The study had ethical approval from the NHS Ethical Committee (NRES South East Coast, Surrey), the local Research and Development offices and the Administration of Radioactive Substances Advisory Committee (ARSAC). Participation required provision of written informed consent to all study procedures.
Participants
The study aimed to acquire complete datasets (including one T1-weighted MRI scan, two [13N]ammonia scans and two [15O]water PET scans) in eight healthy volunteers. Participants were recruited internally though King’s College London’s recruitment system. Inclusion required that participants were aged 18 or older and were able to provide written informed consent in English. Exclusion criteria included the standard contraindications to PET and MRI, including pregnancy. Absence of pregnancy in female participants was confirmed by a negative urine pregnancy test on arrival to the PET scanning visit.
MRI
MRI scans were performed at the Centre for Neuroimaging Sciences, King’s College London, UK on a General Electric MR750 3 Tesla MRI scanner. A T1-weighted structural MRI scan based on the ADNI protocol (voxel size 1.05 x 1.05 x 1.20 mm, TE 3.016 ms; TR 7.312 ms matrix 256 x 256; FoV 270 mm; inversion time 400 ms) was acquired for co-registration of the participants’ PET images.
Radiochemistry
Aqueous [13N]ammonia was produced on a CTI RDS 112 biomedical cyclotron via the 16O(p,α)13N nuclear reaction. The target contained 8 mL H2O with 5 mM ethanol according to Wieland et al [22].
[15O]water: Oxygen-15 was produced in the form of [15O]oxygen gas by the bombardment of enriched [15N]nitrogen gas containing 1-2.5% oxygen gas via the 15N(p,n)160 nuclear reaction. [15O]water was subsequently obtained by passage with hydrogen over a platinum catalyst according to Berridge et al [23].
PET image acquisition
PET scans were acquired at St Thomas’ Hospital, King’s College London on a GE Discovery 710 PET-CT scanner with 3D acquisition and list mode. Each participant underwent two PET scanning sessions, performed in the morning and afternoon of the same day. Each of the two scanning sessions consisted of an initial low dose CT scan to enable correction for tissue attenuation of radioactivity, a dynamic [15O]water scan (5 minutes), and a dynamic [13N]ammonia scan (30 minutes). There was a break of approximately one hour between the two sessions, during which lunch was provided, and an appropriate gap (at least 5 half-lives) between subsequent scans to avoid residual counts (i.e. at least 10 minutes following the [15O]water scans and 50 minutes following the [13N]ammonia scan) .
At the start of the PET scan visit, a cannula was inserted in a vein in the arm for radiotracer injection. After application of local anaesthetic, an arterial line was inserted into the radial artery and flushed every 20 minutes with heparinised saline (20 IU/mL of heparin in sterile 0.9% w/v sodium chloride) until removal at the end of PET scanning. Just before the start of each scanning session, 6 mL of arterial blood was taken to measure baseline blood ammonia levels.
Participants were positioned in the PET-CT scanner, with head movement minimised via a moulded headrest and head strap. The arterial cannula was connected to an automated blood sampling system (Allogg ABSS, www.allogg.se, Sweden) using a 150cm PTFE coated tubing (inner diameter 1mm). CT scout (0.015 mSv) and CT attenuation correction (0.05 mSv) scans were acquired. 15O-water (target dose at time of administration: 960 MBq, 1.10 mSv) was injected through the venous cannula over 10 seconds. PET image acquisition started 10 seconds before the start of [15O]water injection and continued for a total of 5 minutes. Arterial blood collection via the fluid analyser commenced 70 seconds before [15O]water injection and 60 seconds before the start of scan acquisition and continued for the 5 minute scan duration, to a total of 25 mL. Additionally, a single 2 mL arterial blood sample was manually drawn at 4 minutes into the scan.
After completion of the [15O]water scan the arterial line was flushed with heparinised saline. At least 20 minutes after the end of the 15O-water scan (25 minutes after [15O]water injection), [13N]ammonia (target dose at time of administration: 550 MBq, 1.5 mSv) was injected through the venous cannula. PET image acquisition started 10 seconds before the start of [13N]ammonia injection and continued for 30 minutes. Arterial blood collection via the fluid analyser commenced 70 seconds before [13N]ammonia injection and 60 seconds before the start of scan acquisition and continued for 15 minutes, to a total of 75 mL. In addition, 6 manual arterial blood samples of 10 mL each were drawn at 4, 6, 8, 12, 20 and 30 minutes after scan start during the [13N]ammonia scans, which were used for whole blood, plasma and metabolite analysis.
In the second session, a minimum of one hour later, both the [15O]water and [13N]ammonia scans were repeated using identical acquisition protocols.
Ammonia and metabolite analysis
The method used for separation of ammonia and metabolite from plasma samples was based on the method published by Keiding et al [11] and is described below.
Levels of non-radioactive ammonia in arterial blood were determined from samples collected before radiotracer collection. These samples were collected in K-EDTA tubes (pre-tested and confirmed as ammonia-free) and transported on ice within 20 minutes of collection to the hospital laboratory for standard analysis.
Unless stated otherwise, all water used in these metabolite analyses was passed through ion exchange resin and 0.22 μm membrane filtered to produce water with a specific resistance of 18.2 micro-ohms using a Milli-Q Ultrapure water purification system manufactured by Millipore Corporation.
Plasma was separated from whole blood by centrifuging at 3000 x g for 3 minutes at room temperature (RT). Levels of radioactive metabolites in plasma were estimated through solid phase extraction, based on the methods of Keiding et al. [17]. In preparation for solid phase extraction, one cartridge was filled with 0.6 mL Dowex 1X8-50 anion exchange resin and pre-treated with 6 mL 0.75 M sodium acetate solution. A second cartridge, connected in series via an Agilent Bond Elut adapter, was filled with 0.35 mL AG50W-X8 cation exchange resin and pretreated with 3.5 mL 0.8 M Tris-acetate solution. The third cartridge which connected to the second cartridge in the same way via adapter was filled with 0.35mL AG50W-X8 cation exchange resin and pretreated with 3.5 mL Milipore water.
For extraction, 0.5 mL of the supernatant protein-free plasma was loaded onto the first cartridge followed by washing with 3 mL of Milipore water through the cartridge stack and flushed with 10 mL of air. The eluent from the first cartridge passed through the second cartridge and third cartridge, which were subsequently washed with 7 mL of Milipore water followed by 10 mL of air. The third cartridge was washed with 7 ml Milipore water and followed by 10 mL of air. All eluates were collected with a 25 mL pot. With this method, the radioactivity measured on the first cartridge corresponded to [13N]glutamate, on the second cartridge corresponded to intact [13N]ammonia, on the third cartridge corresponded to [13N]glutamine, and the pot corresponded to [13N]urea .
A 10-detector gamma-counter (Wizard2 2470, Perkin-Elmer) cross-calibrated to the PET scanner was used to measure radioactivity concentrations in whole blood (0.5 mL per sample), plasma (0.5 mL per sample) and metabolite fractions (3 mL for urea and full cartridge contents for other fractions). All samples were counted for 3 minutes on a fixed energy window (358-664 keV) with software cross-talk correction and in-house volumetric geometry correction. The samples and cartridges were corrected for weight to calculate the total radioactivity of blood sample analysed. All sample data were background and decay corrected to scan start time prior to data analysis.
Image processing
[15O]water PET list mode data was unlisted to 26 frames (1 x 10 sec, 10 x sec, 6 x 10 sec and 9 x 20 sec). [13N]ammonia PET list mode was unlisted to 47 frames (1 x 10 sec, 10 x 5 sec, 6 x 10 sec, 3 x 20 sec, 27 x 60 sec). All PET images were reconstructed to a 2562 matrix with 47 slices with 0.98 x 0.98 x 3.27 mm voxel size, 3D iterative reconstruction (GE “VuePoint”, 4 iterations, 24 subsets, 4mm FHWM Gaussian post-filter), scatter correction and inter- and intra-frame decay correction. Images were reconstructed with CT attenuation correction (attenuation corrected, AC) and without (non-attenuation corrected, NAC).
Frame-by-frame motion correction was performed on dynamic PET data using the NAC image to derive the rigid-body motion parameters which were applied to the paired AC image (first 9 frames ignored to avoid low counts). Regions of interest (ROI) were defined by the “Hammers_mith Atlas” [24, 25] (83 regions) in MNI stereotaxic space. Non-linear warps from MNI to subject space were defined using the unified segmentation algorithm [26] in SPM8 (www.fil.ucl.ac.uk/spm) on each subject’s T1 MRI. Resliced atlases for each subject were then co-registered to a summed PET image (sum of total scan duration of motion corrected AC image ignoring first 60 seconds) for each PET scan via the MRI.
For both the [15O]water and [13N]ammonia scans, time activity curves (TACs) were extracted from the co-registered Hammers_mith atlas [24, 25] (ignoring ventricular and white matter regions). Using each subject’s co-registered probabilistic grey matter mask from the segmented MRI, TACs were extracted using the mean voxel value within the region or a weighted mean for cortical regions using each subject’s grey matter probabilistic mask.. Whole-brain grey matter and white matter weighted mean TACs were also defined.. A total of 79 regions were explored (77 atlas ROIs plus global grey and white matter).
Blood Data Processing
For the [13N]ammonia scans, arterial whole blood input functions were created from decay-corrected continuous blood samples with manual samples used for cross-calibration to scanner and interpolation to scan end. Plasma-over-blood ratio was calculated as the mean of the manual plasma and whole blood sample ratios for each subject (Supplementary Figure 2). Parent fraction data (ratio of [13N]ammonia to total 13N activity) was fitted to a biexponential curve for each subject as used by Keiding et al (2006) [11]. Parent plasma input functions (i.e. [13N]ammonia in plasma only) for the kinetic modelling were created by multiplying the whole-blood input function by plasma-over-blood ratios and the biexponential curve fitted to the parent fractions. To account for delay between the blood sampling detector and PET scan whole blood and parent plasma input functions were delay corrected by visually matching the blood rise with the grey matter TAC, with decay correction.
Kinetic analysis
Regional cerebral blood flow (CBF) was calculated from the [15O]water TACs using the 5-parameter free diffusion model as described by Meyer (1998) [27] applied to each time activity curve. In brief, a nonlinear least squares fit method was used to simultaneously estimate the 5 free parameters of this 1-tissue compartment model: CBF, k’2 (15O wash-out), blood fraction, and delay and dispersion of the blood curve between the brain and sampling point (in addition to the visual delay correction described above).
Ammonia is a freely diffusible tracer and as such has been used to quantify perfusion in myocardium [28] and brain [29]. Though ammonia is rapidly trapped in tissue, in order to index GS activity, the kinetic parameters describing the uptake of [13N]ammonia by GS must be distinguishable from those reflecting CBF. The model chosen for primary analysis of [13N]ammonia scans was an irreversible two tissue compartment model (2TCM) as used in Keiding et al., 2006 [11]. To confirm the model choice a nonlinear spectral analysis approach was used to identify the most appropriate tissue uptake model [30]. In brief, the data was fitted to a number of candidate PET compartmental models with increasing numbers of parameters. In this case, a reversible 2TCM [Supplement Figure 1] was the most complex model considered, with increasingly simpler models defined by setting k4, k3, k2 to zero (i.e. 4 candidate models). The blood fraction contributing to the TAC for each region was also included as a free parameter.
Each compartmental model was fitted using a weighted least squares method with weighting inversely proportional to the variance of each frame determined by frame duration and radioactive decay: , where is the decay rate constant, and and are the frame duration and frame mid-point time respectively for frame .
Additional macroparameters from the 15O and 13N scans were calculated to compare with the results of Keiding et al (2006) [11]. These parameters are not directly of interest to the identification of the k3 parameter, but were obtained solely for the purpose of comparison. PSBBB (flow independent permeability-surface area product of the blood brain barrier to [13N]ammonia) was calculated as
where CBF is calculated from the [15O]water scan (assuming a 100% extraction fraction), and K1 from the [13N]ammonia scan. Extraction Fraction (EF) was also calculated as the simple ratio of K1 to CBF. Net metabolic clearance of [13N]ammonia in blood into intracellular [13N]glutamine, Kmet, was calculated using the Patlak graphical method using the complete data, with a t* of 20 minutes [32]. PSmet (flow-independent permeability-surface area product of conversion of ammonia to intracellular glutamine) was calculated as
Finally, metabolic flux of ammonia molecules from blood to glutamine in tissue, Fluxmet, (as described by Keiding et al 2006 [11]), was calculated as
where A is the measured concentration of endogenous ammonia in the blood.
Statistical Analysis
Identifiability of the k3 rate-constant from the [13N]ammonia data was evaluated by testing if the estimated parameter value, relative to estimated error, was significantly greater than zero (using the one-sided t-test). For the irreversible 2TCM model (4 parameters) with 47 frames, this corresponds to a proportional estimate parameter error of 39%. In addition, optimal model selection on the [13N]ammonia data was assessed using the Akaike Information Criterion (AIC) [31].
Kinetic parameter repeatability between the test-retest scans was assessed using mean fractional difference (VAR), absolute fractional difference (AbsVAR), and intraclass correlation coefficient (ICC) using a two-way random model for consistency [33]. For 8 subjects, the threshold for a significantly positive ICC is 0.58 at the at the p< 0.05 level. VAR and AbsVAR were calculated for N subjects as a percentage:
Image registration, TAC extraction, blood data processing, kinetic modeling and statistical analyses were performed in Matlab (www.mathworks.com). Data are presented as mean ± s.d. unless otherwise stated.