Development of the[18F]Fluorodeoxyglucose Method:

A Serendipitous Journey From Bench to Bedside

Louis Sokoloff, NIMH

Presented at Brookhaven National Laboratory

October 19, 2012 as the New York Section

of the American Chemical Societyhonors BNL Chemistry Department

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After the war’s end, Bush actively cam-paigned in support of the idea that a rich nation like the USA could and should, in its own best interests, support scientists to conduct basic research of their own choosing and curiosity, and this would ultimately prove beneficial to the country and to humanity. The many fortuitous circumstances in the development of the 18FDG method and its applications are, I believe, an excellent example of the validity of his vision. As a participant in this history, I would like to briefly review the background and history of its development.

In 1948 Seymour Kety and Carl Schmidt published their nitrous oxide method for the measurement of average rates of blood flow and oxygen consumption in the brain as whole in human beings. Applications of this method demonstrated changes in the brain’s energy metabolism in conditions in which conscious-ness was depressed, but failed to detect any

changes in conditions in which normal brain functions were certainly altered, such as normal sleep, performance of arithmetical calculations, and a number of other conditions. This was in contrast to findings in other organs in which altered functional activities were clearly associated with changes in oxygen consumption and blood flow. A popular explanation for the failure to find the same in brain was that the energy required for specific functions in the brain was negligible compared to the energy consumed by the baseline housekeeping functions of the brain. Kety and his group, however, hypothesized that specific functions are localized to specific regions of the brain, and that when they are altered, their effects on metabolism and blood flow are localized only to those regions and diluted out in measurements of average rates of blood flow and energy metabolism in the brain as a whole. What was needed, we believed, was a

Congratulations

Local changes in blood flow in brain associated with visual stimulation Functional activity is localized to specific cells, e.g., neurons, which do not have their own private

blood flows. Blood flow supplies a wider area surrounding the cells, and, furthermore, cerebral blood flow is markedly affected by chemical constituents in the blood. Energy metabolism, however, is confined only to cells, and could, therefore, be expected to provide more specific and better spatial localization within the brain. Glucose is an essential and the almost exclusive substrate for the brain’s energy metabolism, and its consumption is almost completely stoichiometric with the brain’s oxygen

Congratulations to Joanna Fowler and the Chemistry Laboratory at the Brookhaven National Laboratory for this honor being bestowed on them by the American Chemical Society. It is well deserved. I regret that health issues prevent my direct participation in this celebration, but I hope that this videotape will be an acceptable substitute.

In 1944 Vannevar Bush, President Roosevelt’s Science Advisor during World War II, sent him a memorandum which contained the following passage below.

method to measure energy metabolism and/or blood flow locally in the appropriate anatomical regions of the brain.

There was at that time no obvious approach to measuring local brain energy metabolism. Kety, however, had previously worked on the principles of inert gas exchange between blood and tissues which included a theoretical basis for a method for measuring local rates of blood flow in the brain. Kety, and a group of us that included William Landau, Walter Freygang, Lewis Rowland, and myself, applied these principles to develop a method for measuring local cerebral blood flow. We used the freely diffusible, radioactive gas 131I-labeled trifluo-roiodomethane as the tracer because it is chemically and biologically inert in tracer amounts, and its radioactive label facilitated assay of its concentrations in the blood and brain tissues that were required by the method. Blood concentrations were measured by standard scintillation counting. Local brain tissue concentrations were measured by a

specially developed quantitative autoradiograph-ic technique applied to frozen brain sections, which, of course, limited use of the method to animals. The autoradiograms displayed images of the label’s distribution within the brain slices as well as those of calibrated standards from which local tissue tracer concentrations could be determined by densitometry. The local tissue blood flows could then be computed from the tissue and blood concentrations. Fortuitously, with the procedure adopted, the local tissue tracer concentrations imaged in the autoradio-grams closely reflected the local rates of blood flow, the darker the image, the greater the blood flow. Applications of the method clearly showed that blood flow does indeed increase and could be visualized in the functionally activated regions of the brain. For example, visual stimulation with light flashes increased blood flow in those regions involved in vision that was clearly visible in the autoradiograms.

Eyes Patched Eyes Open & Stimulated with Light Flashes

The Endless Frontier

(Excerpted From Scientific Advisor Vannevar Bush’s Recommendation to President Roosevelt in 1944)

– SUMMARY OF THE REPORT

– SCIENTIFIC PROGRESS IS ESSENTIAL

Progress in the war against disease depends upon a flow of new scientific knowledge. New products, new industries, and more jobs require continuous additions to knowledge of the laws of nature, and the application of that knowledge to practical purposes. Similarly, our defense against aggression demands new knowdledge so that we can develop new and improved weapons. This essential, new knowledge can be obtained only through basic scientific research.

Functional Activation of Local Cerebral Blood Flow in Visual Pathways

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Sols and Crane had previously reported that 2-deoxyglucose could be phosphorylated to 2-deoxyglucose-6-phosphate by glucose hexokinase, the enzyme that catalyzes the phos-phorylation of glucose to glucose-6-phosphate, the first step in the glycolytic pathway of glucose metabolism. The 2-deoxyglucose-induced coma could not. however, be due to competitive inhibi-tion of glucose utilization at this step because the coma could not be reversed by glucose administration. In 1959, I learned about studies by Wick, Tower, and their respective cowork-ers that clarified the mechanism. Both glucose and deoxyglucose cross the blood-brain barrier and are converted in the brain by hexokinase to their hexose-6-phosphate derivatives. Nor-mally, the glucose-6-phophate is isomerized by glucose-6-phosphate isomerase to fructose-6-phosphate, which is then metabolized down the glycolytic pathway to pyruvate and lactate, which are then oxidized to CO2 and H2O.

2-Deoxyglucose-6-phosphate can also bind to the glucose-6-phosphate isomerase, but be-cause it lacks the hydroxyl group in its 2-carbon position, it cannot be isomerized, and other enzyme activities that might remove it are negli-

gible in brain. The 2-deoxygluose-6-phosphate, therefore, accumulates in the brain, and with pharmacological doses of 2-deoxyglucose rises to levels high enough to competitively inhibit glucose-6-phosphate conversion to fructose-6-phoshate, thus blocking glucose utilization and causing coma. On learning that its phosphorylat-ed product remains and accumulates in brain, it occurred to me that radioactive 2-deoxyglucose, in tracer doses too low to block glycolysis, might be used with the quantitative autoradiographic technique to measure local rates of glucose phosphorylation and thus glucose utilization in the brain. Differences in enzyme kinetics between glucose and 2-deoxyglucose phos-phorylation by hexokinase would, of course, have to be addressed. My lab was then fully engaged in studies of biochemical mechanisms of action of thyroid hormones, and so I filed the idea away for future study.

In 1964 Martin Reivich joined us at NIH where he modified the [131I]trifluoroiodomethane auto-radiographic local blood method for use with a non-volatile 14C-labeled tracer. He used [14C]anti-pyrine as the tracer which required adapting the quantitative autoradiographic technique for use

Kinetic Model and Operational Equation of 2-[14C]Deoxyglucose Method

Chemical Structure of Glucose, 2-Deoxyglucose, and 3-O-Methylglucose

consumption. I, therefore, made an attempt to use radioactive glucose and the quantitative autoradiographic technique to measure local rates of glucose utilization in the brain, but failed because the labeled products of glucose metabolism, carbon dioxide and water, were lost too rapidly from the brain.

In 1957 I learned that high doses of the glucose analogue, 2-deoxyglucose, caused a coma very much like insulin-induced hypoglycemic coma.

D-GLUCOSE 2-DEOXY-D-GLUCOSE 3-O-METHYL-D-GLUCOSE

with 14C. The availability of quantitative 14C-autora-diography resurrected the idea of using 14C-labeled 2-deoxyglucose to measure local cerebral glucose utilization. Shortly after he returned to the University of Pennsylvania in 1966, Reivich and I initiated a collaboration to develop such a method. The initial experiments were done with brain slices in his lab, and they confirmed that the approach was feasible. We then developed a model and operational equa-tion that were based on the one used for the local blood flow technique but with the addition of a term for metabolic trapping of the tracer. The model was theoretically sound but technically impractical because it required simultaneous measurements of local cerebral blood flow along with the 2-[14C]deoxygluose uptake and also included some parameters that were difficult to quantify.

Again serendipitously, in 1968 I took the oppor-tunity to spend a sabbatical year in the Laboratory of General and Comparative Biochemistry in the College of France in Paris. I joined an ongoing project on the peroxidase-catalyzed iodination of tyrosine residues in proteins, a model system for thyroxine biosynthesis in the thyroid gland. The reaction exhibited very atypical enzyme kinetics which required me to become relatively proficient in enzyme kinetics. This new knowledge led me to consider a new approach to the 2-deoxyglucose method, one based on enzyme kinetics. Soon after returning to NIH, studies based on this idea were initiated in collaboration with Charles Kennedy, Michel des Rosiers, and others at NIH, and Reivich at Penn.

By 1974 a model based on enzyme kinet-ics was designed from which an operational equation was derived and a practical method using 14C autoradiog-raphy developed. The method was first applied in rats and monkeys.

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As with the autoradiographic blood flow meth-ods, the optical densities for the various tissues in autoradiograms closely reflected the rates of glucose utilization, The autoradiograms, therefore, provided visual representations of the relative

rates of local glucose utilization within the brain. Applications of the method demonstrated dramatic changes in glucose utilization in brain regions with altered local functional activities that were easily visualized in the autoradiograms.

Functional Imaging with 2-[14C]Deoxyglucose in the Visual Cortex

ACS: “Visualizing Brain Chemistry in Action”

Both Eyes Open Both Eyes Patched Right Eye Patched

The manual densitometry of the autoradiograms that the method required for full quantification was tedious and time-consuming. We, therefore, acquired a computerized scanning microdensitome-ter which scanned the films and stored the local optical densities in a computer. Charles Goochee and Wayne Rasband in our lab developed a program that used these stored optical densities to compute the local rates of glucose utilization and then to reconstruct and display brain images in color with the local rates of glucose utilization encoded in a scale based on the visible spectrum, the lower the rate of glucose utilization, the lower the wave-length. The program was used in collaborative studies of vi-sual functions in the monkey carried out with Mort Mishkin’s Laboratory of Neuropsychology at NIMH. It was first presented publicly at the Annual Meeting of the Society for Neuroscience in St. Louis in 1978 and was sensationally received. In fact, Chemical and Engineering News featured it on the cover of its issue reporting on the meeting.

After its successful use in the laboratory, Martin Reivich raised the question of adapting the [14C]deoxyglucose method for use in man. I was skeptical because it required autoradiog-raphy of brain slices, not possible with humans. Reivich then told us of a single-photon scanner that Dave Kuhl had developed at. Penn.. This instrument consisted of an array of scintillation counter detectors that scanned the human brain in vivo for !-radiation and reproduced images of the local isotope concentrations in cross-sections of the brain. Deoxyglucose, however, contains only hydrogen, oxygen, and carbon. Hydrogen has no !-emitting isotopes, and oxygen and carbon have !-emitting isotopes, but with half-lives that seemed too short for organic synthesis of labeled deoxyglucose. Serendipi-tously, I belonged at that time to a wine-tasting club of NIH biochemists. One of them, Peter Goldman, was working on enzyme actions on fluorinated analogues of natural substrates, and I knew of his finding that glutamic decarboxylase,

the enzyme that decarboxylates glutamate to form the natural neurotransmitter !-aminobutyric acid, GABA, could also convert fluoro-glutamate to fluoro-GABA. The explanation was that because the fluorine atom is so small, when it replaces a hydrogen atom in a non-critical position in the substrate, the enzyme acts on it just like on the natural substrate. This suggested that we use a !-emitting fluorinated analogue of 2-deoxyglucose. On searching the handbooks of radioisotopes in my office, we found 18F, a !-ray emitter with a 110 minute half-life. Now the problem was how to synthesize 18F-labeled-fluorodeoxyglucose.

Reivich returned to Penn with this idea, and he and Kuhl organized a meeting at Penn to discuss the possibility of using 18F-labeled fluorodeoxyglucose with Kuhl’s scanner. Al Wolf and Joanna Fowler, radiochemists from the Brookhaven National Laboratory, were invited and attended. The first issue to be considered was where to insert the 18F in the deoxyglucose molecule. Because the phosphorylation by hexo-kinase is on the 6-carbon, it was reasoned that the 18F should be placed on a carbon as far from the 6-position as possible. A fluorine bonded to the 1-carbon would be unstable, and so we decided on the next most distant carbon, the 2-carbon. Wolf and Fowler were confident that they could synthesize 2-18F-2-deoxyglucose-D-glucose (2-18FDG). We asked them, however, to first synthesize 14C-labeled 2-FDG so that we could examine if it was phosphorylated by brain hexokinase and behaved like 2-[14C]deoxyglu-cose in the rat brain. They did so, and we used it to confirm that 2-FDG was as good a tracer for glucose metabolism in brain as 2-deoxyglucose.

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Autoradiograms of Rat Brains Labeled with 14C-Labeled Glucose Analogs

Wolf, postdoctoral Tatsuo Ido, and Fowler succeeded in developing a synthesis of 2-18FDG, produced it in their lab in Brookhaven, and sent it to Penn where Reivich and Kuhl successfully used it with Kuhl’s Mark IV scanner to measure, for the first time, regional rates of glucose utilization in the human brain.

Mark IV Scans of Regional Cerebral Glucose Utilization in the Human Brain

PET 18FDG Scans of Human Brain

18FDG Studies of Drug Abuse 2-[18F]Fluorodeoxyglucose (18FDG) Scan of Human Brain Done with with Kuhl’s MARK IV Single Photon Scanner

Brookhaven National Laboratory

Soon afterward, Kuhl moved to UCLA in Los Angeles and took Michael Phelps and Ed Hoffman with him. Both had been gradu-ate students with Michael Ter-Pergossian at Washington University where they developed the first Positron Emission Tomographic (PET) scanners, and they soon acquired a PET scanner at UCLA. 18F is a positron emitter; and its !-ray emissions are derived from the interaction of the emitted positrons with electrons in the absorbing

medium which results in two annihilation !-rays of equal energies radiating from the tissue at approximately 180° with respect to each other. Coincident counting of these !-rays enables much better spatial resolution and quantitative accuracy than single photon tomography. Phelps, Hoffman, and Kuhl then proceeded to adapt the 18FDG method for use with PET scanning, and they successfully applied it in a variety of normal and pathological human conditions.

The publication of many applications of the 18FDG method by the UCLA group and the commercial availability of PET scanners led to a proliferation of centers using the 18FDG method. The Chemistry Laboratory of the Brookhaven National Laboratory soon acquired one, and under the leadership of first Al Wolf and, subsequently, Joanna Fowler, it became one of the world’s leading PET centers. It carried out many outstanding studies, including the classic studies by Joanna Fowler, Nora Volkow, and their collaborators, in which they used the 18FDG method to identify the sites and some of the mechanisms of drug addiction in the brain of addicted patients.

The legendary French chemist, Louis Pasteur, in studies of wine-production, had found that glycolysis, the metabolic pathway that produces ethanol from glucose in yeast, is inhibited by oxygen. This so-called Pasteur Effect also occurs in mammalian tissues in which the glycolytic conversion of glucose to pyruvate and/or lactate is negatively regulated by oxygen consumption. The German biochemist, Otto Warburg found that this negative regulation is impaired in tumor tissues, and be-cause of this defect, glucose utilization in tumor cells is less inhibited by aerobic oxygen metabolism. Consequently, glucose is consumed more rapidly in tumor tissues than in normal tissue, and the more transformed and malignant the cells, the higher the rate of glucose utilization. Giovanni di Chiro at NIH was the first one to exploit this trait to localize and grade the malignancy of brain tumors in human patients by means of 18FDG and PET.

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Localization of Brain Tumor with 18FDG and PET

This biochemical abnormality in tumor tissue is now the basis of the wide use of the 18FDG method to diagnose, localize, and stage a variety of primary and metastatic malignant tumors. 18FDG, I have been told, is now the most com-monly used radiochemical in nuclear medicine and is of great value in the practice of oncology and clinical medicine.

When the 2-[14C]DG method was first presented at the Annual Meeting of the American Society for Neurochemistry in 1944, the eminent neurochemist Norman Radin remarked about what a crazy idea it was to use an inhibitor of a metabolic pathway to measure the rate of flux through that pathway. It was indeed a crazy idea, and yet the Federal government supported all the basic research at NIH, Penn, Brookhaven, and UCLA that led to the development of the 2-[14C]DG and 18FDG methods. Who could have foreseen that pursuing so basic a question of whether functional activity in brain uses energy would eventually lead to such practical benefits to the practice of medicine and to mankind? What a confirmation of Vannevar Bushs’s wisdom!

Glioblastoma

The Endless Frontier

(Excerpted From Scientific Advisor Vannevar Bush’s Recommendation to President Roosevelt in 1944)

– SUMMARY OF THE REPORT

– SCIENTIFIC PROGRESS IS ESSENTIAL

Progress in the war against disease depends upon a flow of new scientific knowledge. New products, new industries, and more jobs require continuous additions to knowledge of the laws of nature, and the application of that knowledge to practical purposes. Simi-larly, our defense against aggression demands new knowdledge so that we can develop new and improved weapons. This essential, new knowledge can be obtained only through basic scientific research.

NOTES

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