A Comparison of 11 C-Labeled l -DOPA and l -Fluorodopa as Positron Emission Tomography Tracers for the Presynaptic Dopaminergic System

Abstract

¹¹C-labeled 3,4-Dihydroxy-phenyl-l-alanine (L-DOPA) and l-fluorodopa were used as tracers for the functional state of the presynaptic dopamine system in anesthetized monkeys with positron emission tomography, The radiotracer disposition in brain tissue and plasma were studied and effects induced by pharmacologic challenges were evaluated, 6R-l-erythro-5,6,7,8-tetrahydrobiopterin (6R-BH₄) increased the striatal influx rate constant, e.g., striatal K_i for l-[β-¹¹C]DOPA, but it induced no effect on the K_i-value using l-[β-¹¹C]-6-fluorodopa. Studies of radiolabeled tracer and metabolites in plasma showed substantial differences between the two tracers. At baseline conditions, 60% unchanged l-[β-¹¹C]DOPA was detected in plasma 50 minutes after tracer injection and the 3-O-methylated fraction accounted for 25% of total radioactivity. For l-[β-¹¹C]-6-fluorodopa, the relation was inverse; about 25% unchanged tracer and 60% 3-O-methyl metabolite were present in plasma after 50 minutes. A site-specific ¹¹C-labeling in the carboxylic position in the molecules revealed a significant specific retention of radioactivity in striatum with l-[carboxy-¹¹C]-6-fluorodopa but not with l-[carboxy-¹¹C]DOPA. The 3-O-methyl metabolite of l-DOPA is known to pass the blood-brain barrier and may interfere with the calculation of the K_i value using a brain reference region. Thus, extensive 3-O-methylation in circulation of the fluorinated analog could obscure the detectability of potential functional change in striatal K_i of the tracer when using a reference tissue model for calculation.

Keywords

Positron emission tomography Tolcapone Monkey

Positron emission tomography (PET) and radiolabeled 3,4-Dihydroxy-phenyl-l-alanine (l-DOPA) have been used to study presynaptic dopaminergic function in vivo for several years. l-DOPA has been radiolabeled with different positron- emitting nuclides, ¹¹C-labeled l-DOPA in β-position, l-[β-¹¹C]DOPA, and ¹⁸F-labeling in 6 position on the aromatic ring giving the analogue l-6-[¹⁸F]fluorodopa (Fig. 1), are commonly used today. These tracers have both been shown to reveal the presynaptic dopaminergic function in healthy and pathologic states of humans (Garnett et al., 1983; Hartvig et al., 1991; Leenders et al., 1986; Tedroff et al., 1992a). However, divergent estimates of the specific striatal utilization have been indicated using l-[β-¹¹C]DOPA and l-6-[¹⁸F]fluorodopa, e.g., regarding a possible increased dopamine synthesis rate in schizophrenia as compared to healthy volunteers (Dao-Castellana et al., 1997; Hietala et al., 1995; Lindstrom et al., 1998; Reith et al., 1994).

FIG. 1.

¹¹C-labeled tracer used in the study. (A) 3,4-dihydroxy-l-phenylalanine and (B) 3,4-dihydroxy-6-fluoro-l-phenaylalanine. *Indicates labeling in β-position and **indicates in the carboxylic position.

Some earlier comparisons of the two tracers imply a similar distribution and metabolism (Firnau et al., 1986; Firnau et al., 1987). Other studies indicate differences with respect to the affinity for the metabolic enzymes, especially the peripheral formation rate of the major metabolite the corresponding 3-O-methyldopa and the blood-brain clearance of the tracers (Cumming et al., 1995; Cumming et al., 1987; Melega et al., 1990).

The comparisons compose not only possible differences due to the effects of introduction of fluorine into the aromatic ring but methodologic differences as well. The use of different radionuclides, such as ³H compared to ¹⁴C, may yield large differences in specific radioactivity, and tritium exchange during the radioanalysis procedure could influence the outcome of the measurements. To evaluate differences and similarities of the l-DOPA and its 6-fluoroanalogue as tracers for the presynaptic dopaminergic system PET experiments were performed in anesthetized monkeys with both molecules labeled with ¹¹C in β-position. Effects induced by pharmacologic challenges were studied with respect to the radiotracer influx rate constant, K_i, in striatum and radioactivity disposition in plasma. Additional experiments were also performed with site-specific ¹¹C-labeling in carboxylic position, and the effect on the calculated K_i value was studied.

MATERIALS AND METHODS

Animals and drugs

Female rhesus monkeys (Macaca mulatta) 20 to 30 years of age weighing 5 to 11 kg from the Primate Research Laboratory of Uppsala University PET Centre were used after an overnight fast. The monkeys were housed in groups of four to eight animals for at least 4 months before the study. The studies were approved by the Animal Ethics Committee of Uppsala University (permission C03/92, C289/94, and C338/97). Anesthesia was induced with 100 mg intramuscular ketamine (Ketalar, Parke Davis, Morris Plains, NJ, U.S.A.). After transport of the anesthetized monkey to the investigation area of the Uppsala University PET Centre, a venous catheter was inserted into each hind leg of the monkey, one for administration of anesthetic drugs and blood sampling, and the other for administration of the radioactive tracer.

Tracheal intubation was followed by muscle relaxation with 5 mg/kg/h intravenous atracurium (Tracrium, Wellcome, London) and mechanical ventilation with 30% oxygen in air. Anesthesia was maintained with a dose of 8 to 12 mg/kg/h intravenous propofol (Diprivan, ICI). Vital signs including blood pressure, heart and respiratory rate, and body temperature were monitored throughout the investigation. In all studies, recovery from anesthesia was unremarkable.

6R-l-erythro-5,6,7,8-tetrahydrobiopterin (6R-BH₄) obtained from the Suntory Institute for Biomedical Research (Osaka, Japan) was dissolved in a phosphate buffer (pH 7.4), and a fresh solution was prepared every hour. The infusion of 5 mg/kg/h was started 60 minutes before the PET scan using a syringe pump (Harvard apparatus, model 22, South Natick, MA, U.S.A.).

The peripherally and centrally active catechol-O-methyl trans ferase inhibitor 3,4-di hydro-4-methyl-5-ni trobenzophenone (tolcapone, Ro 40-7592, Roche, Basel) (Zurcher et al., 1990) was dissolved in equal parts of polyethyleneglycol (Macrogolum 400, Apoteket AB, Gothenburg) and sterile water and given as a bolus dose of 10 mg/kg 15 minutes before injection of the radiotracer.

Radiochemical synthesis

The ¹¹C radionuclide was produced by a ¹⁴N (p,α) ¹¹C reaction using the MC17 cyclotron (Scanditronix, Uppsala) at the Uppsala University PET Centre and obtained as [¹¹C]carbon dioxide. The synthesis of l-[β-¹¹C]DOPA (Bjurling et al., 1990) and l-6-[β-¹¹C]fluorodopa (Bjurling et al., 1991b) was performed using a combination of organic synthesis methods and a multi enzymatic procedure. The synthesis of the corresponding carboxy-¹¹C-labeled tracer was performed in a similar manner (Bjurling et al., 1991a).

After purification by semipreparative liquid chromatography, the fraction containing ¹¹C-labeled tracer was collected and adjusted to pH 4.5. The final solution was filtered through a 0.22-μm filter. The identity and radiochemical purity of ¹¹C-labeled tracer were confirmed by liquid chromatography and by comparison with authentic reference compounds. The radiochemical purity was always higher than 98%. The specific radioactivity was at least 2 GBq/μmol at the time of injection and the radioactive dose of the tracer injected intravenously to the monkey was in the 90 to 200 MBq range corresponding to 9- to 20-μg tracer.

Positron emission tomography

Studies of brain radioactivity distribution were performed with the monkey positioned with the head in an eight-ring PET (GE 2048-15B Plus, GE 4096-15WB General Electric Medical Systems, Uppsala). Radioactivity concentrations were recorded in 15 transaxial slices 6.8 mm apart and with an inplane resolution of 5 mm (Holte et al., 1989) and 6 mm (Rota Kops et al., 1990), respectively. Fixation was achieved by using plugs attached to the auditory passage of the monkey. The head was adjusted to the detectors of the tomograph with the middle slice parallel to the orbitomeatal line.

In all studies, a series of sequential ¹¹C-labeled tracer injections were made, always separated by at least 120 minutes. The first scan served as the baseline condition for the following scans, in which additional pharmacologic treatments were made. The monkeys remained in the scanner between sequential scans. Scanning began at the start of tracer injection and continued for 60 minutes with time frames of 15×60 seconds and 15×180 seconds. PET images were reconstructed from data collected from each time-frame, corrected for tissue attenuation of 511-Ke V gamma radiation measured with an external 68Ge ring (Ostertag et al., 1989), and filtered with a 4.2-mm Hanning filter.

Realignment of PET scans and definition of region of interests

Summation images were constructed for each scan using data collected 20 to 60 minutes after tracer injection by the use of computer software supplied with the tomograph. A realignment method was used when necessary (>0.5 mm head movement) to prevent movements between scans from causing artifactual increases or decreases in the calculated K_i of the tracer (Anderson, 1995). Briefly, the method is based on using a mask of one reference data set (e.g., baseline) made from voxels with high signal-to-noise ratio in comparison to a sample data set (e.g., perturbation). The result of the comparison is a matrix against which both data sets are resliced. After this procedure, an average image was formed using the summation images from the two realigned data sets. All regions of interest (ROI) were drawn in this averaged summation image. ROI were drawn on visual inspection of the images, and the position of the ROI was compared to a brain atlas made from cryosectioning of a rhesus monkey brain. A circular ROI with diameter of 0.8 cm was used to delineate each striate. Left and right striate values were averaged. The whole set of ROI was then transferred to the two realigned data sets and used in the calculations.

Calculations of PET data

Time-activity curves were generated and used to reconstruct images according to the mathematical background as described previously (Patlak and Blasberg, 1985; Tedroff et al., 1992b). A large region of nondopaminergic brain tissue situated posterior to the corpus striatum (about 8 cm2) was delineated as reference tissue and input function for the reconstruction. This method generates a linear description of the striatal radioactive uptake and the slope, denoted influx rate constant or K_i, was determined by linear regression analysis using weighted least-square analysis from data collected 5 to 60 minutes of normalized time after tracer injection. Each data point was used after correction by a weight factor by the empirical formula for calculation of the standard deviation for a given pixel (Carson et al., 1986). The calculated l-[β-¹¹C]DOPA K_i using the tissue-slope method has been validated to represent the rate for transport and conversion of the tracer to [¹¹C]dopamine within the ROI studied (Lindner et al., 1995; Miwa et al., 1992; Tedroff et al., 1992b).

Plasma sample preparation and high-performance liquid chromatographic analysis

Venous blood samples were collected at 5, 25, 45, and 50 minutes after tracer injection, and centrifuged at 4000 rpm for 2 minutes. Proteins were precipitated with 45 μl of 5 mol/L perchloric acid in 0.8 mL of plasma. After centrifugation for 3 minutes at 15,000 rpm, the supernatant was filtered through an acetate filter (0.22 μm, Dynagard^®, Microgon Inc, U.S.A.) yielding ≈0.5 mL of sample. High performance liquid chromatography analysis of the composition of the ¹¹C-derived radioactivity was performed as described previously with minor modifications (Lindner et al., 1995). Before injection into the chromatographic system a 15 μL standard solution yielding 0.1−0.2 mmol/L of reference compounds was added to the sample. Separation of ¹¹C-labeled tracer and derived labeled metabolites was performed on a C-18 column (Ultracarb ODS20, 3 μm 100×4.6 mm i.d., Phenomex, Scanditec). The composition of the mobile phase was 0.1 mol/L acetate-citrate, 1 mmol/L edetic acid, 5 mmol/L sodium heptanesulphonate, and 12.5% methanol. The pH was 3.7 in the final buffer. The recovery of radioactivity from plasma after protein precipitation was 82 ± 7% and in the chromatographic step 101 ± 8%, n = 26.

l-DOPA, dopamine, homovanillic acid, 3-O-methyl-l-DOPA, and 3,4-dihydroxy-phenylacetic acid were obtained from Sigma Chemical Company (St Louis, MO, U.S.A.). 6-Fluoro-3,4-dihydroxy-phenyl-l-alanine was obtain from Research Biochemicals International (U.S.A.), and fluorodopamine, fluoro-homovanillic acid, 6-fluoro-3-O-methyl-l-DOPA, and 6-fluoro-3,4-dihydroxy-phenylacetic acid were kindly provided by Dr. Philippe Damhaut, Université Libre de Bruxelles, Belgium. Organic solvents were of analytical grade and purchased from Merck (Darmstadt, Germany). Edetic acid was obtained from KEBO AB (Stockolm, Sweden), whereas 1-heptanesulphonic acid as the sodium salt was purchased from Aldrich-Chemie (Steinheim, Germany).

Statistical analysis

The paired t-test was used to evaluate induced changes from pharmacologic challenge in comparison to baseline conditions. A one-way analysis of variance with repeated measure was performed to evaluate the striatal K_i value increase after adding the 6R-BH₄ infusion to the tolcapone-treated monkeys. Regression-analysis was performed for analysis of significant slope-values of the carboxy-labeled tracer. STATISTIC A for Windows (StatSoft) version 5.0 was used and the level of significance was set to P < .05. All values are represented as mean ± SD.

RESULTS

Baseline studies of l-[β-11C]DOPA and l-6-[β-11C]fluorodopa striatal Ki

Sequential PET scans without any challenge, i.e. test-retest values, revealed data shown in Table 1. The coefficient of variation obtained were similar for the two tracers, 3% to 4% for l-[β-¹¹C]DOPA and 4% to 5% for l-6-[β-¹¹C]fluorodopa.

TABLE 1.

Test-retest scans, baseline conditions

	l-[β-¹¹C]DOPA				l-6-[β-¹¹C]fluorodopa
	Monkey 1		Monkey 2		Monkey 3		Monkey 4
	Striatal K₁(min⁻¹)	Change%	Striatal K₁(min⁻¹)	Change%	Striatal K₁(min⁻¹)	Change%	Striatal K₁(min⁻¹)	Change%
1^st scan	0.0117		0.0139		0.0099		0.0146
2^nd scan	0.0111	−5.1	0.0151	8.6	0.0091	−8.1	0.0155	6.2
3^rd scan	0.0113	−3.4	0.0146	5.0	0.0096	−3.0	0.0141	−3.4
Mean	0.0114		0.0145		0.0095		0.0147
SD	0.0003		0.0006		0.0004		0.0007
% CV	2.7		4.1		4.2		4.8

The striatal K_i-value measured with l-6-[β-¹¹C]fluorodopa were 11 ± 8% lower than the striatal K_i-value measured with l-[β-¹¹C]DOPA in the same monkey on the same day (paired t-test; P < .01), Table 2.

TABLE 2.

Baseline striatal K₁ values of l-[β-¹¹C]DOPA and l-6-[β-¹¹ C]fluorodopa

	l-[β-¹¹C]-DOPA	l-6-[β-¹¹C]-fluorodopa	Intra-individual difference% (mean value)
Mean striatal K_i/(min⁻¹)	0.0136	0.0120	−11*
SD	0.0025	0.0020	8
%CV	18	17

Sequential scans were performed in each monkey (n = 13) with both tracer on the same day.

P < 0.05; paired t-test.

Effect of 11C-labeling in carboxylic position on the striatal Ki

The calculated K_i value was lower for both tracers when labeled in the carboxylic position, Table 3. The K_i value of carboxy-labeled tracer decreased as compared to β-labeling with 77% on average for the fluorinated analog and 101% with the l-DOPA tracer. A significant K_i-value was still present on carboxy-labeling of the fluorinated analog (regression analysis; P < .05; Table 3). Typical Patlak curves used for the calculation of striatal K_i values of the β- and carboxy-labeled tracers are shown in Fig. 2.

FIG. 2.

Typical Patlak curves from ¹¹C-labeled l-[¹¹C]-fluorodopa tracer in β-position (filled symbol) and in carboxylic-position (open symbol). CT, radioactivity in striatum; CR radioactivity in reference area.

TABLE 3.

Effect of ¹¹C site-labeling on the measured K₁ value

	l-[¹¹C]DOPA
	¹¹C-labeling in β-position	¹¹C-labeling in carboxylic-position	Difference %
Striatal K₁/(min⁻¹)	Striatal K₁/(min⁻¹)		Difference %
Monkey 5	0.0098*	−0.0005	−105
Monkey 6	0.0121*	0.0002	−98
	l-6-[¹¹C]fluorodopa
Monkey 7	0.0100*	0.0023*	−77
Monkey 8	0.0121 *	0.0029*	−76

P < 0.05; regression analysis.

Effects of 6R-BH4 on l-[β-11C]DOPA and l-6-[β-11C]fluorodopa striatal Ki

6R-BH₄ (5 mg/kg/h) increased the striatal l-[β-¹¹C]DOPA K_i with a mean value of 13 ± 13% (P < .05; Table 4; Fig. 3). In otherwise identical conditions, the striatal K_i value measured for l-6-[β-¹¹C]fluorodopa was not significantly different than the baseline value observed in the same monkey −1 ± 16% (Table 4; Fig. 3)

FIG. 3.

Percent change of striatal K₁ value after pharmacological challenges compared with baseline scan in the same monkey. The bars indicated mean values ± SD. *P < .05 from from baseline. (A), l-[β-¹¹C]DOPA, tolcapone data shown are derived from Hartvig et al., 1992 (B) l-[β-¹¹C]-6-fluorodopa.

TABLE 4.

Effect of pharmacologic challenges on the measured K₁ value

	Baseline	6R-BH₄	Change	Tolcapone	Change	6R-BH4 + tolcapone	Change
			%		%		%
	Striatal	Striatal		Striatal		Striatal
	K₁(min⁻¹) ± SD	K₁(min⁻¹) ± SD	% ± SD	K₁(min⁻¹) ± SD	% ± SD	K₁(min⁻¹) ± SD	% ± SD
l-[β-¹¹C]DOPA	0.0135 ± 0.002	0.0154* ± 0.001	13 ± 13	—	0 ± 2‡	0.0151* ± 0.001	11 ± 6
	n = 8	n = 6				n = 4
L = 6 = [β-¹¹C]fluorodopa	0.0132 ± 0.002	0.0125 ± 0.003	−1 ± 16	0.0178* ± 0.002	32 ± 6	0.0189† ± 0.002	45 ± 12
	n = 10	n = 8		n = 4		n = 6

n = number of monkeys and “change” indicates the mean value of each individual change from its own baseline value.

P < 0.05; paired t-test from baseline in same monkey.

†

P < 0.05; from tolcapone treatment, one-way ANOVA with repeated measure.

‡

Taken from Hartvig et al., (1992).

Effects of tolcapone on the striatal Ki before and after 6R-BH4 infusion

Tolcapone (10 mg/kg/h) increased the striatal K_i of l-6-[β-¹¹C]fluorodopa with an average of 32 ± 6% (P < .01; from baseline; Table 4; Fig. 3). The K_i values were also significantly increased from baseline value after challenge with both 6R-BH₄ and tolcapone (45%; P < .01; Table 4; Fig. 3). Compared with the tolcapone challenge alone, a significant increase of the striatal l-6-[β-¹¹C]fluorodopa K_i was observed in monkeys after challenge with both 6R-BH₄ and tolcapone (13%; P < .05; one-way analysis of variance; Table 4; Fig. 3).

In a previous study with identical experimental conditions as in the present study, tolcapone treatment did not change the measured striatal K_i compared to baseline values with l-[β-¹¹C]DOPA in monkeys (Hartvig et al., 1992). These result are included in Table 4. Compared with the 6R-BH₄ challenge alone, no further effects on the striatal l-[β-¹¹C]DOPA K_i were induced by the challenge with both tolcapone and 6R-BH₄ (P = .31; Table 4; Fig. 3).

Measurement of radioactive metabolites in plasma samples

A difference between metabolite formation rates of the two tracers l-[β-¹¹C]DOPA and l-6-[β-¹¹C]fluorodopa was measured on plasma analysis of tracer and radioactive metabolites (Fig. 4A). At baseline conditions, 60 ± 4% of unchanged l-[±-¹¹C]DOPA was determined in plasma 50 minutes after tracer injection, and the 3-O-methylated fraction accounted for 25 ± 4% of the total radioactivity. The relation was inverse for l-6-[β-¹¹C]fluorodopa; 30 ± 3% of unchanged tracer and 57 ± 7% of 3-O-methyl metabolite was present at 50 minutes.

FIG. 4.

Fractions of unmetabolized tracers (circles) and radiolabeled metabolites in plasma; 3-0-methyl-DOPA fractions (squares) and sum of other metabolites, 3,4-dihydroxy-phenylacetic acid, dopamine, and homovanillic acid fraction (triangles) Filled lines represents studies with l-[β-¹¹C]-6-fluorodopa, n = 2−4. (A) Baseline conditions; (B), Tolcapone treatment.

6R-BH₄ did not induce any discerible effect on the rate of metabolism in plasma of any of the tracers studied (data not shown). Tolcapone changed the relative radioactive fraction of the tracer and its metabolites in plasma as expected and the 3-O-methyl metabolite fraction was reduced to between 0 to 5% of total radioactivity (Fig. 4B). The homovanillic acid fraction also decreased to zero values whereas the 3,4-dihydroxy-phenylacetic acid fraction increased.

DISCUSSION

6R-BH₄, a cofactor for tyrosine hydroxylase, was used as a tool to enhance the dopamine synthesis rate. The constant rate infusion of 6R-BH₄ (5 mg/kg) induced a significant increase of the striatal l-[β-¹¹C]DOPA K_i that was in accordance with earlier studies showing the modulating properties of 6R-BH₄ on the dopamine synthesis rate in rats (Tsukada et al., 1994) and in monkeys (Tedroff et al., 1997; Tsukada et al., 1996). The proposed mechanism of action is that 6R-BH₄ induces a higher formation rate of endogenous l-DOPA, which in turn regulates the AADC activity (Misu et al., 1996; Opacka-Juffry and Brooks, 1995; Tedroff et al., 1997; Torstenson et al., 1997). The 6R-BH₄-induced increase in aromatic l-amino acid decarboxylase (AADC) activity and has been verified in an AADC activity assay in rats in which the conversion of l-DOPA to dopamine is measured (Torstenson et al., in manuscript).

The dopamine releasing effect induced by 6R-BH₄ (Koshimura et al., 1990) could theoretically decrease the apparent influx rate constant due to a release of the trapped radioactivity in the striatum. This influence on the calculated value is less likely because PET studies with a potent dopamine releasing agent, e.g., amphetamine, did not decrease the measured striatal K_i for l-[β-¹¹C]DOP A (Hartvig et al., 1997).

In contrast to the increase striatal K_i value measured with l-[β-¹¹C]DOPA, 6R-BH₄ induced no significant change of the measured striatal K_i value compared to the baseline with l-6-[β-¹¹C]fluorodopa.

¹⁸F-Iabeled l-6-fluorodopa has been evaluated with a variety of models using different methods to calculate the utilization rate of the tracer in vivo with PET. This includes methods using metabolite corrected arterial plasma input function also regarding the 3-O-methyl fraction competition for the transport enzyme (Gjedde et al., 1991; Kuwabara et al., 1993; Kuwabara et al., 1995), as well as the tissue reference ratio model (Patlak and Blasberg, 1985; Cumming and Gjedde, 1998).

Estimation of the striatal K_i for l-[β-¹¹C]DOPA with PET has been performed with a reduced three-compartment model (Hartvig et al., 1991; Patlak and Blasberg, 1985; Tedroff et al., 1992b) using a reference region in the brain with negligible AADC activity. This model might be sensitive to radiolabeled metabolites formed in the periphery that pass the blood-brain barrier and interfere unequally with the reference and the target region (Sawle et al., 1994).

l-6-[¹⁸F]fluorodopa has been shown in vitro to have a low affinity for the catechol-O-methyl transferase (COMT) enzyme (Creveling and Kirk, 1985), and this was one of the reasons for choosing 6 position labeling instead of, e.g., 2 position (Cumming et al., 1988). Studies of radiolabeled tracer and metabolites in plasma have indicated that native l-DOPA has higher affinity than the fluoroanalogue for the COMT enzyme (Firnau et al., 1987; Firnau et al., 1988). The present study showed an extensive 3-O-methyl metabolite formation of the fluoroanalogue, which the l-[β-¹¹C]DOPA tracer did not exhibit. This higher rate of 3-O-methyl-6-fluorodopa formation corresponds well with studies on ¹⁸F-Iabeled F-DOPA and ³H-labeled DOPA in rats (Cumming et al., 1995).

The 3-O-methyl metabolite enters the brain in substantial amounts (Doudet et al., 1991; Reith et al., 1990) and hence will interfere in the calculation of the striatal K_i value using a brain reference area (Sawle et al., 1994). A significant contribution of radioactivity from this metabolite in the brain would decrease the calculated specific use in the target tissue due to an increased background radioactivity in the tissue-reference model as predicted in the original method description by Patlak and Blasberg (1985). This influence of different peripheral metabolite formation rates is also found in the baseline striatal K_i value of the two tracers where the l-[β-¹¹C]DOPA tracer showed a 11 % higher value as compared to l-6-[β-¹¹C]fluorodopa. Previously, a 48% baseline difference between the two tracers was reported (Hartvig et al., 1992). However, the monkeys in that study received either l-[β-¹¹C]DOPA or l-6-[β-¹¹C]fluorodopa so the interindividual variation of about 20% shown for both tracers in baseline striatal K_i value (Table 2) might explain these divergent estimates.

The 3-O-methyl metabolite fraction was reduced to less than 5% of total plasma radioactivity, and the striatal K_i value of the ¹¹C-labeled fluoroanalogue was increased with 32% after COMT inhibition with tolcapone. This corresponds well with previous results in monkeys and humans with l-6-[¹⁸F]fluorodopa and PET after COMT inhibition (Doudet et al., 1997; Sawle et al., 1994). The striatal K_i values measured with ¹¹C-labeled l-DOPA was shown not to be dependent on COMT inhibition in identical PET studies as in the present study (Hartvig et al., 1992; Tedroff et al., 1992b). The COMT inhibition in the present study did not induce any additional change in the striatal l-[β-¹¹C]DOPA K_i which strengthens the observation.

Pretreatment with COMT inhibition and 6R-BH₄ infusion increased the K_i value of the fluoroanalogue by 45% on average compared with 32% without COMT inhibition. This 6R-BH₄ induced increase was in parallel with the increase (+13%) measured with l-[±-¹¹C]DOPA as tracer without COMT inhibition. Hence, only after COMT inhibition was it possible to detect the stimulation of the K_i value of fluorodopa produced by 6R-BH₄ using the present tissue-slope method.

¹¹C-labeling of l-DOPA and l-fluorodopa in the carboxylic-position in the molecule (Fig. 1) would imply that the ¹¹C-label is lost as [¹¹C]CO₂ in the brain due to the activity of AADC, no radioactivity accumulates in the striate and the measured K_i value should approach a zero value. Site-specific ¹¹C-labeling in carboxylic position reduced the radioactivity accumulation for both tracers. However, the reduction differed between the two tracers. The striatal K_i value measured with the l-[β-¹¹C]DOP A tracer approached zero whereas the fluoroanalogue did not reach a zero-value (average 77% decrease). Radiolabeled tracer or formed metabolites appeared to be specifically retained in the striatum when using the fluoroanalogue giving a measurable rate constant. One hypothesis is that peripherally formed 3-O-methyl metabolite has affinity for the AADC enzyme without any conversion to 3-O-methylfluorodopamine and hence is retained in the striatum. This hypothesis is strengthened by a visualization of the striatum after administration of the radiolabeled 3-O-methyl fluorometabolite (Wahl et al., 1994).

Correction for the 3-O-methyl fraction influence in the calculation of the K_i value of the fluoroanalogue is cumbersome and is supported by the massive literature in the area (Cumming and Gjedde, 1998). Large differences in hepatic COMT activity exist within species (also sex differences) (Cumming et al., 1993; De Santi et al., 1998) and also between species (Guldberg and Marsden, 1975) which implies difficulties in introducing standard corrections in the calculations of PET data. COMT inhibition in connection with the PET study would be the only way to avoid a possible 3-O-methyl metabolite interaction in the brain with the AADC enzyme.

There may also be an affinity difference of the two radiotracers for the AADC enzyme in the brain. In vitro studies in rats have shown Km-values of 101 ± 22μmol/L for l-6-[¹⁸F]fluorodopa and 228 ± 7μmol/L for D,l-[¹⁴C]DOPA (Cumming et al., 1988). The l-6-[β-¹¹C]fluorodopa K_i value increased by 32% after COMT inhibition and the baseline difference between the tracers was only 11%. This might indicate that the fluoroanalogue has a higher formation rate to fluorodopamine in vivo than the endogenous molecule to dopamine. This is still a matter for further studies.

In conclusion, the two studied β-¹¹C-labeled molecules l-DOPA and l-fluorodopa have different metabolism and distribution. The extensive 3-O-methyl metabolite formation of the fluoroanalogue seems to obscure the 6R-BH₄ induced effect on dopamine synthesis rate as evaluated with PET. It seems feasible to compare quantitative data from clinical and research studies performed with the two tracers evaluated with tissue-slope method if COMT inhibition is performed for the fluoroanalogue. Careful interpretation of data, however, should be made when including a new pharmacologic challenge in a study that would give new influences not easily foreseen.

Footnotes

Abbreviations used

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