J. Endocrinol. Invest. 14: 509-528, 1991

REVIEW ARTICLE

Positron emission tomography applied in the study of pituitary adenomas C. Muhr and M. Bergstrom Department of Neurology, Akademiska Hospital, Uppsala, Sweden

INTRODUCTION

to define tumor extent, existance of cystic and necrotic regions and especially relationships between tumor and adjacent anatomical structures. CT and MRI are morphological techniques and only indirectly and in special cases can conclusions be drawn on functional and biochemical factors . Although there are optimistic features of MR-spectroscopy indicating the use of this technique for the study of biochemistry, these expectations are far from fullfilled . MR-spectroscopy can be used for the study of phosphorus peaks, which might indirectly relate to energy turnover, but the biochemical possibilities are limited due to the intrinsic insensitivity of the technique. With PET, however, wide new fields are opened and, for the first time it appears to be possible to directly, noninvasively, study the function and biochemistry of the normal pituitary gland and pituitary adenomas in vivo.

Positron emission tomography (PET) is a new in vivo tracer and imaging technique that has evolved from tomographic image reconstruction started with computed tomography. The pioneering works of Dr. Ter-Pogossian and co-workers (1) on the external detection of distribution of short-lived, positron emitting radionuclides, were combined with an elaborate detector configuration and a sophisticated computerized image reconstruction algorithm to make an instrument that was the equivalent of a CT scanner in the field of nuclear medicine (2). This new instrument, the PET-scanner, promised much for the future with the enormous potential of being able to utilize practically all biologically active molecules in the in vivo study of biochemistry and organ function. Now, with a delay of about 15 years, we see that the major limiting factor for the wide application of the technique, i.e. the development of the special type of chemistry necessary, is being overcome and the technique is starting to become a most important tool in the study of normal and pathological conditions in the human body (3,4). The study of pituitary adenomas and the normal pituitary gland in man has been hampered by the inaccessibility of these tissues. Except for the pathological and histological studies on operative samples and postmortem material, only indirect studies have been possible. Hormonal evaluations and perturbation studies have thus played an important role. The introduction of CT and MRI has had a major impact on the diagnosis and characterization of anatomical factors and these methods are presently routinely used in patients with suspected pituitary adenomas. With these techniques it is possible

UTILIZA TION OF SHORT LIVED RADIONUCLIDES The key element in positron emission tomography is the utilization and external detection of short-lived positron emitting radionuclides. With 11C, 13N, 150 or 18F as radionuclides it is in principle possible to label almost all biologically active or functionally related molecules . Sugars, different amino acids or pharmaceutical agents contain carbon atoms at different positions in the molecules and by refined chemistry this carbon can be replaced by 11C to produce a radiolabelled molecule . The production of these so called bio-isotopes can only be done in an accelerator where high energy protons or deuterons are directed onto a small gas container. The accelerated protons will split, for example, the nucleus of nitrogen to produce 11C. The highly reactive 11C will combine with oxygen in the target to generate 11CO or 11C0 2 which is passed through a thin tube to the chemistry box for further syntheses. The major limitation in positron emission tomogra-

Key-words: Positron emission tomography, bromocriptine treatment, pituitary adenomas, prolactinomas, amino acid metabolism, dopamine receptor. Correspondence: Dr. Carin Muhr, Department of Neurology, University Hospital, S-751 85 Uppsala, Sweden.

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potential of PET is illustrated by the fact that with the rapid incorporation of radiolabelled key molecules into precurser molecules of great complexity it is possible within the short time available to label about 70% of all registered pharmaceutical drugs and a very large number of substances utilized in metabolism, receptor interaction, enzyme systems and various other biochemical actions. This list of potentially labelled compounds is now being rapidly extended. The short half-life imposes a restriction on the type of biological problem that can be studied in that the kinetics of the tracer substance must be fast enough to allow significant interaction with the studied biological factor during 30-90 minutes. It is, thus, generally possible to study metabolism of nutrition substrates but the diffusion of some macromolecules across the blood-brain-barrier with equilibration times of several hours may be impossible to examine. The rapid decrease of the radioactivity is, however, also an advantage due to the fact that the radiation

phy relates to the fact that the available radionuclides are very short lived. 11C has a half-life of 20 minutes, which means that every 20 minutes half of the radioactivity disappears. After 1 hour 1/8 of the radioactivity remains and after 2 hours 1/64 is left. The half-lives of the other radionuclides are 150: 2 minutes, 13N: 10 minutes and 18F: 2 hours. Other radionuclides such as iodine and bromide can potentially also be used in PET, but their production requires a very large accelerator. The total time available from production of a radioactive gas such as 11CO to the injection of the labelled tracer substance is in practice 5-10 halflives, i.e. with 11C 1.5-3 hours. In order to allow rapid syntheses, special procedures have been developed based on the initial rapid synthesis of key chemical elements such as 11C-methyl-iodide. A complicated molecule containing a methyl group can thus be labelled with 11C by an exchange reaction which substitutes a hydrogen for the labelled methyl group in methyl-iodide. Another route for rapid chemistry utilizes 11C-cyanide. The very wide

Fig. 1 - The detector system of the positron camera consists of a ring of detectors. During radioactive decay of the radionuclide, two opposing gamma rays are emitted and recorded by a pair of detectors. A computer analyzes the data and reconstructs a tomographic image which is shown on an image display. The patient is lying on the examination bed and the radiolabeled substance is injected into one arm. During the examination time of about 30 min•illIi.iI.IIIi~lIIl1!1!1iil utes several blood samples are taken to record plasma radioactivity.

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dose to the patient is kept low and that repeat studies in the same patient can be made within a few hours without disturbances from the previous injection. Furthermore, the specific activity attainable is inversively related to the decay time and the short-lived radionuclides therefore allow very high specific activities. This is of fundamental importance in the study of potentially toxic substances or in the study of very delicate receptor systems. The amount of injected unlabelled compound in conjunction with the radiolabelled substance can be kept at very low levels, micro- to picomoles.

POSITRON CAMERA INSTRUMENTATION The radioactively labelled substance is injected intravenously into the patient and carried by the blood to the organ of interest. The radionuclides disintegrate throughout the whole phase of distribution , active accumulation and transport, receptor binding and metabolic transformation. At each radioactive decay a positron , the equivalent of an electron except for its positive charge, is emitted with high energy. This particle collides with electrons in the immediate surroundings and successively looses energy until almost brought to rest within approximately 1 mm. The positron, being the antiparticle of the electron , cannot exist at rest together with an electron but interacts with it to form two opposedly-directed gamma rays with high energy. The gamma rays readily penetrate the tissue and are emitted out of the body and detected externally with the aid of a special detector system (Fig. 1). The positron camera system consists of a gantry containing a ring with several hundred to several thousand detectors, a patient couch similar to that of a CT scanner, and a powerful computer system with a picture processing system. The two opposed gamma rays emitted as a result of the disintegration of one radionuclide are recorded simultaneously by two separate detectors in the detector ring. The information on which detectors were signalling is fed into the computer and , based on the position of the detectors, a line is defined through the object connecting the two detectors and through the position of the disintegration. The accumulation of 100,000 to 1 million "counts", each representing a line through the object intersecting a position of decay, is sufficient for the computer to calculate a cross section image of the radioactivity distribution within the object. This image is presented on a TV screen as a grey-scale or color image where the color coding is directly representative of the radioactivity concentration in the object.

Uptake (nCllml)

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Plasma Time (minutes) Fig. 2 - With an interactive cursor, regions of interest are selected to represent areas for numerical determinations (A). The computer automatically calculates time-activity curves for the chosen regions (B). For the analyses, often plasma radioactivity curves are used.

Special software allows the interactive outlining of regions of interest using a cursor and the calculation of the radioactivity concentration within this region of interest (Fig. 2). All modern positron cameras contain several de-

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tector rings, thus simultaneously recording up to 15 slices. At present the slice thicknesses can be about 5 mm and the spatial resolution 4-5 mm. One implication of this is that two structures must have a spacing of at least 7 mm to be separated and the smallest object in which a correct measurement can be obtained has a diameter of about 10 mm. Finer details can be visualized although a correct representation of the radioactivity concentration may not be attained (5) . The normal pituitary gland , for example, is well visualized with a modern positron camera, especially since there are no biologically active tissues in the immediate surroundings. The absolute measurements in the pituitary gland might be slightly degraded but still adequate for the study of how modulating substances influence the pituitary function. The PET study is usually done as a dynamic examination sequence . A sequence for the measurement is preprogrammed and activated at the time of injection of the tracer. The camera then accumulates counts during predetermined times, such as every 10 seconds during the first 2 minutes after injection, every minute during the following 10 minutes and thereafter every 5 minutes. Such a procedure results in a set of images, each representative of a certain time after injection .

be used in combination with the kinetics of the region of interest to extract the functional or biochemical parameter. Examples of analyses of metabolism and receptor binding are given below.

Evaluation of metabolism The measurement of amino acid metabolism is an important factor in the evaluation of functional aspects of the normal pituitary gland and pituitary adenomas because of its relation to protein synthesis. With PET it is possible to study only one labelled amino acid at a time and it is thus not possible to achieve a true measurement of protein synthesis expressed as Ilmol protein synthesised per gram of tissue per time unit. It is, however, likely that the entrapment of one amino acid into proteins is proportional to protein synthesis and that the influences of modulatory factors will not completely disrupt the proportions of the different amino acids incorporated into proteins. Thus amino acid metabolism related to one specific amino acid has some value as an indicator of cellular activity coupled to protein synthesis. PET can record the distribution and concentration of the labelled radionuclide but not its chemical form and it is , therefore , not possible to discern whether the radioactivity remains as unmetabolized amino acid, is incorporated in proteins synthesized from the amino acid or whether it is bound in a metabolite . For this reason it is necessary for the evaluation of the results to choose amino acids with a high fraction of protein incorporation and with low metabolism along routes other than protein synthesis (8). For this purpose we have used 11C-labelled L-methionine which also has the advantage of being rapidly transported across both the bloodbrain barrier and across cell membranes. Furthermore, it has been postulated that transmethylation processes quantitatively playa minor role and that proteins in the plasma only contribute a limited fraction of the radioactivity if the examination time is kept below 30 minutes (9-12). Thus the radioactivity in tumor or normal pituitary tissue exists during the examination time primarily in either of two forms: bound in proteins or as unmetabolised L-methionine. The distribution in this type of tissue is fast enough that equilibration is reached between unmetabolized methionine in plasma and in the tissue. Thus the dynamics of the tracer substance in the tissue has two components, one part which during the experiment is directly proportional to the concentration of free methionine in plasma and one part representing methionine continuously bound and entrapped in proteins , in proportion to the amount of free metionine times the rate of metabolism . This re-

KINETIC STUDIES Positron emission tomography studies are usually performed as dynamic studies. The reason for this is that in most cases the short examination time is not sufficient for achieving a steady state condition and a "snapshot in time" will not properly represent the desired functional parameter but will relate to various other factors such as distribution, blood flow, contribution from the blood pool etc. Exceptions include the study of glucose metabolism using the irreversibly trapped tracer 18F-fluorodeoxyglucose and the use of 150-water for the study of blood flow (6, 7). For the dynamic examinations a dynamic imaging sequence is used, starting at the time of injection of the tracer. A region of interest is outlined in one of the images or in an average image and the computer calculates the radioactivity concentration in each of the separate images in the dynamic sequence (Fig. 2). The tracer concentration in the chosen region plotted against time represents the dynamics of the tracer accumulation. Depending on choice of tracer and type of analysis, the dynamics of plasma radioactivity, obtained through blood samples taken during the PET-examination , or a reference region with known properties, can

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lationship can be illustrated graphically with the technique of Gjedde (13) and Patlak et al. (14): The tracer concentration Ci(t) in the tissue at each time point is divided by the radioactivity concentration in plasma Cp(t) R(t) = Ci(t) / Cp(t) = Cif(t)/Cp(t)+Cib(t)/Cp(t) = Ki+Cib(t)/Cp(t) By this procedure a ratio R(t) is obtained that is the sum of the ratio of free methionine in tissue (Cif) to methionine in plasma plus bound methionine in tissue (Cib) divided by methionine in plasma. After a short time an equilibrium is achieved between free methionine in tissue and free methionine in plasma with a constant ratio of ki. The ratio R(t) is then plotted against a modified time T according to T = JCp(t)dt/Cp(t) This time normalization changes the time so that the exposure of the tissue to plasma radioactivity becomes proportionally equal at each time point. This plot is thus equivalent to the plot that would have resulted from a constant plasma radioactivity with time and with a relative magnitude of 1. An example of this type of plot is given in Figure 3, illustrating the different fates of the stereoenentiomers of methionine; L-methionine which is trapped into proteins and D-methionine which is not. This plot is composed of two distinct phases. In the initial distribution phase the equilibration of free methionine in tissue takes place. During the second phase the curve is the sum of a constant level ki: freely distributed methionine divided by plasma methionine, plus a continuously increasing level which is equal to the fractional incorporation of methionine. This fractional value multiplied by the concentration of unlabelled methionine in plasma in Jlmol/ml gives a quantitative value of the rate of incorporation of methionine expressed in Jlmol/g/min. In the example above the fractional incorporation of L-methionine in the pituitary adenoma as obtained from the PET-analysis is 0.10 min- 1. The plasma L-methionine level was 20 Jlmol/I. Thus the methinone metabolic rate is 2.0 Jlmol/kg/min in the pituitaryadenoma. In the brain tissue the methionine metabolism is 0.4 Jlmol/kg/min.

Ci I Cp

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Fig. 3 - A) The enentiomers of methionine, L-methionine and O-methionine, show both high uptake directly after injection, indicating high perfusion. In the late images L-methionine uptake in the adenoma is high as an indication of tracer trapped in protein synthesis. O-methionine shows, however, low uptake because of absence of trapping. B) The kinetic plot of Patlak et al demonstrates that L-methionine is continuously trapped whereas O-methionine only shows equilibration.

Receptor binding Because PET does not discriminate between chemical forms in which the radionuclide is bound , special demands have to be put on the ligands used for receptor studies. The receptor affinity has to be very high as it is not possible, as in in vitro recep-

tor studies, to wash away non bound ligand . For beta-receptor determinations it is possible to use ligands such as cyano-pindolol with an equilibrium kD of less than 0.1 nmolar but not metoprolol with a ko measured in Jlmolar. For dopamine D 2 -receptor

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binding it is possible to use both N-methylspiperone ko=O.2 nmolar and Raclopride ko=3.8 nmolar. With a higher ko the ratio of bound to unbound ligand .is too unfavorable. It is in principle possible to perform a receptor study with PET very much in the same way as in vitro, by using a few different specific activities and constructing a Scatchard plot (15). The tracer concentration in plasma is , however, not constant and therefore more elaborate evaluation procedures involving compartment analysis should be used (1621). An approximate and simple way of analysis can be used with the aid of the graphical technique of Gjedde and Patlak et al. (13, 14), which simulates an equilibrium condition . The amount of free ligand can, in the case of brain structures and evaluation of dopamine D2-receptor binding, be approximated by the amount measured in the cerebellum, an area almost devoid of dopamine Drreceptors (17). In the case of the pituitary gland and pituitary adenomas, however, the cerebellar concentration can not be used for an adequate estimation of free ligand because of the significant differences in perfusion, blood volume and tissue composition. We have therefore chosen to repeat the study after the injection of 3 mg haloperidol iv, an amount sufficient to block most of the Drreceptors (Fig . 4) . As in a Scatchard plot the total/free ligand is plotted against concentration of free ligand at the different specific activities . From the plot the equilibrium ko and the receptor concentration can be deduced. In vitro studies have demonstrated that the receptor dissociation constant ko differs slightly among pituitary adenomas of different type. Thus, Bevan et al. (22) and Koga et al. (23) demonstrated that nonse-

Fig. 5 - With the active S-enentiomer of raclopride, significant binding is observed in the prolactinoma and in striatum (upper images). The inactive R-form lacks binding to the O[ receptors and is used to evaluate the non-specific binding (lower images).

creting pituitary adenomas had higher ko than prolactin-secreting adenomas (24). It is feas ible, however, that a potential drug effect is best related to the binding potential which is related to both the number of receptors and their affinity (16) . An adequate description of the receptor binding can thus be attained without separate determination of ko and we have chosen to simplify the examination protocol and perform a receptor study using only two PET examinations. One study is performed using maximum specific activity. From this study, after equilibration , the amount of total binding is determined. A second study is performed to evaluate the amount of free ligand. This can be done in 'two different ways, either after the blocking of the receptors with haloperidol , or alternatively using the inactive stereoisomer of raclopride. This inactive form, the rform, is distributed in the same way as the active sform but is not bound to the receptor. (Fig. 5). In the example, the total binding in the adenoma compared to cerebellum at equilibrium is 3.7 and the nonspecific binding is 1.2. Thus the value of receptor binding denoted by Mintun et al. (16) as receptor binding potential = 2.5.

CI I Ccbl

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Fig. 4 - The labeled OTantagonist IIC-raclopride is strongly taken up in the prolactinoma. The tumor-to-cerebellum ratio increases with time and reaches a constant level after about 50 minutes. After haloperidol protection, a repeat study shows considerably lower uptake in the adenoma. The tumor-to-cerebellum ratio is constant with time.

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Fig. 6 - A) The D2 -antagonist raclopride is strongly taken up in the prolactinoma (upper left). After haloperidol protection a repeat study shows considerably lower uptake in the adenoma (upper right). Subtraction of the two images results in an image representing specific dopamine D;rbinding. B) A similar set of images shows high D2-binding in the striatum in the same patient.

Amino acid metabolism Diagnosis using IIC-methionine. The PET summation images obtained with 11C-L-methionine represent a fairly good estimation of the amino acid metabolism, at least concerning the relative proportions between different regions in the images. The high uptake of 11C-L-methionine in intracranial tumors has provided means to diagnose brain tumors with greatly improved sensitivity as compared to CT (35, 36). The usually very high metabolism in the adenoma tissue makes it easy to discriminate. The high contrast between viable tumor and bone structures sometimes makes it easier to determine the tumor extent with PET than with CT, especially in cases with invasive growth into the bone. Furthermore, discrimination between tumor tissue and aneurysm is simplified by comparing images obtained early and late after the injection. In the early image the radioactivity concentration is high in the blood whereas at the late stage it is low. An early image thus gives an indication of blood volume which is high in the aneurysm, whereas in the late image the lack of metabolism in the aneurysm will show up as low uptake. Presently the inferior spatial resolution limits the

A pictural illustration of the dopamine D2-receptor binding is given in Figure 6 where the upper left image represents the total uptake of the dopamine D2 -receptor antagonist raclopride. A repeat study after protection of the receptors through an i.v injection of 3 mg haloperidol results in an image showing only nonspecific binding, upper right. By subtraction of the nonspecific binding from the total uptake an image is obtained representing specific dopamine DTreceptor binding.

APPLICATION TO PITUITARY ADENOMAS We have used positron emission tomography since 1982 to evaluate the characteristics of pituitary adenomas (25-34). These studies have primarily focused on the use of metabolic and receptor binding substances with the aim of developing a better diagnosis, an improved characterization, a high sensitivity in the follow up of treatment and a better understanding of how medication acts on the adenomas. The studies have in many cases been beneficial to the patients through the extra information gained.

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up at 14 months of treatment only a small remnant of the tumor was seen.

use of PET for a refined analysis of spatial relations. For the discrimination of viable tumor tissue from cystic or necrotic areas the PET images are of special value. In Figure 7 a proper characterization of the tumor condition in a macro-prolactinoma under bromocriptine treatment could not be made by use of CT or MRI in spite of several attempts with different pulse sequences and use of contrast material. After an initial positive response, with considerable lowering of PRL/s and tumor volume decrease, this tumor had suddenly increased in size during the bromocriptine treatment. "Escape" from treatment and active tumor growth was suggested from the CT and MRI findings. PET demonstrated unequivocally that the tissue in the adenoma was metabolically inactive and thus allowed the exclusion of active tumor growth. A therapeutic, transsphenoidal fine needle aspiration (37) was performed at which a few ml of a thick fluid was withdrawn . The patient's vision improved instantly during the procedure and the tumor size decreased. The patient was kept on bromocriptine treatment and the tumor continued to decrease and at follow

Distribution and metabolism. The kinetic studies combined with an analysis like the one described above allow more information to be gained from the PET studies with 11C-labelled methionine. We can see in all adenomas with solid tumor tissue that the methionine is rapidly taken up in the tissue. Distribution in the tissue takes place during the first few minutes after the iv injection, while metabolic entrapment dominates later. An improved understanding of the fate of the labelled methionine is obtained by using dual studies with the two stereo-isomers of methionine (30,38). The D-enantiomer flows with the plasma and is transported into the adenoma tissue in the same way as the L-form, but is not further metabolized. Thus an equilibrium with plasma is achieved. The L-form is further metabolized and incorporated into proteins (Fig. 3). These two enantiomers of methionine thus demonstrate distinctly different patterns in the kinetic plots . D-methionine rapidly reaches a constant level in the plot

Fig. 7 - A) PET with L-methionine in a prolactinoma examined before and during bromocriptine treatment. A significant reduction of the metabolism is noted already 5 days after start of treatment. 2.5 months after start of treatment the tumor demonstrates very low metabolism. A few weeks later the tumor suddenly rapidly increased in size. Repeat PET showed almost total absence of metabolism. B) MRI performed 2.5 months after start of treatment (upper left) shows significant reduction of tumor size. At 4 months after treatment (upper right) a significant increase in tumor size is noted as compared to 2 months after treatment. With prolonged treatment the tumor regresses and one year after start of treatment (lower) only a small tumor rest remains.

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as an indication of equilibration between the tumor and plasma compartments without entrapment. Lmethionine shows, after the initial curved portion during the first few minutes, a linear increase with time as a reflection of the entrapment of L-methionine in metabolic processes. This type of plot is in fact obtained in all types of adenomas, both secreting and nonsecreting, but with different rate constants. In an adenoma with remaining perfusion, but with minimal metabolic activity, the plot obtained with L-methionine mimics that of D-methionine. In comparison, conditions in the normal brain tissue are distinctly different due to the existance of the blood-brain-barrier (BBB). Also in some gliomas a remaining aspect of the BBB affects the dynamics of the accumulation (39). The slow transport of amino acids across the BBB probably makes it difficult to separate transport from metabolic entrapment during the relatively short examination time. With L-methionine a linear increase with time is obtained as a reflection of this transport mechanism coupled with metabolic entrapment. That entrapment does not fully explain the accumulation has been shown by Ishiwata et al. (12), who demonstrated that 60 minutes after the injection of 11 C-Lmethionine, 30% of the tracer substance in the tissue was not bound to proteins. Furthermore a definite transport limitation exists across the BBB as evidenced by the stereospecificity. The transport affinity for D-methionine is a factor of 2.5 lower than that for L-methionine (39). The transport system across the BBB has a lower capacity than that across the cell membrane such as in pituitary ade-

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Fig. 8 - Amino acid metabolism in the tumors plotted against serum prolactin levels, demonstrate a good correlation with increased metabolism in relation to serum prolactin. The nonsecreting adenomas have a metabolism which correspond to the intercept of the regression line.

nomas and can be competed with by other amino acids sharing the same transport system. A 3-fold increase in plasma levels of leucine, for example, decreased the transport of 11C-L-methionine across the BBB into the brain by approximately 30% (40).

Study of different adenoma types. The metabolic activity of pituitary adenoma cells serves a multitude of purposes: cellular proliferation and repair, exchange of substances with surrounding cells, hormone synthesis and secretion. The relative proportions between the metabolic demands for these different processes are not easily elucidated. In the PET studies with L-methionine, however, we have found significant differences between the prolactinomas and clinically nonsecreting adenomas. As a group the prolactinomas showed a 50% higher amino acid metabolism than the nonsecreting adenomas, but in cases with very high serum prolactin levels the amino acid metabolism can be up to 3 times higher than in the clinically nonsecreting adenomas. The GH adenomas and TSH adenomas could not be distinguished from the nonsecreting adenomas with respect to the magnitude of amino acid metabolism. In view of the fact that the serum levels of prolactin in a patient with a prolactinoma can be several thousand Ilg/1 whereas a patient with acromegaly has a serum level of GH amounting to a maximum of a few hundred Ilg/I, it is understandable that the prolactinomas are metabolically more active than the other types of adenomas. When the amino acid metabolism of the prolactinomas, measured by PET, is plotted against the serum

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prolactin level, a clear linear correlation is obtained (Fig . 8). The intercept at low prolactin levels is close to the amino acid metabolism of the nonsecreting adenomas. It is thus feasible to see the metabolic demand of pituitary adenomas as consisting of two portions: a basal demand for the maintainance of the cellular function and integrity and a portion attributed to the activity of hormone synthesis and secretion . In some prolactinomas with extremely high serum prolactin levels the metabolism related to hormone synthesis constitutes the major part of the metabolism .

that this leads to a later tumor shrinkage or development of cystic or necrotic areas. Quantitatively we have observed reductions in amino acid metabolism amounting to 40-60% during the first few hours after the i.m. injection of bromocriptine (31). A week or more after the start of treatment the reduction in metabolism was of the order of 80% . In the prolactinomas this reduction in metabolism was in all cases later, within several weeks or a few months, accompanied by significant tumor shrinkage or development of necrosis or cysts (42-46). The fact that the amino acid metabolism is so rapidly affected by medication and seems to be well related to the cellular response , makes PET an important tool in the evaluation of treatment effects in pituitary adenomas . In most prolactinomas , treatment with dopamine agonists works well and it is not necessary to use such an elaborate examination technique as PET. In some instances, however, we have demonstrated the extra beneficial use of PET in prolactinomas . In one case described above , PET was of great value for clarifying the characteristics of a prolactinoma which increased in volume during bromocriptine treatment. In another case a true resistant prolactinoma was investigated both with and without bromocriptine treatment. In this case no effect of bromocriptine could be recorded with PET, which further confirmed the resistant character of the tumor. We could also demostrate that this tumor did not respond to somatostatin analogue treatment either. In GH-secreting pituitary adenomas the beneficial

Evaluation of drug effects. In patients with prolactinoma, dopamine agonist treatment in most cases results in a rapid reduction of serum prolactin levels (41). Concomitantly we observe a significant reduction in amino acid metabolism (31). It is interesting to see that when amino acid metabolism is plotted against serum prolactin levels during bromocriptine treatment, it seems as if initially the decrease in amino acid metabolism correlates well with the portion attributed to hormone synthesis and secretion (Fig . 9). Thus the reduction in amino acid metabolism initially fits wetl with the line showing the relation between amino acid metabolism and serum prolactin levels in untreated patients. With prolonged treatment, however, the amino acid metabolism starts to decline to levels even lower than those seen in nonsecreting adenomas (Fig. 10). It is feasible to assume that in this latter phase the metabolism is lowered beyond the level necessary for the maintainance of cellular integrity and

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PET and pituitary adenomas

use of bromocriptine in a subgroup of patients was confirmed with PET through measurement of amino acid metabolism. Thus a 30-40% reduction in amino acid metabolism was seen in those patients who responded with a reduction in serum GH levels. In the patients who did not respond hormonally, no effect was either recorded with PET. In one patient previously operated on who had a large recurrence, bromocriptine treatment had no effect on amino acid metabolism or on serum GH levels. Nor was any response seen with somatostatin analogue treatment at a dose of 150 f.,lg/day. The dose was successively increased to 250 f.,lg/day, still without response. When the treatment dose was increased to 1,200 f.,lg/day the amino acid metabolism in the tumor and the serum GH levels started to decline. A further reduction in amino acid metabolism down to 40% of the pretreatment level was observed when the patient was given a continous subcutaneous infusion of somatostatin 1,500 f.,lg/ day with a pump. A significant reduction in tumor size was observed in this patient after 2 months of treatment with the highest dose. Monitoring of serum hormone levels in patients with hormonally active tumors, gives an indirect measure of the activity of the tumor that is in most instances sufficient for the follow up of treatment. It can, however, be of value to have a direct measurement in the tumor, as is possible with PET. This is especially true when serum hormone levels are only slightly elevated or normal and the tumor's influence on serum hormone levels cannot be separated from the contribu-

tion from the normal puituitary gland. The follow up of treatment with PET should be of great value, for example, in the group of patients with clinically hormonally inactive or nonsecreting pituitary adenomas. In a group of clinically nonsecreting adenomas, the amino acid metabolism was evaluated before and during treatment with bromocriptine. Only in a small fraction of the tumors could a slight decrease in amino acid metabolism be recorded. Even with treatment periods extending up to 2 years the maximum effect on amino acid metabolism was 20%. In the few patients with a slight response of amino acid metabolism, however, a slight clinical improvement was also noted with improvement of neurological symptoms and of visual field. This correlates with the observations by others (22,47,48) of a slight clinical effect of bromocriptine in non-secreting pituitary adenomas. In a different small group of patients with non-secreting adenomas no effect could be recorded after treatment with somatostatin analogue. One patient with a clinically non secreting adenoma was treated with LHRH-agonist. In this patient an initial clear increase in amino acid metabolism was noted amounting to 30-40%. During prolonged treatment and follow up the elevated amino acid metabolism decreased and remained at 15% above pre-treatment levels. (Fig. 11). The a-subunit increased considerably from a normal level of 3.7 f.,lg/I to 40 f.,lg/I during the first 3 weeks. This level gradually decreased during 5 months to 14 f.,lg/I. This observation of increased amino acid metabolism in the

Fig. 10 - Amino acid metabolism in a prolactinoma is high before treatment. Already 2 hours after the im injection of Parlodel LA@, a 40% reduction in metabolism is observed and 7 days after the start of treatment an 80% reduction in metabolism is observed.

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Fig . 11 - Patient with a nonsecreting adenoma. The amino acid metabolism is increased by 30% after the initiation of treatment with LHRH-analogue. With prolonged trea tment a sustained elevation of the metabolism is observed in parallel with an increased serum a.-subunit.

Glucose metabolism Glucose metabolism in pituitary adenomas have been evaluated using 18F-fluorodeoxyglucose. This tracer has the interesting property of being irreversibly trapped in the initial steps of glucose metabolism at a rate that is proportional to glucose utilization (6, 49). The proportionality constant by which the PET-data must be multiplied , the so called

tumor during treatment with LHRH-analogue is of fundamental importance as it shows that the medication can have a direct effect on the metabolism in this type of tumor, i.e. the tumor cells have retained the regulatory mechanisms of LHRH . The inhibitory effect observed in the normal pituitary during prolonged treatment was, however, not observed in this tumor.

PET metabolism comparing C-l1-methionine and F-18-FDG

........0 !U ~

....&::

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4

Methionine FOG

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...... ~

3

0

8

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Prolactinoma

Chordoma

520

Fig. 12 - The ratio of tumor-to-brain tissue in a group of pituitary tumors examined with 18F-fluorodeoxyglucase and with I1C-L-methionine. The tumor-to-brain ratio is higher with methionine than with deoxyglucose in all the adenomas but not in the chordomas.

PET and pituitary adenomas

effects with this type of treatment. The factors behind the high glucose metabolism as observed also in nonsecreting pituitary adenomas is still unexplained. It is not likely that this metabolism is primarily related to growth in view of the slow growth of these tumors. As a comparison the glucose metabolism in meningiomas is considerably lower. One possibility, although not substantiated, is that this high metabolism as found especially in some non secreting adenomas is correlated to oncocytic changes, observed in about 50% of the nonsecreting adenomas. It is possible that the overexpression of mitochondria seen in oncocytomas results in disturbances in the glucolytic and respiratory pathways. The 18F-fluorodeoxyglucose is trapped in the hexokinase enzyme and in these tumors an overrepresentation of the enzyme could relate to increased metabolism or a dissociation between the enzyme level and metabolism.

DOPAMINE RECEPTOR BINDING IN VIVO STUDY IN PITUITARY ADENOMA

1,5

•• 1,0

II •••

• 0,5

• I

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Dopamine receptor binding

Fig. 13 - The table demonstrates dopamine o2-receptor binding in pituitary adenomas as determined with PET. The prolactionomas

show high 0,,- binding, the non-secreting adenomas show lower and the TSH-adenomas show minimal o,,-receptor binding.

lumped constant is numerically close to 2 and takes into consideration the different transport rates and enzyme affinities of glucose and fluorodeoxyglucose. The glucose metabolism has in other tumors been demonstrated to relate well to growth (50, 51) but might in hormonally active tumors also be assumed to correlate to hormone secretion. All untreated pituitary adenomas that we have examined with 18F-fluorodeoxyglucose have demonstrated high glucose metabolism, similar to or higher than in normal brain tissue. The ratio of tumor to brain tissue uptake is, however, less favourable than with methionine, making the latter tracer substance better suited for delineation of the tumor and for evaluation of tumor viability. Furthermore the variability in the group of nonsecreting adenomas seems to be greater with 18F-fluorodeoxyglucose than with 11C-Lmethionine. In the limited group of patients we have examined with 18F-fluorodeoxyglucose, there does not seem to be the same good correlation between tracer uptake and hormone secretion as we have seen with methionine. Thus in one patient with a pro- . lactinoma, the glucose metabolism was lower than in one of the nonsecreting adenomas, whereas the amino acid metabolism in the same prolactinoma was much higher than in all nonsecreting adenomas (Fig. 12). With dopamine agonist treatment, which in this patient was clinically effective, the reduction in glucose metabolism paralleled the reduction in amino acid metabolism, indicating the generality of cellular

521

With the use of two different dopamine D2-antagonists labelled with 11C, we have characterized the dopamine receptor binding in patients with pituitary adenoma (26-28, 32, 33). The indication for these studies have been the availability of an adequate medical treatment in prolactinomas and some GHsecreting adenomas using dopamine agonists. A prerequisite for a successful treatment with this type of agent is the existence of dopamine Drreceptors on the cell surface through which the antitumoral action is assumed to occur. The dopamine receptors have also been shown to play an important role in the regulation of the pituitary gland function, notably prolactin synthesis and secretion. With PET the dopamine D2-receptor binding is measured in vivo under the present hormonal and functional status of the patient. This means that only available free receptors are measured and that receptors occupied by circulating dopamine in the portal system are hidden in the study.

Receptor binding in different adenoma types. The dopamine D 2-receptor studies as performed with PET, i.e. with one study performed with high specific activity and one after the protection of the receptors with haloperidol, result in quantitative values of receptor binding potential. This measure is equivalent to Bmax/ko and is under equilibrium conditions equal to the ratio of bound/free ligand (16). When the dopamine D2 -receptor binding is determined in different types of pituitary adenomas, significant differences are noted as presented in Figure 13. The prolactinomas are characterized by high dopamine receptor binding, quantitatively in the same range as in the dopamine receptor-rich cor-

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Muhr, and M. Bergstrom

pus striatum. Among the prolactinomas there is, however, quite a wide range, with some tumors having half the receptor binding of the average of the group. The nonsecreting adenomas had on the average a receptor binding that was only 1/4 of that measured in the prolactinomas. The variations were also considerable in this group, with some tumors approaching the prolactinomas in D2-receptor binding and some tumors in which no receptor binding could be observed. The few GH-secreting adenomas included in the study showed intermediate D2receptor binding and the TSH-secreting adenomas demonstrated low D2-binding. These values for receptor binding in the different groups of adenomas correspond quite well with studies performed in vitro in operation samples (23).

known which factors regulate the process. The variable level of non specific binding makes it impossible to use only one unprotected study for the assessment of receptor binding.

N-methylspiperone versus raclopride The two dopamine Drantagonists raclopride and N-methylspiperone differ in some properties which are of importance in PET studies. The receptor equilibration dissociation konstant ko is more favourable for N-methylspiperone which in principle should mean a higher ratio of specific to non specific binding. This advantage is, however, upset by the more rapid metabolism of N-methylspiperone. Furthermore N-methylspiperone undergoes significant binding to serotonin receptors. This is demonstrated in Figure 14 where the same patient with a metastatic prolactinoma was examined with the two ligands (32) . With N-methylspiperone the cerebral cortex shows higher uptake that the central white matter, indicating binding to serotonine receptors. With raclopride a more even distribution over the brain is observed, indicating an even level of non specific binding. The Drreceptor binding in the tumor is well visualized with both ligands.

Nonspecific binding The non specific uptake of dopamine-antagonist as measured with PET after protection with haloperidol, demonstrates quite variable levels. Uptake of raclopride in the tumor to almost the same degree as in the striatum has been found in some nonsecreting adenomas. In these patients we·could, however, show that this high uptake in the tumor was almost entirely related to non specific binding. We could not find any relation between the amount of non specific binding and type of adenoma. It is likely that the variation in non specific binding in some way reflects the tissue composition but it is still not

Receptor modulation during treatment An important aspect to consider during treatment is the extent to which the number of free receptors is affected by the drug. One of the main advantages of

Fig. 14 - Patient with a metastatic praiactinoma in the franta-parietal region. The high dopamine binding in the tumor makes it well visible with the two dopamine.

522

PET and pituitary adenomas

Fig. 15 - Patient with a prolactinoma evaluated with raclopride be-

Fig. 16 - 'Evaluation of the whole body distribution of 11 C-Iabeled

fore and during bromocriptine treatment. The initially very high amount of O;rreceptor binding is decreased by 30%, 3.5 hours after the injection of Parlodel LAR. 13 hours after the injection a further decrease in the free receptor is observed. Even with prolonged treatment a considerable amount of free receptors persists.

tamoxifen in a Rhesus monkey. A complete coverage of the monkey body with multiple tomographic slices allows reformatting to visualize sagittal (left) and coronal (right) body sections.

PET as an in vivo technique is that it allows repeat studies in the same patient. We have thus examined dopamine D2-receptor binding at different times after the initiation of bromocriptine treatment in a few patients with prolactinomas . These patients were given an intramuscular injection of bromocriptine (Parlodel LAR 50 mg) . Prior to and at about 3.5 and 13 hours after the injection, the amount of free receptors was determined using PET and 11C-raclopride (Fig . 15). The initially very high D2-binding was decreased by about 30% 3.5 hours after the injection. At the later follow up at 13 hours a further reduction amounting to 50% was noted . The pharmacokinetics of bromocriptine in this preparation is such that the highest plasma concentration is reached 3-4 hours after the injection . After this peak the plasma concentration is gradually decreased so that 13 hours after the injection the plasma level is lower. The 30% decrease in free receptors at 3.5 hours after the injection probably primarily represents receptor occupancy by the therapeutic dose of bromocriptine . It is interesting to note that this degree of receptor occupancy is sufficient for a ther-

apeutic effect with a significant reduction of plasma prolactin levels and, as we have shown earlier, a significant reduction in amino acid metabolism . At the later follow up at 13 hours after injection , the number of free receptors is further decreased in spite of a lower plasma level of bromocriptine. The receptor dissociation of bromocriptine is too fast to explain the decrease in free receptors by bromocriptine attached to the receptors . Rather it is suggested that the decrease in free receptors is related to secondary phenomena such as modification of receptor properties after activation , perhaps a reduction of new synthesis of receptors etc. These phenomena are covered by the notation "receptor down regulation ". In one of the patients, the number of free recepotors was evaluated 6 weeks after the start of treatment, 2 weeks after the second injection of bromocriptine (100 mg Parlodel LAR®). At this time the amount of free receptors was 50% of the pretreatment value . Thus with bromocriptine treatment it seems as if a considerable amount of free receptors persist even during prolonged treatment. The amount of free dopamine Drreceptors in the

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striatum is high in all cases and it was also of interest to evaluate how the medication affected these receptors. In the patients above, the amount of free D 2-receptors in the striatum measured with PET was unaffected by the medication both at 3 .5 hours and at 13 hours after the injection. In a few patients the amount of free receptors in the striatum was evaluated after longer times of treatment. Even after one year of treatment and with oral doses of 50 mg bromocriptine/ day no effect on the free D 2-receptors in the striatum was observed.

the pituitary adenoma (25). The drug is also distributed in the extracranial soft tissues but the uptake in the brain tissue is low. A high uptake is noted in all adenomas regardless of type although the prolactinomas show a slightly higher value than the nonsecreting adenomas. If a repeat study is performed in the prolactinomas after the protection of the receptors with haloperidol, a difference is seen as compared to the native study. At this low tracer concentration , approximately 20% of the drug available in the tissue in the prolactinoma during the first hour is bound to the dopamine D2-receptors.

Drug distribution

Other ligands For the further in vivo characterization of pituitary adenomas and the normal pituitary gland, a number of possible ligands are being made available. The initial preliminary studies have thus been made and in a few instances patient studies have also been made with other substances, selected as being of interest for a better understanding of the behavior of pituitary adenomas and as tools for evaluation of the prerequisites for therapeutic possibilities. Studies have started with an 11C-labelled somatostatin analogue which will preferentially be used to study GH-secreting adenomas (52, 53) (Fig . 17). A labelled beta-adrenergic antagonist will be used to characterize the betaadrenergic receptors and initial studies have been made to characterize the central and peripheral types of benzodiazepine receptor systems. Using labelled deprenyl , an agent which irreversibly binds to mono-

PET is the method of choice to study the body distribution of labelled pharmaceuticals with high accuracy. In the preliminary studies performed prior to the application of a radiotracer in man , we always perform a drug distribution study in the monkey. (Fig. 16). Thus we have studied the whole body distribution of beta-adrenergic agents, antiestrogens and antiprogestins, dopaminergic agents, substances interferring with enzyme systems etc. With labelled radiopharmaceuticals it is possible not only to demonstrate the distribution and accumulation of drug in an organ of interest but also to study how different functional conditions and other pharmaceuticals influence the drug distribution and binding.

Bromocriptine After the injection of 11C-labelled bromocriptine, a rapid and significant accumulation is observed in

Fig . 17 - Three sec tions in a patient with an ACTH-secreting adenoma investigated with I1 C-L-methionine (upper) and I1 C-labeled somatostatin (lower) . A high metabolism is observed in this tumor which seems to lack somatostatin receptors. Th is was further substantiated by the total lack of response to somatostatin treatment.

524

PET and pituitary adenomas

amineoxidase B (MAO-B), we have demonstrated high levels of MAO-B in nonsecreting pituitary adenomas. Different opioids such as encephaline, metencephaline and DALA have been used in Rhesus monkey examinations and have demonstrated the possibility of studying uptake in the pituitary (54).

clinical methionine studies, we have performed 8-10 patient studies in one day, 8-10 hours. These studies have been made by two persons, one technician running the camera and aiding in the patient set up and one person doing the injections and taking and analyzing the blood samples . With a simplified analysis the results of the previous study have been evaluated while running the next patient. During such a day, 3-5 syntheses have been made by the chemists and the activity of each have been divided to cover 23 patients. Thus we are confident that in a clinical protocol it is possible to examine 8-10 patients per day. The research protocols are usually much more complex and allow 4-5 examinations per day.

PET AS A CLINICAL TOOL For a proper perspective on PET, not only as a sophisticated and flexible research tool, but also as a facility used in the clinical handling of patients, it is important to relate the type of information attainable to the impact on patient handling and to the cost of a PET examination. In our experience, PET at present, offers valuable information in the characterization and diagnosis of pituitary adenomas that in some cases can not be deduced from other examinations. This importance of PET is likely to increase in the future . This includes definition of tumor extent, appreciation of viability, differential diagnosis and characterization of receptor systems where in selected cases PET can give complementary information of decisive importance for the proper handling of patients. Of further importance is the potential of PET to monitor accurately the effect of medical treatment which allows much shorter treatment periods for an assessment and decision on whether to continue a certain drug regimen. In this context we should like to point out the importance of improving the accuracy of the follow up studies. Thus standardization of the conditions with respect to physiological influences such as noise and light might be of importance especially in glucose-metabolism studies and standardization of meals and thus plasma levels of nutritients might be of importance in all metabolic studies . Ensuring proper repositioning to allow the evaluation of the same section with the same angulation in follow up studies could be of vital importance (55). The cost of a PET examination is higher than that of CT or MRI. The cost of the PET equipment, including the positron camera and cyclotron, is approximately twice as high as that for MRI. The personnel necessary for a PET study, including chemists and technicians, is about twice that necessary for an MRI study. This implies that with equal number of studies per week the cost of a PET study would be twice that of an MRI study. How many PET studied is it possible to perform and analyze per day? This is very much dependent on which type of tracer and which type of experiment is performed. The demand on clinically motivated studies is so high in our institution that we have to rationalize the studies and make them fit a routine practice. Thus, during the days committed to

FUTURE ASPECTS From our experiences and from those of other groups working with positron emission tomography it is obvious that PET is an important tool in the research on various normal and pathological conditions . This is perhaps especially true in the field of endocrinology where the complex interactions make a direct in vivo technique of utmost value . Furthermore, endocrinologists have the training and experience to fully appreciate the functional and biochemical information attainable with PET. With the new improved positron camera devices with spatial resolutions approaching a few millimeters and with a more developed synthesis technique giving a multitude of labelled substances for monitoring biochemical parameters, completely new areas of research will be opened. We have so far used PET in the study of pituitary adenomas. We believe that the study of other endocrine disorders of the hypothalamo-pituitary axes will be of even greater importance. It is not possible today to analyze the functional aspects of the hypothalamus directly in man. Only indirect tests and evaluation of serum hormonal levels are possible. With PET it will be possible directly to have an insight into the functional and biochemical aspects of the hypothalamus and thereby disclose its role in different types of disease such as polycystic ovarian syndrome, hormone disturbances with possible involvement primarily from the hypothalamus, infertility, obesity, anorexia nervosa, sexual disturbances etc. It is likely that this new technique of PET may allow a penetration of the biochemistry of the hypothalamus and the pituitary, and thus aid in disclosing the cause of several diseases and hormonal disturbances, some of which we may today even attribute to other organs. Similarly, with the whole body positron cameras the functional connection between the hypothalamus-pituitary and the end organs such as thyroid, adrenals, ovaries etc. can be even better elucidated.

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ACKNOWLEDGEMENTS

In: Greitz T. (Ed.), The metabolism of the human brain studied by positron emission tomography. Raven Press, New York, 1985, p. 223.

This work was supported by grants from the Swedish Medical Society and the Swedish Cancer Society. The very im.portant contributions from the members of the Uppsala PET group, especially professor PO Lundberg, professor Kjell Bergstrom and the skilled chemists headed by professor Bengt Langstrom are greatly appreciated.

11. Bustany P., Chatel M., Derlon J.M., Darcel F., Sgourpoulus P., Soussaline F., Syrota A Brain tumor protein synthesis and histological grades: a study by positron emission tomography (PET) with C11-L-methionine. J Neuro-Oncol. 3: 379, 1986.

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46.

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