### Subjects

Seven healthy, nonsmoker, male volunteers (72 ± 9.5 kg, age 26 ± 4.5 years) from a previous clinical protocol were included in this study [7]. All subjects were free of current medical or psychiatric illnesses and had no history of drug or alcohol abuse. Subjects were instructed to avoid monoamine-rich food 3 days before and alcohol consumption 1 week before imaging. Vital signs (blood pressure, heart rate) were recorded before [^{11}C]befloxatone injection and at the end of the scan. The study was approved by the regional ethics committee for biomedical research at the Bicêtre Hospital. Each subject gave a written informed consent.

### Brain image acquisition

PET scans were performed on an ECAT EXACT HR + scanner (Siemens Medical Solutions, Knoxville, TN, USA). One transmission scan was acquired before intravenous bolus injection of [^{11}C]befloxatone (290 ± 24.4 MBq). The specific activity was 35.2 ± 14.9 GBq/μmol, which is within the range of the values usually obtained from the synthesis of [^{11}C]befloxatone [11] and sufficient to rule out any mass effect. Even when considering a conservative *B*
_{max} value of 99 pmol/mL (as found *in vitro* in the human cerebellum [14]), the *B*
_{max} occupancy is well below 1% in each subject. Dynamic three-dimensional images were acquired over 60 min in 29 frames, whose duration ranged from 10 s to 5 min. Images were reconstructed with filtered back projection.

Each subject also underwent brain T1-weighed three-dimensional magnetic resonance imaging (MRI) using a 1.5 T Signa scanner (GE Healthcare, Milwaukee, WI, USA). MRI images were acquired with a voxel size of 0.938 × 0.938 × 1.3 mm^{3} and a thickness of 1.3 mm in axial slices.

### Measurement of [^{11}C]befloxatone in arterial and venous plasma

Arterial blood samples were drawn from the radial artery of each subject in 10-s intervals until 2 min, followed by samples at 3, 4, and 5 min and then every 5 min until the end of the scan at 60 min. About 27 samples were drawn for each subject. For each blood sample, the decay-corrected concentration of [^{11}C]befloxatone was measured in whole blood and plasma. Plasma was obtained by centrifugation. Plasma-free fraction was not measured. Venous blood samples were simultaneously drawn from the cubital vein of the opposite arm, using the same number of samples and time schedule as the arterial ones. As for arterial blood, plasma was separated from total venous blood. Arterialization of venous blood was achieved by heating the arm with hot water bags, starting 30 min before ligand injection until the end of the scan. The bags were replaced every 20 to 30 min. Four or five times during each scan, blood gases (pO_{2} and pCO_{2}) were measured in five subjects.

The whole plasma time-activity curves were taken as input functions because in a previous series of (yet unpublished) four human scans from our laboratory, chromatograms obtained with high-performance liquid chromatography (HPLC) did not show any detectable radiometabolites in the plasma, even at late time points. The technique we used to measure the amount of unchanged radiotracer in the plasma was similar to that described in [14]. Specifically, after deproteinization with acetonitrile, the samples were centrifuged and the supernatant was injected directly into the HPLC column. A reverse-phase μBondapak C18 column (300 × 7.8 mm, 10 μm; Waters, Milford, MA, USA) was eluted applying a gradient from 20% acetonitrile in 0.01 M phosphoric acid up to 80% in 5.5 min, to 90% at 7.5 min, and returning to 20% at 7.6 min with a total run length of 10 min. The flow rate of the eluent was maintained at 6 mL/min. Befloxatone was eluted with a retention time of 6 min. Data acquisition and analysis were carried out using Winflow software (version 1.21, JMBS Developments, Grenoble, France).

### Brain image analysis

The summed PET image, obtained by averaging all frames, was first coregistered to the individual MRI using SPM5 (Wellcome Department of Cognitive Neurology, London, UK). MRI and PET images were then normalized to the Montreal Neurologic Institute (MNI) space using the transformation parameters from the MRI images. A template of regions of interest (ROIs) [15] was used to extract brain time-activity curves for the following 13 regions: anterior and posterior cingulate cortices, caudate, putamen, thalamus, pons, cerebellum, hippocampus, parahippocampus, and the prefrontal, parietal, temporal, and occipital cortices. Each region was obtained from the weighted average of the left and right region. Image analysis and kinetic modeling were performed with Pmod 3.1 (Pmod Technologies, Zurich, Switzerland).

### Calculation of distribution volume

Because [^{11}C]befloxatone is a reversible MAO-A inhibitor [11], kinetic analysis was performed using one- (1TCM) and two-tissue compartment (2TCM) model to calculate the total distribution volume (*V*
_{T}), which equals the ratio at equilibrium of the concentration of radioligand in the brain to that in the plasma [16]. *V*
_{T} is the sum of the binding in the specific compartment (*V*
_{S}) and in the nondisplaceable compartment (*V*
_{ND}). In theory, *V*
_{T} may change not only as a function of *V*
_{S} but also of *V*
_{ND}. However, in kinetic modeling studies, *V*
_{ND} is commonly assumed to be constant across different brain regions and across different subjects of the same species. Thus, variations of *V*
_{T} are considered to reflect variations of *V*
_{S} values. For [^{11}C]befloxatone in particular, *in vivo* studies in primates showed that, after pharmacological blockade, the residual radioactive concentrations were very low and identical in all brain structures [11]. The 2TCM analysis was performed in two ways, (1) without constraining the rate constants and (2) fixing the ratio *K*
_{1}/*k*
_{2} to the value obtained in the whole brain, to improve the identifiability of parameters. The individual rate constants were estimated with the weighted least square method and with the Marquardt optimizer. Brain activity was corrected for its vascular component using the measured whole blood concentrations, assuming that the cerebral blood volume is 5% of the total brain volume [17]. The delay of radiotracer arrival between the radial artery and the brain was corrected by fitting the whole brain. The plasma input function was modeled using a linear interpolation of the [^{11}C]befloxatone concentrations before the peak and a tri-exponential fit of concentrations after the peak. Kinetic modeling was also performed at the regional level using the graphical Logan plot (Logan_{voi}), Ichise's multilinear analysis (MA1_{voi}), and a standard spectral analysis (SA_{voi}).

#### Logan plot

The Logan plot is a model-independent graphical method for reversible tracers [18]. It performs a linearization of the data so that, after a certain time (*t > t**), the slope can be related to the *V*
_{T}, according to

\frac{{\displaystyle {\int}_{0}^{\mathit{t}}{C}_{\mathrm{T}}\left(\tau \right)\mathit{d\tau}}}{{C}_{\mathrm{T}}\left(t\right)}={V}_{\mathrm{T}}\frac{{\displaystyle {\int}_{0}^{\mathit{t}}{C}_{\mathrm{P}}\left(\tau \right)\mathit{d\tau}}}{{C}_{\mathrm{T}}\left(t\right)}+c

(1)

where *C*
_{T} represents the tissue concentration and *C*
_{p} the plasma activity.

The Logan plot is a computationally quick and robust technique. It may, however, underestimate *V*
_{T}, especially in the case of small and noisy regions [19].

#### Multilinear analysis

The multilinear analysis (MA1) is a modification of the Logan plot developed to remove noisy measurements (*C*
_{T}) from the independent variables [20], according to the following equation:

{C}_{\mathrm{T}}\left(t\right)=-\frac{V}{b}{\displaystyle {\int}_{0}^{t}{C}_{\mathrm{p}}\left(\tau \right)\mathit{d\tau}}+\frac{1}{b}{\displaystyle {\int}_{0}^{t}{C}_{\mathrm{T}}\left(\tau \right)\mathit{d\tau}}

(2)

where *V* is the total distribution volume and *b* is the intercept of the Logan plot that becomes constant at *t > t**. This approach has been shown to minimize *V*
_{T} estimation bias in the case of noisy measurements [20].

#### Spectral analysis

This technique is based on a time-invariant single input/single output model used to identify tissue kinetic components [21]. As with the Logan plot, SA does not require prior knowledge of the number of compartments in the system. The tissue concentration, *C*
_{T}(*t*), is obtained by convolving the plasma time-activity curve, *C*
_{P}(*t*), with the sum of *M + 1* exponentials, as follows:

{C}_{\mathrm{T}}\left(t\right)={\displaystyle {\sum}_{j=0}^{M}C\mathrm{p}\left(t\right)}\otimes {\alpha}_{\mathit{j}}{e}^{-{\beta}_{j}t}={\displaystyle {\sum}_{j=0}^{M}{\alpha}_{j}{\displaystyle \underset{0}{\overset{t}{\int}}C\mathrm{p}\left(\tau \right)}}{e}^{-{\beta}_{j}\left(t-\tau \right)}\mathit{d\tau}

(3)

where α_{
j
} and *β*
_{
j
} are assumed to be real-valued and nonnegative.

*V*
_{T} was calculated from the estimated spectrum as

{V}_{\mathrm{T}}={\displaystyle \sum _{j=1}^{M}\frac{{\alpha}_{j}}{{\beta}_{j}}}

(4)

In the present study, the *β*
_{
j
} grid was defined with a maximum value of 100 and a logarithmic distribution of *β*
_{
j
}, *j =* 1, 2… *M*, using a spectral range from 0.01 to 1. SA was performed using the SAKE software [22].

Finally, we sought to determine whether *V*
_{T} values could be obtained from voxel-wise analysis. We created parametric images using the Logan plot (Logan_{voxel}), multilinear analysis (MA1_{voxel}), and standard spectral analysis (SA_{voxel}). For Logan_{voxel} and MA1_{voxel}, the PET frames used for regression were selected on the basis of the start time obtained from the time-activity curve of the whole brain. For all parametric images, regional values were obtained by averaging the voxel values within each region.

### Statistical analysis

Goodness of fit by nonlinear least squares analysis was evaluated using the Akaike information criterion (AIC) and model selection criterion (MSC). The most appropriate model is that with the smallest AIC and the largest MSC score. Goodness of fit by the one- and two-compartment models was compared with *F* statistics [23]. A value of *P* < 0.05 was considered significant. The identifiability of kinetic variables was expressed as a percentage of the rate constant and calculated as standard error obtained from the diagonal of the covariance matrix [24]. Standard error for *V*
_{T} was calculated from the covariance matrix using the generalized form of error propagation equation [25], where correlations among parameters are taken into account. Smaller values indicate better identifiability.

For each subject, regional *V*
_{T} values obtained with 2TCM at the regional level were considered as the gold standard. Comparisons to the *V*
_{T} calculated with Logan_{voi}, MA1_{voi}, SA_{voi}, Logan_{voxel}, MA1_{voxel}, and SA_{voxel} were performed with a repeated measures analysis of variance (ANOVA) for *V*
_{T} values in the various regions. Statistical analyses were performed with SPSS (version 17 for Windows; SPSS Inc., Chicago, IL, USA).