Endogenous compounds labeled with radionuclides of short half-life-some perspectives.

In the article, the strategy and synthesis of some endogenous compounds labeled mainly with (11) C are presented. There are some examples illustrating how endogenous labeled compounds in connection with positron emission tomography have unique properties to describe various biological processes, and a few examples of the use of tracers labeled with (13) N and (15) O are also discussed. Labeled endogenous compounds may be an important asset to describe the conditions and the status of biological systems and might therefore be a key for the future search of individualized medicine.

News ways of understanding the complex biology of diabetes using PET.

The understanding of metabolic disease and diabetes on a molecular level has increased significantly due to the recent advances in molecular biology and biotechnology. However, in vitro studies and animal models do not always translate to the human disease, perhaps illustrated by the failure of many drug candidates in the clinical phase. Non-invasive biomedical imaging techniques such as Positron Emission Tomography (PET) offer tools for direct visualization and quantification of molecular processes in humans. Developments in this area potentially enable longitudinal in vivo studies of receptors and processes involved in diabetes guiding drug development and diagnosis in the near future. This mini-review focuses on describing the overall perspective of how PET can be used to increase our understanding and improve treatment of diabetes. The methodological aspects and future developments and challenges are highlighted.

Positron emission tomography in clinical islet transplantation.

The fate of islets in clinical transplantation is unclear. To elude on this positron emission tomography combined with computed tomography (PET/CT) was performed for 60 min during islet transplantation in five patients receiving six transplants. A fraction of the islets (23%) were labeled with 18F-fluorodeoxyglucose ([(18)F]FDG) and carefully mixed with unlabeled islets just prior to intraportal transplantation. The peak radioactivity concentration in the liver was found at 19 min after start of islet infusion and corresponded to only 75% of what was expected, indicating that islets are lost during the transplantation procedure. No accumulation of radioactivity was found in the lungs. A nonphysiological peak of C-peptide was found in plasma during and immediately after transplantation in all subjects. Distribution in the liver was heterogeneous with wide variations in location and concentration. Islets found in areas with concentrations of >400 IEQ/cc liver tissue varied between 1% and 32% of the graft in different subjects. No side effects attributed to the PET/CT procedure were found. Clinical outcome in all patients was comparable to that previously observed indicating that the [(18)F]FDG labeling procedure did not harm the islets. The technique has potential to be used to assess approaches to enhance islet survival and engraftment in clinical transplantation.

Imaging dopamine receptors in the human brain by positron tomography.

Neurotransmitter receptors may be involved in a number of neuropsychiatric disease states. The ligand 3-N-[11C]methylspiperone, which preferentially binds to dopamine receptors in vivo, was used to image the receptors by positron emission tomography scanning in baboons and in humans. This technique holds promise for noninvasive clinical studies of dopamine receptors in humans.

[(11)C]-PIB imaging in patients with Parkinson's disease: preliminary results.

[(11)C]-PIB positron emission tomography ([(11)C]-PIB PET) is a sensitive marker of amyloid in Alzheimer's disease (AD), but its specificity has not been fully evaluated. Vascular amyloid-beta deposition is common in Parkinson's disease (PD) and alpha-synuclein, the major component of the Lewy bodies in PD, forms amyloid fibrils. We investigated five apparently cognitively normal PD patients with [(11)C]-PIB PET. The results were compared to 16 patients with AD and six healthy controls from a previous study. [(11)C]-PIB retention was not significantly increased in our patients who all had early stage PD. Further studies of more advanced PD patients are warranted.

Determination of metabolic stability of positron emission tomography tracers by LC-MS/MS: an example in WAY-100635 and two analogues.

A method is presented for determination of microsomal metabolic stability of potential positron emission tomography (PET) tracers by LC-MS/MS in the lower nm range. The PET tracers used for the study were the serotonin receptor antagonist WAY-100635 and two potential tracer analogues. The sensitivity permitted the substrates to be directly collected from PET radiolabelling batches, containing very low amounts of substance (0.3-7 microg), for subsequent metabolic stability incubations. Sample preparation was minimal, with addition of internal standard, acetonitrile and a fast centrifugation step, as a result of the low protein concentration of the microsome solutions. Linearity (R2 > or = 0.99), precision (inter-assay R.S.D. < 7%) and accuracy (bias < or = 8%) for the tested concentration range 0.5-5 nM proved to be well within accepted limits. No significant differences in metabolic rates were detected using substrates from cold (non-labelling) chemistry syntheses and PET labelling batches, indicating the validity of using substrates from the latter source. A para-methoxy-benzamide analogue (MeO-WAY) displayed a significantly lower rate of metabolism compared to WAY-100635, whereas a para-iodo-benzamide analogue was more susceptible to metabolic transformation. LC-MS/MS Analysis of formed metabolites from WAY-100635 and MeO-WAY suggested similar metabolic pathways, with hydroxylation, demethylation and dearylation reactions. The main metabolic route in humans, amide hydrolysis, was not observed with the rat liver microsome assay.

Whole-body (11)C-5-hydroxytryptophan positron emission tomography as a universal imaging technique for neuroendocrine tumors: comparison with somatostatin receptor scintigraphy and computed tomography.

Neuroendocrine tumors (NETs) can be small and situated almost anywhere throughout the body. Our objective was to investigate whether whole-body (WB) positron emission tomography (PET) with (11)C-5-hydroxytryptophan (5-HTP) can be used as a universal imaging technique for NETs and to compare this technique with established imaging methods. Forty-two consecutive patients with evidence of NET and a detected lesion on any conventional imaging (six bronchial, two foregut, 16 midgut, and two thymic carcinoids; one ectopic Cushing's syndrome; four gastrinomas; one insulinoma; six nonfunctioning endocrine pancreatic tumors; one gastric carcinoid, one paraganglioma; and two endocrine-differentiated pancreatic carcinomas) were studied. The WB-(11)C-5-HTP-PET examinations were compared with WB-computed tomography (CT) and somatostatin receptor scintigraphy (SRS). Tumor lesions were imaged with PET in 95% of the patients. In 58% of the patients, PET could detect more lesions than SRS and CT and equal numbers in 34%, whereas in three cases, SRS or CT showed more lesions. In 84% (16 of 19 patients), PET could visualize the primary tumor compared with 47 and 42% for SRS and CT, respectively. The surgically removed PET-positive primary tumor sizes were 6-30 mm. To conclude, this study indicates that WB-(11)C-5-HTP-PET can be used as a universal imaging method for detection of NETs. This study also shows that WB-(11)C-HTP-PET is sensitive in imaging small NET lesions, such as primary tumors, and can in a majority of cases image significantly more tumor lesions than SRS and CT.

Positron emission tomography studies in patients with locally advanced and/or metastatic breast cancer: a method for early therapy evaluation?

PURPOSE: To investigate if sequential positron emission tomographic (PET) scans with the glucose analog 18F-2-fluoro-2-deoxy-D-glucose (18FDG) and/or L-methyl-11C-methionine (11C-methionine) in patients with breast cancer could provide early information on the efficacy of polychemotherapy. PATIENTS AND METHODS: Sixteen patients with breast cancer (11 with locally advanced tumors, three with recurrent disease in the contralateral breast, two of them with distant and regional metastases, and two with distant metastases) underwent a baseline and two follow-up PET scans after the first and third/fourth polychemotherapy course. Tumor response was determined clinically/radiographically after three/four polychemotherapy courses. RESULTS: Five patients were investigated with 18FDG, seven with both 11C-methionine and 18FDG, and four with only 11C-methionine before polychemotherapy. 11C-methionine presented a more distinct visualization of primary/contralateral breast cancers in five of seven patients when compared with 18FDG. Twelve of 16 patients demonstrated a response using conventional methods after the third/fourth course of polychemotherapy. Eight of these 12 clinical responders had a significant decrease in tracer uptake at the first PET scan performed 6 to 13 days after the first polychemotherapy course, and these reductions were further augmented after the third/fourth course and corresponded to the conventional therapy evaluation (clinical examination, computed tomography [CT], ultrasonography, and mammography). CONCLUSION: Our data indicate that PET may be of clinical value in predicting response to chemotherapy in patients with locally advanced breast cancer and/or metastatic disease earlier than any other method used.

Positron emission tomography with 5-hydroxytryprophan in neuroendocrine tumors.

PURPOSE: Carcinoid tumors, especially those of midgut origin, produce serotonin via the precursors tryptophan and 5-hydroxytryptophan (5-HTP). We have evaluated the usefulness of positron emission tomography (PET) with carbon-11-labeled 5-HTP in the diagnosis and treatment follow-up evaluation of patients with neuroendocrine tumors. PATIENTS AND METHODS: PET using 11C-labeled 5-HTP was compared with computed tomography (CT) in 18 patients (14 midgut, one foregut, one hindgut carcinoid, and two endocrine pancreatic tumors [EPT]). In addition, 10 of 18 patients were monitored with PET examinations during treatment. RESULTS: All 18 patients, including two with normal urinary 5-hydroxyindole acetic acid (U-5-HIAA), had increased uptake of 11C-labeled 5-HTP in tumorous tissue as compared with normal tissue. Liver metastases, as well as lymph node, pleural, and skeletal metastases, showed enhanced 5-HTP uptake and PET could detect more lesions than CT in 10 patients and equal numbers in the others. Tumor visibility was better for PET than for CT due to the high and selective uptake of 5-HTP with a high tumor-to-background ratio. Binding studies indicated an irreversible trapping of 5-HTP in the tumors. Linear regression analyses showed a clear correlation (r = .907) between changes in U-5-HIAA and changes in the transport rate constant for 5-HTP during treatment. CONCLUSION: PET with 11C-labeled 5-HTP demonstrated high uptake in neuroendocrine gastrointestinal tumors and thereby allowed improved visualization compared with CT. The in vivo data on regional tumor metabolism, as expressed in 11C-5-HTP uptake and transport rate, provided additional information over conventional radiologic techniques. The close correlation between the changes in 11C-5-HTP transport rate and U-HIAA during medical treatment indicates the potential of 11C-5-HTP-PET as a means to monitor therapy.

Developments in PET for the detection of endocrine tumours.

Positron emission tomography (PET) supplies a range of labelled compounds to be used for the characterization of tumour biochemistry. Some of these have proved to be of value for clinical diagnosis, treatment follow-up, and clinical research. (18)F-fluorodeoxyglucose PET scanning is now a widely accepted imaging approach in clinical oncology, reflecting increased expression of glucose transporters in cancerous tissue. This tracer, however, does not show sufficient uptake in well-differentiated tumours such as neuroendocrine tumours. Endocrine tumours have the unique characteristics of taking up and decarboxylating amine precursors. These so-called APUD characteristics offer highly specific targets for PET tracers. Using this approach, radiopharmaceuticals such as [(11)C]-5-hydroxytryptophan and [(11)C]-L-dihydroxyphenylalanine for localization of carcinoid and endocrine pancreatic tumours, 6-[(18)F]-fluorodopamine and [(11)C]-hydroxyephedrine for phaeochromocytomas, and [(11)C]-metomidate for adrenal cortical tumours have been developed. Functional imaging with PET using these compounds is now being employed to complement rather than replace other imaging modalities. Development of new PET radiopharmaceuticals may in the future allow in vivo detection of tumour biological properties, such as malignant potential and responsiveness to treatment.

Meningioma treated with interferon-alpha, evaluated with [(11)C]-L-methionine positron emission tomography.

PURPOSE: In meningioma patients with postoperative residual masses, recurrent or primarily inoperable tumors, positron emission tomography (PET) with [(11)C]-L-methionine was used to evaluate treatment efficacy of IFN-alpha. EXPERIMENTAL DESIGN: Twelve patients were treated with IFN-alpha at a dose of 1.5-5 million IU s.c. daily. PET, computed tomography, and/or magnetic resonance imaging were performed in all patients before and, at regular intervals, during IFN-alpha treatment. The ratio of tumor hot-spot uptake to cerebellar uptake or to cortex uptake was calculated. This ratio estimates the relative methionine accumulation in the tumor and presumably the proliferative activity in the tumor. RESULTS: During IFN-alpha treatment, PET demonstrated a mean relative percentage of reduction in the uptake ratio (MRelR) of 22.3% in the meningiomas. In nine patients who were considered responders, defined as patients with a positive MRelR, the MRelR was 30.4%. For the three nonresponders, defined as patients with a negative MRelR, the MRelR was -1.8%. Three patients were followed for a long time: two patients for 8 years and one patient for 4 years and 6 months; the two patients followed for 8 years are still on IFN. The volumes of these tumors were constant or showed a slight decrease. No correlation was found between histopathological diagnosis (PAD) WHO grading I-III of meningiomas and response to IFN-alpha treatment. CONCLUSIONS: PET was judged a useful method to predict which patients are suitable for long-term treatment with IFN-alpha and also for dose finding. In five patients treated from 9 months to 8 years, IFN-alpha seemed to be an effective oncostatic drug. The clinical usefulness of IFN-alpha, taking adverse reactions into account, must be evaluated in a larger series of patients.

Increased neurokinin-1 receptor availability in the amygdala in social anxiety disorder: a positron emission tomography study with [11C]GR205171.

The neurokinin-1 (NK1) receptor is abundantly expressed in the fear circuitry of the brain, including the amygdala, where it modulates stress and anxiety. Despite its proposed involvement in psychopathology, only a few studies of NK1 receptor availability in human subjects with anxiety disorders exist. Here, we compared NK1 receptor availability in patients with social anxiety disorder (SAD; n = 17) and healthy controls (n = 17) using positron emission tomography and the radiotracer [11C]GR205171. The Patlak Graphical plot using a cerebellar reference region was used to model the influx parameter, Ki measuring NK1 receptor availability. Voxel-wise statistical parametric mapping analyses revealed increased NK1 receptor availability specifically in the right amygdala in SAD patients relative to controls. Thus, we demonstrate that exaggerated social anxiety is related to enhanced NK1 receptor availability in the amygdala. This finding supports the contribution of NK1 receptors not only in animal models of stress and anxiety but also in humans with anxiety disorders.

Middle cerebral artery occlusion and reperfusion in primates monitored by microdialysis and sequential positron emission tomography.

BACKGROUND AND PURPOSE: In a previous investigation concerning the hemodynamic and metabolic changes over time displayed by sequential positron emission tomography (PET) in a middle cerebral artery (MCA) occlusion/reperfusion primate model, a metabolic threshold for irreversible ischemia could be identified (reduction of metabolic rate of oxygen [CMRO(2)] to approximately 60% of the contralateral hemisphere). To evaluate the potential of microdialysis (MD) as an instrument for chemical brain monitoring, the aim of this subsequent study was to relate the chemical changes in MD levels directly to the regional metabolic status (CMRO(2) above or below the metabolic threshold) and the occurrence of reperfusion, as assessed by PET. METHODS: Continuous MD (2 probes in each brain) and sequential PET measurements were performed during MCA occlusion (2 hours) and 18 hours (mean) of reperfusion in 8 monkeys (Macaca mulatta). Energy-related metabolites (lactate, pyruvate, and hypoxanthine) and glutamate were analyzed. The MD probe regions were divided into 3 categories on the basis of whether CMRO(2) was below or above 60% of the contralateral region (metabolic threshold level) during MCA occlusion and whether reperfusion was obtained: severe ischemia with reperfusion (n=4), severe ischemia without reperfusion (n=4), and penumbra with reperfusion (n=5). RESULTS: The lactate/pyruvate ratio, hypoxanthine, and glutamate showed similar patterns. MD probe regions with severe ischemia and reperfusion and probe regions with severe ischemia and no reperfusion displayed high and broad peaks, respectively, during MCA occlusion, and the levels almost never decreased to baseline. Penumbra MD probe regions displayed only slight transient increases during MCA occlusion and returned to baseline. CONCLUSIONS: This experimental study of focal ischemia showed that the extracellular changes of energy-related metabolites and glutamate differed depending on the ischemic state of the brain during MCA occlusion and depending on whether reperfusion occurred. If MD proves to be beneficial in clinical practice, it appears important to observe relative changes over time.

Ligands for in vivo imaging of nicotinic receptor subtypes in Alzheimer brain.

The neuronal nicotinic acetylcholine receptors (nAChR) are involved in functional processes in brain including cognitive function and memory. A severe loss of the nAChRs has been detected in brain of patients with Alzheimer's disease (AD). There is a great interest to image nAChRs noninvasive for detection of receptor impairments even at a presymptomatic stage of AD as well for monitoring outcome of drug treatment. (S) [11C]Nicotine, has so far been the only nAChR ligand used in positron emission tomography (PET) studies for visualizing nAChRs in human brain. In order to develop PET/SPECT nAChRs ligands for detection of subtypes of nAChRs nicotine analogues, epibatidine and A-85380 compounds have been characterized in vitro and investigated in vivo. Epibatidine and A-85380 have been found to have higher specific signals and more favorable kinetic parameters than nicotine and its analogues. The epibatidine and A-85380 compounds can also be radiolabeled with high specific radioactivity, show affinities for the nAChRs in the pM range and readily cross the blood-brain barrier. In addition they reversibly bind to the nAChRs and show low non-specific binding and moderately fast metabolism. Due to a probably high alpha4beta2 nAChR selectivity combined with low toxicity, the A-85380 analogs presently seem to be the most promising nAChR ligand imaging of subtypes of nAChRs in human brain.

Brain dopaminergic mechanisms in Parkinson's disease evaluated by positron emission tomography.

The meso-striatal dopamine neurons, essential for the automated control of movements, are primarily affected in patients with P.D. Direct study of the role of this pathway in states of disease has not been possible until recently and the application of PET for the in vivo investigation of dopaminergic mechanisms may serve to demonstrate the potential of the technique. One basic idea has been to work out methods to investigate multiple aspects of dopaminergic function, i.e. presynaptic mechanisms such as re-uptake sites and synthesis of neurotransmitter as well as postsynaptic such as receptor properties. Furthermore, efforts have been made to evaluate dopamine degradating enzymes. Preclinical PET-investigations have regularly been performed in Rhesus monkeys and the hemiparkinsonian model produced by infusing MPTP into one internal carotid artery has been of great value to characterize new 11C-labelled tracers. Today 11C--(+)-nomifensine is used to give a measure of dopamine re-uptake sites, probably reflecting nerve terminals. 11C-labelled L-dopa has now been introduced and can be expected to replace 18F-L-fluorodopa as a physiological tracer for precursor transport and transmitter synthesis. Several ligands are available for the quantitation of dopamine receptors--11C-N-methylspiperone and 11C-raclopride have been used in our studies. 11C-L-selegiline and its "inactive" D-form have been used in clinical PET-studies aimed to evaluate the enzyme MAO-B. A summary of in vivo information of dopaminergic mechanisms in P.D. obtained using the above-mentioned tracers and PET is presented.

Increased dopamine synthesis rate in medial prefrontal cortex and striatum in schizophrenia indicated by L-(beta-11C) DOPA and PET.

BACKGROUND: The aim of the present study was to investigate dopamine synthesis in the brain of drug-free schizophrenic patients, not only in the striatum but also in extrastriatal areas like the prefrontal cortex, brain areas that for a long time has been in focus of interest in the pathophysiology of schizophrenia. METHODS: PET was performed in 12 drug-free (10 drug-naive) psychotic schizophrenic patients and 10 healthy volunteers matched for age and gender using 11C-labelled L-DOPA as the tracer. The time-radioactivity curve from occipital cortex (located within Brodman area 17 and 18) was used as input function to calculate L-DOPA influx rate, Ki images, that were matched to a common brain atlas. A significant overall increase of the Ki values was found in the schizophrenic group as compared with healthy controls. RESULTS: In particular, significantly higher Ki were found in the schizophrenic patients compared to the controls in the caudate nucleus, putamen and in parts of medial prefrontal cortex (Brod 24). The Ki value reflect an increased utilization of L-DOPA, presumably due to increased activity of the amino acid decarboxylate enzyme. CONCLUSIONS: The results indicate that the synthesis of dopamine is elevated within the striatum and parts of medial prefrontal cortex in schizophrenia.

A comparison of 11C-labeled L-DOPA and L-fluorodopa as positron emission tomography tracers for the presynaptic dopaminergic system.

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-[beta-11C]DOPA, but it induced no effect on the K(i)-value using L-[beta-11C]-6-fluorodopa. Studies of radiolabeled tracer and metabolites in plasma showed substantial differences between the two tracers. At baseline conditions, 60% unchanged L-[beta-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-[beta-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-[car-boxy-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.

Positron emission tomographic measurements of cerebral glucose utilization using [1-11C]D-glucose.

Regional CMRglc was measured in seven healthy volunteers with positron emission tomography using [1-11C]D-glucose. Regional CBF was measured using [11C]fluoromethane. The arteriovenous differences of unlabeled glucose and oxygen together with 11C metabolites were also measured. In addition to the loss of [11C]CO2, a loss of acidic 11C metabolites was also detected. A three-compartment model was applied to the tracer data in the time interval 0-24 min. After correction for the loss of 11C metabolites, the tracer method gave an average CMRglc of 26.4 +/- 1.9 (SD) mumol/100 g/min, close to the value obtained with the Fick principle. After correction for the loss of [11C]CO2 only, the tracer method gave 23.6 +/- 2.1 mumol/100 g/min, compatible with (1/6) CMRO2, obtained with the Fick principle. These results and the time course of the loss of acidic 11C metabolites are consistent with the presence of nonoxidative metabolism of glucose that causes an early loss of mainly [11C]lactate after a bolus injection of the tracer. This implies that [1-11C]D-glucose measures the rate of glucose oxidation rather than the total CMRglc. The experiments using [1-11C]D-glucose were compared to five analogous experiments using [U-11C]D-glucose together with [15O]H2O as a flow tracer. After correction for the loss of [11C]CO2, the two glucose tracers gave similar global values of CMRglc and other parameters associated with glucose utilization, but with labeling in the carbon-1 position, the loss of [11C]CO2 was substantially delayed and the contrast between gray and white matter was improved.(ABSTRACT TRUNCATED AT 250 WORDS)

Simultaneous intracerebral microdialysis and positron emission tomography in the detection of ischemia in patients with subarachnoid hemorrhage.

Intracerebral microdialysis (MD) was applied in patients with subarachnoid hemorrhage. The regional CBF, the CMRO2, and oxygen extraction ratio (OER) were measured with simultaneous positron emission tomography (PET). The aim was to directly correlate alterations in dialysate levels of energy-related metabolites (lactate, lactate/pyruvate ratio, hypoxanthine) and excitatory amino acids (EAAs) (glutamate and aspartate) to the energy state in the MD probe region as determined by PET. Regional ischemia was defined according to Heiss et al. and Lassen (Heiss et al., 1992; Lassen, 1966). Whole-brain ischemia was considered present when the OER for the whole brain exceeded the mean whole-brain OER + 2 SD of six reference patients. In general, the presence of whole-brain ischemia and/or regional ischemia within the region of the MD probe was associated with increased levels of energy-related metabolites and EAAs retrieved by MD. Increased levels of energy-related metabolites and EAAs were only occasionally seen when PET did not show any signs of ischemia or when signs of regional ischemia were found remote from the MD probe region. Thus, the energy-related metabolites and EAAs may be used as extracellular "markers" of ischemia. PET may be of use in defining critical ischemic regions (tissue at risk) where the MD probe can be inserted for chemical monitoring.

Brain networks affected by synchronized sleep visualized by positron emission tomography.

Nineteen lightly sleep-deprived healthy volunteers were examined with H2(15)O and positron emission tomography (PET). Scanning was performed during wakefulness and after the subjects had fallen asleep. Sleep stage was graded retrospectively from electroencephalogram (EEG) recordings, and scans were divided into two groups: wakefulness or synchronized sleep. Global flow was quantified, revealing no difference between sleep and wakefulness. A pixel-by-pixel-blocked one-way analysis of variance (ANOVA) was performed after correcting for differences in anatomy and global flow. The sum of squares of the z-score distribution showed a highly significant (P < 0.00001) omnibus difference between sleep and wakefulness. The z-score images indicated decreased flow in the thalamus and the frontal and parietal association cortices and increased flow in the cerebellum during sleep. A principal component (PC) analysis was performed on data after correction for global flow and block effects, and a multivariate analysis of variance (MANOVA) on all PC scores revealed significant (P = 0.00004) differences between sleep and wakefulness. Principal component's 2 and 5 correlated to sleep and revealed distinct networks consisting of PC 2, cerebellum and frontal and parietal association cortices, and PC 5, thalamus.