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 T MRI scanner. A T1-weighted structural MRI scan based on the ADNI protocol (voxel size 1.05 × 1.05 × 1.20 mm, TE 3.016 ms; TR 7.312 ms matrix 256 × 256; FoV 270 mm; inversion time 400 ms) was acquired for co-registration of the participants’ PET images.
Radiochemistry
Aqueous [13N]NH3 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 line was connected to an automated blood sampling system (Allogg ABSS, www.allogg.se, Sweden). 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 3 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 5-minute intervals during the [13N]ammonia scans.
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
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 1 × 8–50 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 second cartridge in same way via adapter was filled with 0.35 mL 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 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 eluate 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.
Image processing
[15O]water PET list mode data was unlisted to 26 frames (1 × 10 sec, 10 x sec, 6 × 10 sec and 9 × 20 sec). [13N]NH3 PET list mode was unlisted to 47 frames (1 × 10 sec, 10 × 5 sec, 6 × 10 sec, 3 × 20 sec, 27 × 60 sec). All PET images were reconstructed to 256 matrix with 47 slices with 0.98 × 0.98 × 3.27 mm voxel size, 3D iterative reconstruction and 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. 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 for each PET scan via the MRI.
Kinetic analysis
For both the [15O]water and [13N]ammonia scans, time activity curves (TACs) were extracted from the co-registered Hammers_mith atlas [24, 25]. Using each subject’s co-registered probabilistic grey matter mask from the segmented MRI, TAC’s were extracted using the mean voxel value within the region, or a weighted mean for cortical regions. This resulted in values from 77 individual ROI’s. A whole-brain grey matter weighted mean TAC was also defined Ventricular and white matter regions were ignored.
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 sample ratios for each subject. Parent fraction data (ratio of [13N]ammonia to total 13N activity) was fitted to a biexponential curve for each subject.[11] A population parent fraction function was created by fitting a biexponential curve to all subject data. 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. Whole blood and parent plasma input functions were delay corrected by visually matching the blood rise with the grey matter TAC, with appropriate decay correction.
Regional cerebral blood flow (CBF) was calculated from the [15O]water TACs using a 5- parameter free diffusion model [27]. In brief, a nonlinear least squares fit method was used to estimate the 5 parameters of this 1-tissue compartment model: K1 (CBF), k2 (wash-out), blood fraction, and delay and dispersion of the blood curve between the brain and sampling point.
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 Fig. 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 residual weights for 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 . Model fit was assessed using the Akaike Information Criterion.[31]
Additional macroparameters from the 15O and 13N scans were calculated to compare with the results of Keiding et al (2006) [11]. 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, and K1 from the [13N]ammonia scan. Net metabolic clearance of [13N]ammonia in blood into intracellular [13N]glutamine, Kmet, was calculated using the Patlak graphical method [32]. PSmet (flow-independent permeability-surface area product of conversion of ammonia to intracellular glutamine) was calculated as
Finally, the cerebral metabolic rate of ammonia, CMRA, was calculated as
where A is the measured concentration of endogenous ammonia in the blood.
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.