-tracer experiments in pigs and human subjects.
23. Hideo Murayama, MD., National Institute of Radiological Science, Chiba, Japan: Detector units and statistical noise in PET.
June
3. Antony Gee, Chief radiochemist, PET Centre, Aarhus University Hospitals: 13N-Ammonia: A tracer of perfusion or metabolism?
10. Anders B. Rodell, PET Centre, Aarhus University Hospitals: Three-Dimensional Visualization in Medical Imaging.
August
26. Dean F. Wong, Professor, MD, PhD, Johns Hopkins University, Baltimore, Maryland, USA: Dopamine transporters in Rett's syndrome, studied with PET.
September
5. Alfredo MartinezColl, Royal North Shore Hospital, St. Leonards, NSW, Australia: Applications of near infrared spectroscopy (NIRS) to brain and muscle.
9. Yoshio Imahori, MD, PhD, Dept. of Neurosurgery, Kyoto Prefectural University of Medicine, Kyoto, Japan: Synaptic Potentiation Visualized by Positron Emission Tomography in the Living Human Brain.
23. Antony Gee, Chief radiochemist, PET Centre, Aarhus University Hospitals: In vivo binding of amphetamine and cocaine analogs in pig brain.
30. Leif Østergaard, PET Centre & Dept. of Neuroradiology P, Aarhus University Hospitals: Can we monitor the ischemic penumbra in stroke with NMR: More on NMR CBF measurements.
October
7. Søren Ballegaard, MD., Klampenborg: Percutaneous neurostimulation for patients with angina pectoris.
10. Christian Spenger, Professor, Bern, Switzerland: Effects of neurotrophic factors on cultured dopaminergic neurons, intended for intracerebral grafting in Parkinsons Disease.
14. Jens Bo Nielsen, Professor in neurophysiology, University of Kiel, Thomas Sindkjær MRC professor, University of Aalborg, Jørgen Feldbæk Nielsen, Research assistant, University of Aalborg & Neurological Dept. F, Aarhus General Hospital: Bloodflow in motor cortex during simple and complex motor tasks involving the leg muscles.
21. Morten Bøttcher, PET Centre & Dept. B, Aarhus University Hospitals: Measurement of blood flow in the myocardium by PET.
28. Susanne Keiding, MD., D.Sc., PET Centre & Dept. V, Aarhus University Hospitals: Liver blood flow measurements: PET-tracer experiments in pigs and human subjects.
November
4. Jane Rygaard Pedersen, MSc., Dept. of Physics, DTU, Lyngby: The readiness field and its location.
11. Koichi Ishizu, MD., Ph.D., PET Centre, Aarhus University Hospitals & Dept. of Nuclear Medicine, Kyoto University, Japan: Pinhole SPECT for small animals and Quantification of CBF with Tc-99m ECD.
25. Donald F. Smith, DSc., Ph.D., PET Centre & Psychiatric Hospital, Aarhus University Hospitals: Types of Fallacies in Science.
December
2. Elisabeth Hedlund Corder, Professor, Ph.D., Institute of Community Health, Odense University: APOE genotype determines risks of AD and death in late age, but not regional glucose metabolism in dementia patients.
9. Donald F. Smith, DSc., Ph.D., PET Centre & Psychiatric Hospital, Aarhus University Hospitals: Fallacies in Science and what to do about them.
16. Peter Brust, Dr.rer.nat.habil., Forschungszentrum Rossendorf, Institüt für Bioorganische und Radiopharmazeutische Chemie, Dresden, Germany: Measurement of DOPA decarboxylase activity in the brain of pigs using FDOPA and PET« and »Serotonin and the Blood-Brain-Barrier.
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