5-Hydroxy- l -[β- 11 C]tryptophan versus α-[ 11 C]Methyl- l -tryptophan for Positron Emission Tomography Imaging of Serotonin Synthesis Capacity in the Rhesus Monkey Brain

Abstract

The purpose of this study was to compare two positron emission tomography (PET) tracers that were developed to follow serotonin (5HT) synthesis by performing sequential PET scanning of the same rhesus monkey (n = 4) on the same day. α-[¹¹C]Methyl-l-tryptophan ([¹¹C]AMT) and 5-Hydroxy-l-[β-¹¹C]tryptophan ([¹¹C]HTP) are substrates in the first and second enzymatic steps, respectively, in the biosynthesis of 5HT. Regional net accumulation rate constants were derived from kinetic (two-tissue compartment model with irreversible tracer trapping) and graphic (Patlak) analyses, using the arterial plasma concentrations as input. The kinetic data analysis showed that the rate constant for the transfer of [¹¹C]HTP into the brain (K₁) was higher than that for [¹¹C]AMT in the striatum and thalamus but was similar in other brain regions. The rate constant for tracer trapping (k₃) was also higher for [¹¹C]HTP than for [¹¹C]AMT in the striatum (0.046 ± 0.024 versus 0.019 ± 0.006 min⁻¹) and thalamus (0.039 ± 0.013 versus 0.016 ± 0.007 min⁻¹). In agreement with previously reported regional HTP accumulation rates, the net accumulation rate constant (K_acc) for [¹¹C]HTP was also higher in these regions than in other brain regions; this is in contrast to the uniform distribution of [¹¹C]AMT K_acc values. This suggests that the regional net accumulation rates obtained with these two PET tracers will be of different magnitude, which might be related to the activity of each targeted enzyme.

Keywords

biosynthesis positron emission tomography rhesus monkey serotonin

Introduction

Early efforts to determine tissue serotonin (5HT) synthesis rates in small mammals typically involved pharmacological manipulation of the 5HT system followed by the sampling of brain tissue (Brown et al, 1979; Goldman-Rakic and Brown, 1981; Karoum et al, 1984; Shannak and Hornykiewicz, 1980; Vriend et al, 1993). The tissue samples were then analysed for determination of the precursors tryptophan and 5-Hydroxytryptophan (HTP), 5HT itself, and the main metabolite 5-Hydroxyindole acetic acid (HIAA). The radiolabelled compounds α-[¹¹C]Methyl-l-tryptophan ([¹¹C]AMT) (Chandana et al, 2005; Chugani et al, 1998; Fedi et al, 2003; Juhasz et al, 2003; Kagawa et al, 2005; Muzik et al, 1997; Natsume et al, 2003; Nishikawa et al, 2005) and 5-Hydroxy-l-[β-¹¹C]tryptophan ([¹¹C]HTP) (Agren and Reibring, 1994; Agren et al, 1993; Eriksson et al, 2006; Hagberg et al, 2002; Reibring et al, 1992) have been used in positron emission tomography (PET) in humans for probing the first and second enzymatic steps, respectively, in the biosynthesis of 5HT.

[¹¹C]AMT is a [¹¹C]-methyl analogue of the essential amino acid tryptophan, which is a substrate for tryptophan hydroxylase, the first enzyme in the two-step synthesis of 5HT. Partial conversion of [¹¹C]AMT to α-Methylserotonin (AM-5HT) in the brain has been showed in animals (Diksic et al, 1990; Gharib et al, 1999; Shoaf et al, 2000) in serotonergic neurons and nerve terminals (Cohen et al, 1995). However, there seems to be a time delay between uptake and conversion of the tracer into AM-5HT (Gharib et al, 1999; Shoaf et al, 2000). Further, under pathologic conditions, [¹¹C]AMT can occasionally be metabolised by means of the kynurenine pathway and the measurements may thus reflect both pathways (Chugani and Muzik, 2000).

Apart from the radiolabel, [¹¹C]HTP is identical to the endogenous compound that will undergo conversion to 5HT by aromatic l-amino acid decarboxylase (AADC), the second enzyme in the biosynthesis, and then further catabolism by monoamine oxidase A to HIAA. The time-dependent accumulation of radioactivity in the basal ganglia of rats in [¹¹C]HTP/PET is mainly a consequence of the trapping of decarboxylated products (Lindner et al, 1997), thus providing an in vivo index of the activity of the decarboxylating enzyme AADC. In a previous study, analysis of rat brain homogenates revealed that most of the radioactivity (approximately 75%) was associated with [¹¹C]HIAA 40 mins after intravenous [¹¹C]HTP injection (Lindner et al, 1997). The decarboxylating enzyme AADC is ubiquitous; the enzyme is expressed in all monoaminergic cells (serotonergic, noradrenergic, and dopaminergic). Hence, it may be possible to follow AADC activity in all of these neuronal cell types using [¹¹C]HTP.

The endogenous concentrations of tryptophan and HTP in the brain are about equal to (Fernstrom and Wurtman, 1971; Young and Gauthier, 1981), or much less than (Zhu and Juorio, 1995), the half-saturation concentrations for either of the targeted enzymes (tryptophan hydroxylase and AADC). It has been suggested that measurement of either of these enzymatic steps could provide a value that is proportional to 5HT synthesis (Hagberg et al, 2002). In previous [¹¹C]AMT/PET and [¹¹C]HTP/PET studies, the net accumulation rate constant for tracer trapping was stable within an individual and the rank order was consistent with the rank order of regional concentrations of 5HT in the human brain (Chugani et al, 1998; Hagberg et al, 2002). Further, the net accumulation rate constants for tracer trapping obtained in separate [¹¹C]AMT/PET and [¹¹C]HTP/PET studies in humans were found to be correlated (Hagberg et al, 2002; Leyton et al, 2005). The purpose of this study was to directly compare [¹¹C]AMT and [¹¹C]HTP as PET ligands for 5HT synthesis capacity in rhesus monkeys.

Materials and methods

Animals

Four female rhesus monkeys (Macaca mulatta) (Primate Research Laboratory, Uppsala University), aged 15 to 20 years and weighing 6 to 13 kg, were allowed only water from the evening before the PET study. The Animal Ethics Committee of Uppsala University approved the protocol (C 117/04).

Radiochemistry

[¹¹C]AMT and [¹¹C]HTP were synthesised as described previously (Bjurling et al, 1989; Chakraborty et al, 1996). Radiochemical purity was greater than 95%. The mean specific radioactivity levels of [¹¹C]AMT and [¹¹C]HTP at the time of injection were 17 ± 10 and 15 ± 6 GBq/μmol, respectively. The average injected [¹¹C]AMT radioactivity dose was 292 ± 125 MBq, corresponding to a total amount of injected unlabelled AMT of 20 ± 8 nmol. The average injected [¹¹C]HTP dose was 284 ± 157 MBq and contained 19 ± 3 nmol of unlabelled HTP.

Positron Emission Tomography System

The studies were performed using the Hamamatsu SHR-7700 (Hamamatsu Photonics KK, Hamamatsu, Japan). The 16 detector rings of this instrument provide a 114 mm axial FOV and 31 two-dimensional image planes for every 3.6 mm distance. The transaxial resolution is 3 mm in 2D mode (Watanabe et al, 1997). Transmission scans with rotating ⁶⁸Ge sources were used to correct the emission scans for the attenuation of 511keV photons through the tissue and head supports. Images were reconstructed using a 4 mm Hanning filter after correction for attenuation and scattered radiation.

Positron Emission Tomography Studies

After induction of anaesthesia with 100 mg intramuscular ketamine (Ketaminol, Vetpharma, Zurich, Switzerland), the animals were transported to the investigation site at Uppsala Imanet. A venous catheter was inserted in both hind legs of each monkey, one for administration of the anaesthetic agent propofol (8 to 12 mg/kg/h, Propofol-Lipuro, B/Brown) and one for administration of the radioactive tracer. After tracheal intubation, muscle relaxation was achieved with 5 mg/kg/h intravenous atracurium (Tracrium, GlaxoSmithKline). Ventilation was supported with 30% oxygen in air. Heating pads were used to maintain body temperature at 37°C to 38°C. A femoral artery catheter was inserted for blood sampling. PET scans with [¹¹C]AMT and [¹¹C]HTP were performed sequentially on the same day within the same monkey at least 2 h apart. The first PET scan was performed at least 2 h after ketamine administration. Radioactivity in the brain was measured in a series of sequential frames of increasing duration (from 15 secs to 3 mins) for a total of 60 mins after injection of the tracer. An automatic blood sampling system was used to measure radioactivity in arterial whole blood for the first 2 to 3 mins after radiotracer administration. Thereafter, 4 to 7 manual arterial blood samples were drawn at 5, 10, 15, 20, 30, 40, and 60 mins. The radioactivity concentration in blood and plasma was measured in a γ-counter (Well-type, NaI(Tl) scintillation counter), which was cross-calibrated against the PET scanner. Plasma-to-blood radioactivity concentration ratios determined from the manual samples were used to transform the blood radioactivity—time curve measured by the automatic sampling system into the full plasma radioactivity—time curve. Because plasma radioactivity after [¹¹C]AMT is mainly associated with unchanged tracer (Gharib et al, 1999), [¹¹C]AMT plasma/blood samples were directly counted on the γ-counter. For [¹¹C]AMT, the generated total plasma radioactivity—time curve was then used in the model to represent the plasma input function (C_inp). For [¹¹C]HTP, the remaining volume of each manual plasma sample collected at 5, 20, 40, and 60 mins was used to determine the fraction of unchanged radioligand and its radiolabelled metabolites in plasma over time. The metabolite-corrected plasma radioactivity—time curve generated from the plasma curve and the data describing the unchanged fraction of [¹¹C]HTP was then used in the model to represent the plasma input function (C_inp).

Determination of [11C]HTP Metabolism in Plasma

The composition of the [¹¹C]-derived radioactivity was analysed with high-performance liquid chromatography (HPLC) as described previously (Lindner et al, 1995), with minor modifications. Blood samples were centrifuged at 4000 r.p.m. for 2 mins at 4°C to obtain plasma (Beckman Allegra X-22R Centrifuge, Palo Alto, USA). Proteins were then precipitated with 1 mL acetonitrile in 1 mL plasma. After centrifugation for 1 min at 14000 r.p.m. (Eppendorf 5415R centrifuge, Eppendorf AG, Hamburg, Germany), the supernatant was filtered through a nylon membrane (0.2 μm, Corning Inc., Corning, NY, USA). The extent of sample recovery was determined by measuring the radioactivity in the plasma, the filters and the pellet. HPLC analysis was performed using a binary pump system (Gilson, Middleton, USA) with an automated solid phase extraction controller (ASPEC Gilson) connected to a dilutor (Gilson). Before injection of the sample (1.5 mL) into the HPLC column, 10 μL of unlabelled reference compounds (1 mg/mL of HTP, 5HT and HIAA) was added to the supernatant. Separation was performed on a C-18 column (Genesis, 7 μm, 250 × 10 mm² Jones Chromatography). The mobile phase comprised acetonitrile and 50 mmol/L ammonium formiate (pH 3.5). The concentration of acetonitrile was varied during the process to provide graded concentration ratios with ammonium formiate: 0 to 9 mins, concentration ratio 5:95; 9 to 11 mins, ratio 70:30; 11 to 12 mins, isocratic; and then reconditioned with the 5:95 mobile mixture for at least 2 mins. A UV detector (Gilson) was used to detect the unlabelled reference substances at 275 nm. The outlet from the detector was connected to a switching valve on the arm of the ASPEC to enable automatic fraction collection. Seven fractions were collected and their respective radioactivity levels were measured in the γ-counter. A radio detector (Radiomatic 505TR, Packard, USA) was coupled online to the column to verify the fraction times and the radioactivity measurement.

Regions of Interest

Time frames were added to obtain a summation image, on which the regions of interest (ROIs; the frontal, occipital, parietal, and temporal cortices, the cerebellum, the striatum, the thalamus and the whole brain) were outlined, using a rhesus monkey brain atlas for guidance (Paxinos et al, 2000). Where appropriate, the same brain tissue region was delineated in two adjacent sections for both hemispheres, and data from the same region were pooled to obtain the average radioactivity concentration in that region. The total regional radioactivity concentration for each time frame corrected for radioactive decay (C_s, _tiss) was plotted versus time for use in tracer modelling.

Data Analysis

Regional net accumulation rate constants were derived with kinetic and graphic analysis of the regional radioactivity—time curves using the plasma input function. The contribution of blood radioactivity to the measured regional radioactivity was removed, assuming a 3% to 5% blood volume in the ROI. In this study, the blood volume was fixed at 5% for the cerebellum and occipital cortex, 4% for the thalamus and temporal, parietal, and frontal cortices, and 3% for the striatum and whole brain. Values were based on estimates from previous quantitative blood volume measurements using labelled carbon monoxide performed at Uppsala Imanet, and literature values (Eichling et al, 1975; Hagberg et al, 2002; Muzik et al, 1997). The use of fixed blood volumes reduces the number of free parameters (from 4 to 3), which allowed a more precise estimation of the individual rate constants. Kinetic and graphic parameters were derived using software developed in-house implemented in MATLAB 6.5 (The Math Works Inc., Natick, MA, USA).

Kinetic analysis: The compartment model used for describing the kinetics of both [¹¹C]AMT and [¹¹C]HTP in humans is a two-tissue compartment model (2TCM, Figure 1), which assumes irreversible radioactivity trapping in the second tissue compartment (Chugani et al, 1998; Hagberg et al, 2002). The rate constants K₁ (mL plasma/mL tissue/min) and k₂ (min⁻¹) describe the influx and efflux, respectively, for radioligand transport between vascular space and the cell cytoplasm (transport across the blood—brain barrier, the interstitial space, and the cell membrane). Once [¹¹C]AMT is present in the cell cytoplasm it is converted to [¹¹C]AM-5HT by means of two-step enzymatic process (via [¹¹C]AM-HTP), which is characterised by the rate constant k₃ (min⁻¹). For [¹¹C]HTP, the conversion to [¹¹C]5HT is a 1-step enzymatic process. The rate constant k₃ (min⁻¹) describes the trapping of [¹¹C]HTP and its decarboxylated products (i.e. [¹¹C]5HT and the subsequently formed metabolite [¹¹C]HIAA). The operational equation for the 2TCM is

Figure 1

Tracer kinetic models for [¹¹C]AMT and [¹¹C]HTP. The bold components of the irreversible compartment illustrate the main contribution to the PET signal towards the end of the scan.

\begin{array}{l} C_{2 T C M} (t) \\ = (1 - V_{b}) (k_{1} \frac{k_{3}}{k_{2} + k_{3}} C_{inp} (t) \otimes θ (t) \\ + K_{1} \frac{k_{2}}{k_{2} + k_{3}} C_{inp} (t) \otimes e^{- (k_{2} + k_{3}) t}) \\ + V_{b} C_{b} (t) \end{array}

(1)

where V_b is the fractional blood volume for correction of the vascular component, C_inp is the plasma input function, θ(t) is the Heaviside step function, and C_b is the whole-blood radioactivity—time curve. Kinetic parameters were derived by nonlinear regression using a Marquardt—Levenberg least-square minimisation procedure implemented in MATLAB (Levenberg, 1944; Marquardt, 1963). The net accumulation rate constant, K_acc (mL plasma/mL tissue/min), was calculated from the estimated individual rate constants as the ratio

K_{acc} = \frac{k_{1} k_{3}}{k_{2} + k_{3}}

(2)

Graphic Analysis: For a tracer with irreversible kinetics, Patlak et al (Patlak et al, 1983) showed that, in a plot of C_{s, tiss}/C_inp versus ∫^t₀C_inp/C_inp, the data will eventually (t > t*) approach a straight line with a slope (K_pPat) equal to the net accumulation rate constant. When the 2TCM model is valid, the slope will theoretically be equal to K_acc (equation 2). Thus, linear regression of the transformed data (straight line fitted to the data above t = t*) yields this macroparameter. The linear fits in this study were performed between 30 and 60 mins to correspond with previous analyses of human (Hagberg et al, 2002; Muzik et al, 1997) and rhesus monkey data (Lundquist et al, 2006; Shoaf et al, 1998; Shoaf et al, 2000).

Statistical Analysis

A two-way ANOVA for repeated measurements on both factors (ROI and tracer) was used for comparison of the parameters. The null hypothesis was rejected at a 5% significance level (Bonferroni post-test).

Results

Plasma Radioactivity

The time course of the radioactivity concentration in arterial plasma (input function) is shown in Figure 2A. The plasma-to-blood ratio was 0.8 to 0.9 at all measured time points for both tracers. The fraction of unmetabolised [¹¹C]HTP in plasma decreased as the study progressed to 50% at 60 mins (Figure 2B). The major peripheral metabolite was [¹¹C]HIAA; at the end of the scan, 39% of the total plasma radioactivity was associated with this metabolite.

Figure 2

Plasma radioactivity concentrations (the input functions, normalised to accommodate the radioactivity dose and body weight) after injection of [¹¹C]AMT and [¹¹C]HTP (A), and the contribution of [¹¹C]HTP to the total plasma radioactivity (% of total) over time (B). Data points are given as means ± s.e.m. for four monkeys.

Brain Tissue Radioactivity

Positron emission tomography images for the two radiotracers in one rhesus monkey are shown in Figure 3. The whole brain uptake of [¹¹C]AMT and [¹¹C]HTP was low, with only a small percentage of the injected dose present in the brain at peak values. Regional brain tissue radioactivity—time curves obtained after intravenous injection of [¹¹C]AMT and [¹¹C]HTP are shown in Figure 4. For [¹¹C]AMT, there were no substantial differences in radioactivity concentrations among the outlined brain regions. The [¹¹C]AMT radioactivity peaked later than the [¹¹C]HTP-derived radioactivity. For [¹¹C]HTP, the highest tissue radioactivity concentration was observed in the striatum. There were no substantial differences in radioactivity levels among the outlined extrastriatal brain regions for [¹¹C]HTP. The striatal [¹¹C]HTP-derived radioactivity concentration was almost the same as for [¹¹C]AMT, but was higher for [¹¹C]AMT in all other brain regions. This was expected, since the unchanged fraction of [¹¹C]HTP in plasma available for brain uptake is lower than that of [¹¹C]AMT after the same dose of radioactivity (Figure 2A).

Figure 3

PET images for the radiotracers [¹¹C]AMT (A) and [¹¹C]HTP (B) in one rhesus monkey. The images were derived from radioactivity collected between 30 and 60 mins after tracer injection. Intensities were normalised to the injected radioactivity dose. The transaxial plane is at the level of the striatum.

Figure 4

Total radioactivity—time curves in brain tissue (normalised for the radioactivity dose and body weight) after injection of [¹¹C]AMT and [¹¹C]HTR Data points represent means ± s.e.m. for four monkeys.

Kinetic Analysis

Results obtained using the 2TCM are presented in Table 1. Convergence was reached for 58 of 64 fits (four [¹¹C]AMT and two [¹¹C]HTP fits did not converge). Five of the six fits that did not converge came from the same PET experiment. The radioactivity signal was low, which probably contributed to the poor modelling of the data. The estimates of the individual rate constants and the net accumulation rate constant, K_acc, were highly variable for both tracers, as assessed by the relative standard deviation (100% s.d./mean). K₁ was higher for [¹¹C]HTP in the striatum (0.027 ± 0.009 versus 0.022 ± 0.008 mL/mL min) and thalamus (0.025 ± 0.011 versus 0.020 ± 0.008 mL/mL min) than for [¹¹C]AMT (P < 0.05). In other brain regions, similar K₁ values were observed for the two tracers. Compared with [¹¹C]AMT, k₃ was higher for [¹¹C]HTP in the striatum (0.046 ± 0.024 versus 0.019 ± 0.006 mm-¹) and thalamus (0.039 ± 0.013 versus 0.016 ± 0.007 min⁻¹) (P < 0.05). In the same brain regions, K_acc was higher for [¹¹C]HTP than the corresponding [¹¹C]AMT scan value within the same monkey (P < 0.05).

Table 1

Estimates of the individual rate constants (K₁, k₂, k₃), the calculated free distribution volume (DV = K₁/(k₂ + k₃)), and net accumulation rate constant (K_acc) after injection of [¹¹C]AMT and [¹¹C]HTP

Parameter	Tracer	Brain region

		Cerebellum	Frontal cortex	Occipital cortex	Parietal cortex	Striatum	Temporal cortex	Thalamus	Whole brain
K₁ × 10³ (mL mL⁻¹ min⁻¹)	[¹¹C]AMT	20 (6)	17 (6)	23 (11)	21 (7)	22 (8)	22 (5)	20 (8)	19 (6)
	[¹¹C]HTP	21 (8)	18 (6)	22 (12)	21 (6)	27 (9)*	23 (5)	25 (11)*	21 (6)
k₂ × 10³ (min⁻¹)	[¹¹C]AMT	38 (16)	43 (19)	45 (19)	48 (26)	54 (22)	69 (40)	49 (17)	51 (14)
	[¹¹C]HTP	38 (18)	40 (6)	32 (16)	41 (16)	80 (52)	72 (19)	82 (25)	60 (22)
k₃ × 10³ (min⁻¹)	[¹¹C]AMT	10 (4)	20 (9)	13 (6)	18 (9)	19 (6)	19 (8)	16 (7)	16 (6)
	[¹¹C]HTP	13 (8)	25 (16)	14 (6)	13 (9)	46 (24)*	32 (18)	39 (13)*	27 (12)
DV (mL mL⁻¹)	[¹¹C]AMT	0.4 (0.1)	0.3 (0.1)	0.4 (0.1)	0.3 (0.1)	0.5 (0.4)	0.3 (0.1)	0.3 (0.1)	0.3 (0.1)
	[¹¹C]HTP	0.5 (0.1)	0.3 (0.1)	0.5 (0.1)	0.4 (0.2)	0.3 (0.1)	0.3 (0.1)	0.2 (0.1)	0.3 (0.1)
K_acc × 10³ (mL mL⁻¹ min⁻¹)	[¹¹C]AMT	4.0 (1.4)	5.6 (2.6)	5.0 (3.3)	5.5 (2.5)	5.6 (2.2)	4.9 (1.7)	4.9 (2.3)	4.7 (1.9)
	[¹¹C]HTP	5.4 (3.2)	6.3 (2.0)	7.3 (4.1)	4.9 (2.8)	10.1 (3.0)*	6.7 (1.6)	8.0 (3.0)*	6.7 (2.3)

Results are given as means (s.d.) for 3–4 experiments.

P < 0.05 versus [¹¹C]AMT (Bonferroni post-test).

Graphic Analysis

Examples of Patlak analysis of [¹¹C]AMT and [¹¹C]HTP data for one monkey are shown in Figure 5. The data indicate a linear relationship between the apparent partition coefficient (tissue: plasma ratio) for the brain regions and the normalised plasma concentration—time integral for both tracers from 30 to 60 mins of the experiment. The plots show that, with the same magnitude of the plasma input function, the net uptake over time is higher for [¹¹C]HTP than for [¹¹C]AMT.

Figure 5

Patlak plots of the extent of tracer transport into a tissue (tissue:plasma) as a function of the exposure time (min) for [¹¹C]AMT and [¹¹C]HTP in the striatum (top graphs), frontal cortex (middle graphs), and cerebellum (bottom graphs) in one of the studied rhesus monkeys. Open circle: experimental data; solid line: linear fit from 30 to 60 mins after tracer injection; vertical dotted line: corresponds to 30 mins of actual time.

The result using graphic parameter estimation is shown in Table 2. In general, the Patlak fitted values (K_pPat) agreed well with K_acc calculated from the individual rate constants. However, for [¹¹C]AMT, the striatal K_pPat value was lower than the corresponding K_acc value. In the occipital cortex, K_acc for [¹¹C]HTP was higher than K_pPat and showed greater variability. However, the two fits that did not converge for [¹¹C]HTP in the kinetic analysis were from this particular brain region and the mean K_acc was therefore only based on estimates from two monkeys. The Patlak slope was higher for [¹¹C]HTP in the striatum (0.009 ± 0.003 versus 0.004 ± 0.002 mL/mL min) and thalamus (0.008 ± 0.003 versus 0.005 ± 0.002 mL/mLmin) than the corresponding [¹¹C]AMT scan value in the same monkey (P < 0.05).

Table 2

Estimates of the macroparameter K_pPat (Patlak slope, 30 to 60 mins) using the plasma input function after injection of [¹¹C]AMT and [¹¹C]HTP

Parameter	Tracer	Brain region

		Cerebellum	Frontal cortex	Occipital cortex	Parietal cortex	Striatum	Temporal cortex	Thalamus	Whole brain
K_pPat × 10³ (mL mL⁻¹ min⁻¹)	[¹¹C]AMT	4.7 (1.3)	4.7 (1.8)	4.5 (1.4)	4.6 (1.7)	4.0 (1.9)	4.3 (1.4)	4.7 (1.8)	4.3 (1.3)
	[¹¹C]HTP	5.4 (1.8)	5.9 (1.5)	4.8 (2.5)	4.8 (1.6)	9.3 (2.6)*	5.7 (1.1)	8.0 (2.5)*	5.9 (1.8)

Results are given as means (s.d.) for four experiments.

P < 0.05 versus [¹¹C]AMT (Bonferroni post-test).

Discussion

In this paper, a direct comparison of two tracers used in PET for probing the first and second enzymatic steps in the biosynthesis of 5HT is presented.

The only pathway available for blood-borne amino acid entry to the brain is carrier-mediated transport through the blood—brain barrier (Pardridge, 1998). For large neutral amino acids (LNAA: tryptophan, tyrosine, phenylalanine, valine, isoleucine, and leucine), the barrier across cell membranes, including the blood—brain barrier, comprises the sodium-independent L system (Pardridge and Oldendorf, 1977). The entry of plasma HTP into the brain is competitive with l-valine and thus presumably with other large neutral amino acids (Amer et al, 2004). Estimates of the Michaelis—Menten constants for blood—brain barrier transport of labelled AMT have been shown to agree reasonably with those previously measured for tryptophan (Diksic et al, 1991). It has also been shown that there is a highly significant correlation in the rat brain between the permeability surface area products for labelled AMT and those for tryptophan (Diksic et al, 2000). In the present study, the total radioactivity concentration among brain regions (except for the striatum) was 30% to 50% higher for [¹¹C]AMT than for [¹¹C]HTP after the same injected amount of radioactivity to the same monkey. However, as [¹¹C]HTP is subject to decarboxylation by AADC in the periphery, the fraction of unmetabolised [¹¹C]HTP in plasma available for brain uptake would have decreased as the study progressed, which probably influenced the brain concentration of [¹¹C]HTP. The results of the kinetic data analysis showed that the rate constant for the transfer of [¹¹C]HTP into the brain (K₁) was similar to or even higher than for [¹¹C]AMT (striatum and thalamus). The results in Figure 5 also show that the ratio C_s,tiss/C_inp over time is higher for [¹¹C]HTP than for [¹¹C]AMT, which also reflects the higher influx and radioactivity trapping for the former tracer.

Shoaf et al (1998, 2000) and Gharib et al (1999) argued that [¹¹C]AMT cannot be used to measure 5HT synthesis since only a fraction of the AMT taken up into the tissue is converted to AM-5HT. In monkeys, approximately 2% to 4% of the total radioactivity in the dorsal raphe nucleus was [¹⁴C]AM-5HT after 90 mins (Shoaf et al, 2000). These research groups both concluded that [¹¹C]AMT is primarily a measure of brain uptake of tryptophan and cannot be related to 5HT synthesis. A further concern was that tryptophan is also a substrate for protein synthesis. However, in subsequent autoradiographic studies, Diksic and co-workers showed that pharmacological inhibition of tryptophan hydroxylase with p-chlorophenylalanine (Tohyama et al, 2002) or AGN-2979 (Hasegawa et al, 2005) decreased the unidirectional trapping of [¹⁴C]AMT. A protein synthesis inhibitor (cycloheximide) was also examined but this had no effect on [¹⁴C]AMT trapping in brain tissue (Tohyama et al, 2002). These findings suggest that brain trapping of [¹⁴C]AMT relates to brain 5HT synthesis but not to brain protein synthesis.

In the present study, the rank order of the net accumulation rate constant measured with [¹¹C]HTP/PET was in agreement with previous post-mortem regional HTP accumulation rates in the rhesus monkey brain (Brown et al, 1979), namely: highest in the striatum, intermediate in the thalamus and lowest in cortical areas. While there were no substantial differences in K_acc for [¹¹C]AMT among the outlined brain regions, this was consistent with the results of an earlier study in rhesus monkeys (Shoaf et al, 2000). Thus, in contrast to two previous reports (Hagberg et al, 2002; Leyton et al, 2005), the net accumulation rate constants of [¹¹C]HTP and [¹¹C]AMT were not well correlated. Previously, it was observed that the rank order of regional [¹¹C]AMT K-complex (or K_pPat) values in humans matched that of the 5HT content, but the contrast (i.e. magnitude of difference) between brain regions was much lower for K-complex than for 5HT content (Chugani and Muzik, 2000). The authors interpreted the difference to mean that there is a large unmetabolised pool of [¹¹C]AMT trapped in brain tissue. This might also explain the discrepancy between [¹¹C]AMT and [¹¹C]HTP in the regional contrast of the net accumulation rate constant seen in our study.

In this study, the compartmental analysis results for the net accumulation rate constant were compared with those obtained using the graphic method of Patlak. In view of the limited PET scanning time, it was critical for the linearity to appear early enough for accurate parameter estimation from the Patlak slope. In some regions, the Patlak plot linearity appeared later for [¹¹C]AMT than for [¹¹C]HTP. However, the graphically obtained estimates of the net accumulation rate constant for [¹¹C]AMT were quite similar to or somewhat lower (striatum) than the corresponding values of the kinetic analysis. In a previous study using labelled AMT in rhesus monkeys, the final slope of the Patlak curve at 180 mins was small and not statistically different from zero (Shoaf et al, 2000). This limiting, almost horizontal Patlak distribution indicates that the tracer should be treated as reversible during extended measuring times (180 mins). A possible mechanism for the reversible kinetics can be derived from the observation that the rate constant for the [¹¹C]HTP transfer out of the brain (k₂) is substantial. If [¹¹C]AMT follows the endogenous 5HT synthesis pathway, it will first be transformed into [¹¹C]AM-HTP before it is converted into [¹¹C]AM-5HT. It is, thus, likely that [¹¹C]AM-HTP may leave the tissue via the same transport process as [¹¹C]HTP. Such a mechanism might also be a factor behind the lower regional contrast observed for [¹¹C]AMT than for [¹¹C]HTP.

It seems reasonable that the measures obtained with these PET tracers should be different as the tracers target different enzymes that differ in their enzyme activity in the brain (Ichiyama et al, 1968). In recent autoradiographic studies, pharmacological inhibition of tryptophan hydroxylase with p-chlorophenylalanine or AGN-2979 decreased the unidirectional trapping of [¹⁴C]AMT (Hasegawa et al, 2005; Tohyama et al, 2002). In rats (Lindner et al, 1997) and rhesus monkeys (Lundquist et al, 2006), pharmacological inhibition of AADC by NSD1015 decreased the trapping rate constant of [¹¹C]HTP. This suggests that K_acc of [¹¹C]AMT gives a reasonable estimate of the activity of tryptophan hydroxylase while K_acc of [¹¹C]HTP is providing an in vivo index of the activity of the decarboxylating enzyme AADC. In the present study, the extent and distribution of the irreversible binding of [¹¹C]AMT was different from those for [¹¹C]HTP. This also suggests that the two probe compounds provide estimates related to the enzyme activity of the different targeted steps. However, more in vivo validation studies involving drugs inhibiting the enzymes involved in 5HT formation need to be investigated if more general conclusions are to be drawn about the suitability of these tracers for estimating 5HT synthesis. It would be interesting to study the effect of a tryptophan hydroxylase inhibitor on [¹¹C]HTP accumulation in vivo as well as possible reasons for the high tracer accumulation seen in striatal regions. Given the high level of dopaminergic innervation in the striatum, it seems likely that the discrepancy between the tracer accumulation in the striatum and that in other areas is due to the presence of AADC in dopaminergic terminals. Further, the combination of the two tracers in this study might provide a deeper insight into the pathways of tryptophan in the brain tissue. In certain pathologic states, the accumulation of [¹¹C]AMT in tissue may represent metabolites of the kynurenine pathway rather than of 5HT synthesis. Thus, the combination [¹¹C]HTP/[¹¹C]AMT might provide a measure to distinguish between these two metabolic pathways as the two tracers probe different enzymatic steps. Further, in light of the large unmetabolised pool of [¹¹C]AMT in contrast to the rapidly metabolised [¹¹C]HTP, a combination might also be useful when investigating the regulation of amino acid transport across cell membranes and the distribution into different tissue compartments.

In conclusion, the PET tracers [¹¹C]AMT and [¹¹C]HTP might be useful for in vivo monitoring of 5HT synthesis capacity. Additional pharmacological in vivo studies using enzyme-inhibiting drugs in 5HT formation need to be performed, if more general conclusions are to be drawn about their respective predictive capacity of 5HT synthesis. The validated methodologies could be used to address a wide range of issues relevant to the regulation of enzyme activity and mechanisms of psychiatric and neurologic disorders.

Footnotes

Acknowledgements

The authors are grateful to the staff of the Uppsala Imanet Radiopharmaceutical Department, especially Madeleine Svennebäck, for producing [¹¹C]AMT and [¹¹C]HTP. The authors are also grateful to Gudrun Nylén (anaesthesia), Magnus Roman, and Helena Wilking (metabolite analysis) whose excellent technical skills made this work possible.

References

Agren

Reibring

(1994) PET studies of presynaptic monoamine metabolism in depressed patients and healthy volunteers. Pharmacopsychiatry 27:2–6

Agren

Reibring

Hartvig

Tedroff

Bjurling

Lundqvist

Laengstrom

(1993) Monoamine metabolism in human prefrontal cortex and basal ganglia. PET studies using and [beta-11C]L-5-hydroxytryptophan and [beta-11C]L-DOPA in healthy volunteers and patients with unipolar major depression. Depression 1:71–81

Amer

Breu

McDermott

Wurtman

Maher

(2004) 5-Hydroxy-l-tryptophan suppresses food intake in food-deprived and stressed rats. Pharmacol Biochem Behav 77:137–43

Bjurling

Watanabe

Tokushige

Oda

Långström

(1989) Synthesis of β-II-l-tryptophan and 5-hydroxy-l-tryptophan using a multi-enzymatic reaction route. J Chem Soc Perkin Trans 1:1331–4

Brown

Crane

Goldman

(1979) Regional distribution of monoamines in the cerebral cortex and subcortical structures of the rhesus monkey: concentrations and in vivo synthesis rates. Brain Res 168:133–50

Chakraborty

Mangner

Chugani

Muzik

Chugani

(1996) A high-yield and simplified procedure for the synthesis of alpha-[11C]methyl-l-tryptophan. Nucl Med Biol 23:1005–8

Chandana

Behen

Juhasz

Muzik

Rothermel

Mangner

Chakraborty

Chugani

(2005) Significance of abnormalities in developmental trajectory and asymmetry of cortical serotonin synthesis in autism. Int J Dev Neurosci 23:171–82

Chugani

Muzik

(2000) Alpha[C-11]methyl-l-tryptophan PET maps brain serotonin synthesis and kynurenine pathway metabolism. J Cereb Blood Flow Metab 20:2–9

Chugani

Muzik

Chakraborty

Mangner

Chugani

(1998) Human brain serotonin synthesis capacity measured in vivo with alpha-[C-11]methyl-l-tryptophan. Synapse 28:33–43

10.

Cohen

Tsuiki

Takada

Beaudet

Diksic

Hamel

(1995) In vivo-synthesized radioactively labelled alpha-methyl serotonin asa selective tracer for visualization of brain serotonin neurons. Synapse 21:21–8

11.

Diksic

Nagahiro

Chaly

Sourkes

Yamamoto

Feindel

(1991) Serotonin synthesis rate measured in living dog brain by positron emission tomography. J Neurochem 56:153–62

12.

Diksic

Nagahiro

Sourkes

Yamamoto

(1990) A new method to measure brain serotonin synthesis in vivo. I. Theoryand basic data for a biological model. J Cereb Blood Flow Metab 10:1–12

13.

Diksic

Tohyama

Takada

(2000) Brain net unidirectional uptake of alpha-[14c]methyl-l-tryptophan (alpha-MTrp) and its correlation with regional serotonin synthesis, tryptophan incorporation into proteins, and permeability surface area products of tryptophan and alpha-MTrp. Neurochem Res 25:1537–46

14.

Eichling

Raichle

Grubb

Jr Larson

Ter-Pogossian

(1975) In vivo determination of cerebral blood volume with radioactive oxygen-15 in the monkey. Circ Res 37:707–14

15.

Eriksson

Wall

Marteinsdottir

Agren

Hartvig

Blomqvist

Langstrom

Naessen

(2006) Mood changes correlate to changes in brain serotonin precursor trapping in women with premenstrual dysphoria. Psychiatry Res 146:107–16

16.

Fedi

Reutens

Andermann

Okazawa

Boling

White

Dubeau

Nakai

Gross

Andermann

Diksic

(2003) alpha-[11C]-Methyl-l-tryptophan PET identifies the epileptogenic tuber and correlates with interictal spike frequency. Epilepsy Res 52:203–13

17.

Fernstrom

Wurtman

(1971) Brain serotonin content: physiological dependence on plasma tryptophan levels. Science 173:149–52

18.

Gharib

Balende

Sarda

Weissmann

Plenevaux

Luxen

Bobillier

Pujol

(1999) Biochemical and autoradiographic measurements of brain serotonin synthesis rate in the freely moving rat: a reexamination of the alpha-methyl-l-tryptophan method. J Neurochem 72:2593–600

19.

Goldman-Rakic

Brown

(1981) Regional changes of monoamines in cerebral cortex and subcortical structures of aging rhesus monkeys. Neuroscience 6:177–87

20.

Hagberg

Torstenson

Marteinsdottir

Fredrikson

Langstrom

Blomqvist

(2002) Kinetic compartment modeling of [11C]-5-hydroxy-l-tryptophan for positron emission tomography assessment of serotonin synthesis in human brain. J Cereb Blood Flow Metab 22:1352–66

21.

Hasegawa

Kanemaru

Gittos

Diksic

(2005) The tryptophan hydroxylase activation inhibitor, AGN-2979, decreases regional 5-HT synthesis in the rat brain measured with alpha-[14C]methyl-l-tryptophan: an autoradiographic study. Brain Res Bull 67:248–55

22.

Ichiyama

Nakamura

Nishizuka

Hayaishi

(1968) Tryptophan-5-hydroxylase in mammalian brain. Adv Pharmacol 6:5–17

23.

Juhasz

Chugani

Muzik

Shah

Asano

Mangner

Chakraborty

Sood

Chugani

(2003) Alpha-methyl-l-tryptophan PET detects epileptogenic cortex in children with intractable epilepsy. Neurology 60:960–8

24.

Kagawa

Chugani

Asano

Juhasz

Muzik

Shah

Sood

Kupsky

Mangner

Chakraborty

Chugani

(2005) Epilepsy surgery outcome in children with tuberous sclerosis complex evaluated with alpha-[11C]methyl-l-tryptophan positron emission tomography (PET). J Child Neurol 20:429–38

25.

Karoum

Korpi

Linnoila

Chuang

Wyatt

(1984) Reduced metabolism and turnover rates of rat brain dopamine, norepinephrine and serotonin by chronicdesipramine and zimelidine treatments. Eur J Pharmacol 100:137–44

26.

Levenberg

(1944) A method for the solution of certain problems in least squares. Quart Appl Math 2:164–8

27.

Leyton

Diksic

Benkelfat

(2005) Brain regional alpha-[11C]methyl-l-tryptophan trapping correlates with post-mortem tissue serotonin content and [11C]5-hydroxytryptophan accumulation. Int J Neuropsychopharmacol 8:633–4

28.

Lindner

Hartvig

Bjurling

Fasth

Westerberg

Langstrom

(1997) Determination of 5-hydroxy-l-[beta-11C]tryptophan and its in vivo-formed radiolabeled metabolites in brain tissue using high performance liquid chromatography: a study supporting radiotracer kinetics obtained with positron emission tomography. Nucl Med Biol 24:733–8

29.

Lindner

Hartvig

Tyrefors

Hedlund

Langstrom

(1995) An automated liquid chromatographic plasma analysis of amino acids used in combination with positron emission tomography (PET) for determination of in vivo plasma kinetics. J Pharm Biomed Anal 13:353–9

30.

Lundquist

Blomquist

Hartvig

Hagberg

Torstenson

Hammarlund-Udenaes

Langstrom

(2006) Validation studies on the 5-hydroxy-l-[beta-11C]-tryptophan/PET method for probing the decarboxylase step in serotonin synthesis. Synapse 59:521–31

31.

Marquardt

(1963) An algorithm for least-squares estimation of nonlinear parameters. J Soc Indust Appl Math 11:431–41

32.

Muzik

Chugani

Chakraborty

Mangner

Chugani

(1997) Analysis of [C-11]alpha-methyl-tryptophan kinetics for the estimation of serotonin synthesis rate in vivo. J Cereb Blood Flow Metab 17:659–69

33.

Natsume

Kumakura

Bernasconi

Soucy

Nakai

Rosa

Fedi

Dubeau

Andermann

Lisbona

Bernasconi

Diksic

(2003) Alpha-[11C] methyl-l-tryptophan and glucose metabolism in patients with temporal lobe epilepsy. Neurology 60:756–61

34.

Nishikawa

Kumakura

Young

Fiset

Vogelzangs

Leyton

Benkelfat

Diksic

(2005) Increasing blood oxygen increases an index of 5-HT synthesis in human brain asmeasured using alpha-[(11)C]methyl-l-tryptophan and positron emission tomography. Neurochem Int 47:556–64

35.

Pardridge

(1998) Blood—brain barrier carrier-mediated transport and brain metabolism of amino acids. Neurochem Res 23:635–44

36.

Pardridge

Oldendorf

(1977) Transport of metabolic substrates through the blood—brain barrier. J Neurochem 28:5–12

37.

Patlak

Blasberg

Fenstermacher

(1983) Graphical evaluation of blood-to-brain transfer constants from multiple-time uptake data. J Cereb Blood Flow Metab 3:1–7

38.

Paxinos

Huang

Toga

(2000) The rhesus monkey brain in stereotaxic coordinates. San Diego, CA, USA: Academic Press

39.

Reibring

Agren

Hartvig

Tedroff

Lundqvist

Bjurling

Kihlberg

Langstrom

(1992) Uptake and utilization of [beta-11C]5-hydroxytryptophan in human brain studied by positron emission tomography. Psychiatry Res 45:215–25

40.

Shannak

Hornykiewicz

(1980) Brain monoamines in the rhesus monkey during long-termneuroleptic administration. Adv Biochem Psychopharmacol 24:315–23

41.

Shoaf

Carson

Hommer

Williams

Higley

Schmall

Herscovitch

Eckelman

Linnoila

(1998) Brain serotonin synthesis rates in rhesus monkeys determined by [11C]alpha-methyl-l-tryptophan and positron emission tomography compared to CSF 5-hydroxyindole-3-acetic acid concentrations. Neuropsychopharmacology 19:345–53

42.

Shoaf

Carson

Hommer

Williams

Higley

Schmall

Herscovitch

Eckelman

Linnoila

(2000) The suitability of [11C]-alpha-methyl-l-tryptophan as a tracer for serotonin synthesis: studies with dual administration of [11C] and [14C] labeled tracer. J Cereb Blood Flow Metab 20:244–52

43.

Tohyama

Takahashi

Merid

Watanabe

Diksic

(2002) The inhibition of tryptophan hydroxylase, not protein synthesis, reduces the brain trapping of alpha-methyl-l-tryptophan: an autoradiographic study. Neurochem Int 40:603–10

44.

Watanabe

Okada

Shimizu

Omura

Yoshikawa

Kosugi

Mori

Yamashita

(1997) A high resolution animal PET scanner using compact PS-PMT detectors. IEEE Trans Nucl Sci 44:1277–82

45.

Vriend

Alexiuk

Green-Johnson

Ryan

(1993) Determination of amino acids and monoamine neurotransmitters in caudate nucleus of seizure-resistant and seizure-prone BALB/c mice. J Neurochem 60:1300–7

46.

Young

Gauthier

(1981) Effect of tryptophan administration on tryptophan, 5-hydroxyindoleacetic acid and indoleacetic acid in human lumbar and cisternal cerebrospinal fluid. J Neurol Neurosurg Psychiatry 44:323–8

47.

Zhu

Juorio

(1995) Aromatic l-amino acid decarboxylase: biological characterization and functional role. Gen Pharmacol 26:681–96