Identification of imaging biomarkers for the assessment of tumour response to different treatments in a preclinical glioma model.

PURPOSE: Hypoxia-inducible factor 1α (HIF-1α) activity is one of the major players in hypoxia-mediated glioma progression and resistance to therapies, and therefore the focus of this study was the evaluation of HIF-1α modulation in relation to tumour response with the purpose of identifying imaging biomarkers able to document tumour response to treatment in a murine glioma model. METHODS: U251-HRE-mCherry cells expressing Luciferase under the control of a hypoxia responsive element (HRE) and mCherry under the control of a constitutive promoter were used to assess HIF-1α activity and cell survival after treatment, both in vitro and in vivo, by optical, MRI and positron emission tomography imaging. RESULTS: This cell model can be used to monitor HIF-1α activity after treatment with different drugs modulating transduction pathways involved in its regulation. After temozolomide (TMZ) treatment, HIF-1α activity is early reduced, preceding cell cytotoxicity. Optical imaging allowed monitoring of this process in vivo, and carbonic anhydrase IX (CAIX) expression was identified as a translatable non-invasive biomarker with potential clinical significance. A preliminary in vitro evaluation showed that reduction of HIF-1α activity after TMZ treatment was comparable to the effect of an Hsp90 inhibitor, opening the way for further elucidation of its mechanism of action. CONCLUSION: The results of this study suggest that the U251-HRE-mCherry cell model can be used for the monitoring of HIF-1α activity through luciferase and CAIX expression. These cells can become a useful tool for the assessment and improvement of new targeted tracers for potential theranostic procedures.

DTI parameters in neonates with hypoxic-ischemic encephalopathy after total body hypothermia.

BACKGROUND AND PURPOSE: MR imaging provides means for discriminating different patterns of Hypoxic-ischemic encephalopathy (HIE) and may distinguish most severe cases from less severe but is unable to predict long-term outcome. Diffusion tensor imaging (DTI) offers information for a more complete characterization of HIE. The purpose of this study is to compare the modifications of DTI parameters in newborns one week and six months following total-body cooling to healthy controls. METHODS: Forty-seven cooled newborns were studied with MRI, 20 underwent follow-up at 6 months. 12 healthy newborns and nine children at 6 months were enrolled as control groups (HC). Inferior Longitudinal Fasciculus (ILF), Corpus Callosum Fasciculus (CCF), Corticospinal Tract (CST), Optical Tract (OT), Optic Radiation (OR) were generated in all subjects. DTI parameters were evaluated in basal ganglia (BG), thalamus (TH) and tracks. Statistical analysis was performed with MANOVA. RESULTS: In newborns HIE versus HC, there were significantly lower fractional anisotropy (FA) on OR and CST and higher axial diffusivity (AD), apparent diffusion coefficient (ADC) and radial diffusivity (RD) values on CST, BG and TH in HIE-N. At 6 months there were no significant grouping effects. The analysis showed a significant increase of FA, decrease of ADC, AD, RD after 6 months for HIE and HC. CONCLUSIONS: We observed modifications of parameter values in HIE newborns vs HC; however normalization of values at 6 months suggests that changes of parameters cannot be considered early biomarkers for evaluation of therapeutic hypothermia in newborns with moderate HIE and normal conventional MRI.

Exploring dopaminergic activity in ring chromosome 20 syndrome: a SPECT study.

AIM: Several lines of evidence indicate that the dopaminergic system may play a role in the propagation of epileptic seizures and, indeed, DOPA metabolism impairment has recently been demonstrated in PET studies of ring chromosome 20 [r(20)] patients. We conducted a study looking for correlations between r(20) mosaicism, other clinical variables and both pre-synaptic dopamine transporter (DAT) expression and post-synaptic D2 receptor density. METHODS: Five patients with r(20) and epilepsy were enrolled in the study. DAT expression and D2 density were measured by single photon emission tomography (SPECT) imaging with 185 MBq of [123I]ioflupane and [123I]IBZM, respectively, on different days. Linear correlations between r(20) mosaicism, clinical variables and binding of [123I]ioflupane or [123I]IBZM were examined. RESULTS: A significant correlation between seizure frequency and r(20) mosaicism was detected (r=0.903, P<0.05), along with a negative correlation between r(20) mosaicism and binding of [123I]ioflupane in the putamen and in the caudate nucleus (r=-0.692 and r=-807; P<0.05). Seizure frequency was positively correlated with post-synaptic D2 density (r=0.925, P<0.05). CONCLUSION: Striatal neurons are involved in r(20) epilepsy; the relationship found between r(20) mosaicism and DAT expression suggests that drugs acting on the dopaminergic system could have a place in the treatment of this rare form of epilepsy.

Reexamination of glucose-6-phosphatase activity in the brain in vivo: no evidence for a futile cycle.

Glucose-6-phosphatase activity in the rat brain in vivo was estimated by measuring the differential loss of tritium and carbon-14 from the glucose pool labeled by a mixture of [2-3H]glucose and [U-14C]glucose. The results provide no evidence of significant dephosphorylation of glucose-6-phosphate and do not support the hypothesis of a futile cycle involving glucose-6-phosphatase activity in the brain.

Yttrium-90 radioembolization treatment for unresectable hepatocellular carcinoma: a single-centre prognostic factors analysis.

The aim of this study was to evaluate the efficacy and the safety of Y90 radioembolization (Y90-RE) in patients with unresectable hepatocellular carcinoma (HCC) analysing our results and correlating them with independent prognostic factors for overall survival (OS) and for complications. Forty-three patients with advanced inoperable HCC including those with multiple bilobar lesions or portal vein thrombosis (PVT) treated with Y90-RE were reviewed. Treatment efficacy and safety were evaluated. Survival was calculated by the Kaplan-Meier method. Univariate analyses were performed for identifying potential prognostic factors. Radiologic response was evaluated with the modified Response Evaluation Criteria in Solid Tumours (mRECIST) criteria. Clinical toxicities were prospectively recorded. Median overall progression-free survival and OS were 27.7 and 16.8 months, respectively. Longer median OS was revealed in those without PVT (p = 0.0241) and those whose pre-treatment haemoglobin values was higher (p = 0.0471). According with mRECIST criteria, we observed a disease control rate of 69.2 and 61.9% at 3- and 6-month follow-up, respectively. Complications developed in 28 patients (65.1%), among which grade 2-3 events were reported in 17 patients. We noted that activity administered dose presented a correlation with intra-procedural toxicity (p = 0.039259) while common hepatic artery use as release site was associated with a most frequent presentation of remote adverse events. Y90-RE is an alternative treatment with a promising outcome for poor-risk advanced inoperable HCC. PVT and pre-treatment haemoglobin values can be predictors of efficacy. Activity administered dose and arterial release site can be predictors of safety.

Molecular imaging: a new way to study molecular processes in vivo.

Non-invasive imaging of reporter gene expression using different imaging modalities is increasing its role for the in vivo assessment of molecular processes. Multimodality imaging protocols overcome limitations to a single imaging modality and provide a thorough view of specific processes, often allowing a quantitative measurement and direct visualization of the process in a specific target organ or tissue. The use of the right reporter gene for the development of animal models and the characterization of its expression in different conditions and tissues is fundamental for basic, translational and future pharmacological applications of a given model. This paper summarizes the major steps in the development and evaluation of a specific animal model for in vivo molecular imaging studies and describes the first example of an animal model designed for the in vivo assessment of a specific receptor activity and its possible evolution towards multimodality imaging analysis.

The dynamics of estrogen receptor activity.

In the latest few years, the merging of imaging and animal engineering technologies has led to the generation of innovative tools that provide the opportunity to look into the dynamics of specific molecular events in living animals during their entire life under a completely renewed perspective. These tools will have a profound impact not only on basic research, but also on drug discovery and development allowing to depict the activity of any therapeutic agents in all their designed targets as well as in the organs where they may cause undesired effects. Along this research line, our laboratory has recently described the first animal model reporting the state of activity of estrogen receptors (ERs) in real time: the ERE-luc reporter mouse. The application of optical imaging to the ERE-luc has allowed an unprecedented in depth view of estrogen signaling in all of its target tissues. For example, the analysis of the state of activity of ERs in the physiological setting of the estrous cycle has provided compelling evidence that hormone-independent mechanisms are responsible for activating ERs in non-reproductive organs. This discovery may pave the way to a rational basis for the development of novel, more selective and effective treatments for menopause.

Myocardial glucose uptake evaluated by positron emission tomography and fluorodeoxyglucose during hyperglycemic clamp in IDDM patients. Role of free fatty acid and insulin levels.

Myocardial and whole-body glucose metabolism was assessed in 19 insulin-dependent diabetes mellitus (IDDM) patients. A hyperglycemic clamp was performed 1) in the absence of insulin at free fatty acid (FFA) levels of 1.0 mmol/l (test 1); 2) in the absence of insulin at low FFA levels (0.1 mmol/l) by means of a lipid-lowering drug, acipimox (test 2); 3) during insulin infusion to achieve systemic levels of 400 pmol/l and FFA levels of 0.1 mmol/l (test 3); and 4) at the insulin levels of test 3 but increasing FFA to 1.0 mmol/l by means of heparin and intralipid infusion (test 4). Myocardial glucose uptake was measured by positron emission tomography (PET) and 2-[18F]fluoro-2-deoxy-D-glucose. Whole-body glucose uptake was measured in the four conditions by the glucose infusion rate during the PET scanning period. Myocardial glucose uptakes were 40.3 +/- 18.0, 395.5 +/- 139.6, 852.2 +/- 99.1, and 1,388.4 +/- 199.1 mumol.kg tissue-1.min-1 (mean +/- SD) and whole-body glucose uptakes were 10.1 +/- 2.3, 10.1 +/- 3.4, 42.8 +/- 5.8, and 30.5 +/- 5.6 mumol.kg body wt-1.min-1 during tests 1, 2, 3, and 4, respectively. Thus, in IDDM patients without coronary artery disease under the condition of hyperglycemia, an increase of myocardial glucose uptake was obtained either by lowering of FFA levels during hypoinsulinemia or by an increase in FFA levels during hyperinsulinemia. In both conditions no significant changes of whole-body glucose uptake were demonstrated.

Specificity and sensitivity of exercise-induced ST segment elevation for detection of residual viability: comparison with fluorodeoxyglucose and positron emission tomography.

OBJECTIVES: We evaluated the sensitivity and specificity of exercise-induced ST segment elevation for the detection of residual myocardial viability. BACKGROUND: Assessment of residual viability after myocardial infarction is relevant for establishing indication for revascularization. We have previously shown that exercise-induced ST segment elevation is a marker of residual viability. METHODS: We studied 34 patients with a previous Q wave myocardial infarction (anterior in 21, inferior in 13) of whom 18 (group A) had exercise-induced ST segment elevation in more than one lead (mean [+/- SD] 1.8 +/- 0.9 mm, range 1 to 4) and 16 (group B) did not. All patients underwent rest technetium-99m methoxyisobutyl isonitrile single-photon emission computed tomography (SPECT), fluorine-18 (F-18) fluorodeoxyglucose positron emission tomography and coronary angiography. The time elapsed between the infarction and the viability study was 72 +/- 108 days (range 15 to 400) in group A and 516 +/- 545 days (range 14 to 1,800) in group B. RESULTS: The presence and site of previous infarction were confirmed by SPECT studies in all 34 patients. Uptake of F-18 fluorodeoxyglucose within the infarcted area was present in 18 of 18 patients in group A but in only 9 (56%) of 16 in group B (p < 0.01). In patients with an anterior infarction, the sensitivity, specificity and predictive accuracy of exercise-induced ST segment elevation for detection of residual viability were 82%, 100% and 86%, respectively (95% confidence intervals 46% to 83.5%, 59% to 100% and 55.6% to 87.1%, respectively). CONCLUSIONS: Exercise-induced ST segment elevation in infarct-related leads has a high specificity and acceptable sensitivity for detection of residual viability within the infarcted area.

Exercise-induced ischemic arrhythmias in patients with previous myocardial infarction: role of perfusion and tissue viability.

OBJECTIVES: This study sought to investigate whether residual viability of infarcted myocardium may play a role in the pathogenesis of exercise-induced ventricular arrhythmias. BACKGROUND: We previously showed that transient ischemia within partially infarcted areas often precipitates ventricular arrhythmias during exercise that are consistently obliterated by intravenous nitrates. METHODS: We studied 60 patients with chronic stable angina and a previous myocardial infarction. All underwent at least two consecutive exercise stress tests, coronary angiography and stress/rest myocardial perfusion tomography by Tc-99m 2-methoxy isobutyl isonitrile (MIBI). In the last 26 consecutive patients, residual viability was assessed by single-photon emission computed tomography (SPECT) using fluorine (F)-18 fluorodeoxyglucose. Perfusion and metabolic data were evaluated qualitatively by three independent observers in blinded manner. RESULTS: With exercise, 30 patients (group A) consistently developed ventricular arrhythmias (> 10 ventricular ectopic beats/min, couplets, nonsustained ventricular tachycardia); the remaining 30 patients (group B) did not. The severity of coronary artery disease (Gensini score) was similar in the two groups. Postexercise SPECT showed partial reperfusion of an infarcted area in 28 of 30 patients of group A but in only 9 of 30 of group B (p < 0.0001). Uptake of F-18 fluorodeoxyglucose was observed within the infarcted zone in 10 of 13 and 1 of 13 patients in groups A and B, respectively (p = 0.0003). CONCLUSIONS: In patients with myocardial infarction, exercise-induced ventricular arrhythmias appear to be triggered by transient ischemia occurring within a partially necrotic area containing large amounts of viable myocardium. Therefore, occurrence of arrhythmias during exercise may represent a clue to the presence of residual viability within a previously infarcted area.

Reversal of left ventricular diastolic dysfunction after kidney-pancreas transplantation in type 1 diabetic uremic patients.

OBJECTIVE: Diastolic function is frequently impaired in diabetic patients. Our aim was to evaluate the effects of glycometabolic control achieved by pancreas transplantation on left ventricular function in uremic type 1 diabetic patients. RESEARCH DESIGN AND METHODS: Left ventricular systolic and diastolic functions were evaluated using radionuclide ventriculography in 42 kidney-pancreas transplant patients and 26 kidney-alone recipients who had similar clinical characteristics before transplantation. Patients were grouped according to 6, 24, and 48 months of follow-up. Control subjects consisted of 20 type 1 diabetic patients. RESULTS: The left ventricular ejection fraction was normal in all of the patients. However, kidney-pancreas transplant patients with 4 years of graft function had a higher ejection fraction (75.7 +/- 1.8%) than kidney-alone patients with 4 years of graft function (65.3 +/- 2.8%, P = 0.02) and type 1 diabetic patients (61.3 +/- 3.7%, P = 0.004). In patients with 4 years of graft function, normal diastolic parameters were evident in kidney-pancreas but not in kidney-alone or in type 1 diabetic patients (peak filling rate: 4.46 +/- 0.15 end diastolic volume (EDV)/s in kidney-pancreas patients vs. 2.73 +/- 0.24 EDV/s [P < 0.01] and 3.39 +/- 0.30 EDV/s [P < 0.01] in kidney-alone and type 1 diabetic patients, respectively; time-to-peak filling rate: 141.9 +/- 7.8 ms in kidney-alone patients vs. 209.4 +/- 13.5 ms in kidney-alone patients [P < 0.01]; peak filling rate/peak ejection rate ratio: 1.10 +/- 0.04 in kidney-pancreas patients vs. 0.81 +/- 0.08 in kidney-alone patients [P < 0.01]). A significant reduction in diastolic dysfunction rate was observed only in kidney-pancreas patients. CONCLUSIONS: Kidney-pancreas transplantation results in complete insulin independence, a better glycometabolic pattern and blood pressure control, an improvement of left ventricular function, and a reversal of diastolic dysfunction.

Different patterns of local brain energy metabolism associated with high and low doses of methylphenidate. Relevance to its action in hyperactive children.

Rates of local cerebral glucose utilization were measured by means of the quantitative autoradiographic [14C]deoxyglucose technique in conscious rats following the acute administration of methylphenidate hydrochloride (1.25-15.0 mg/kg). Significant dose-dependent alterations in metabolic activity were found in the components of the extrapyramidal system, including the substantia nigra, subthalamic nucleus, and the entopeduncular nucleus, as well as in the lateral habenula. Significant changes were also observed in the nucleus accumbens and olfactory tubercle, but occurred only following administration of low doses of methylphenidate. Comparison of the patterns of metabolic activity observed in this study with those obtained following the administration of other psychostimulant drugs suggests possible substrates for the therapeutic action of methylphenidate in the treatment of hyperactive children.

Cerebellar diaschisis in pontine ischemia. A case report with single-photon emission computerized tomography.

Regional cerebral and cerebellar blood flows were studied by N,N,N'-trimethyl-N'-(2-hydroxy-3-methyl-5-[123I]iodobenzyl)-1,3- propanediamine 2 HCl (I-123 HIPDM) and single-photon emission computerized tomography (SPECT) in a patient with an ischemic lesion of the pons. An asymmetry of perfusion of the cerebellar hemispheres, normal on transmission computerized tomography scan, was demonstrated by SPECT studies in the early acute phase and confirmed 15 days after. This finding may be related to the interruption of the corticopontocerebellar pathways.

Measurement of local cerebral blood flow with [14C]iodoantipyrine in the mouse.

Local cerebral blood flow was measured in the mouse by means of the [14C]iodoantipyrine method. This method has been previously used in the monkey, dog, cat, and rat, but its application to small mammals such as the mouse requires special attention to potential sources of error. The small size of the mouse brain requires special attention to the rapid removal and freezing of the brain to minimize effects of postmortem diffusion of tracer in the tissue. Because of the relatively low diameter/length ratios of the catheters needed for arterial sampling in small animals, substantial errors can occur in the determination of the time course of the [14C]iodoantipyrine concentration in the arterial blood unless corrections for lag time and dead space washout in the catheter are properly applied. Local cerebral blood flow was measured in seven awake mice with appropriate care to minimize these sources of error. The values were found to vary from 48 ml/100 g/min in the corpus callosum to 198 ml/100 g/min in the inferior colliculus. The results demonstrate that the [14C]iodoantipyrine method can be used to measure local cerebral blood flow in the mouse and that the values in that species are, in general, somewhat higher than those in the rat.

Influence of plasma glucose concentration on lumped constant of the deoxyglucose method: effects of hyperglycemia in the rat.

The lumped constant of the deoxyglucose method was determined by the steady-state, model-independent method in the brain of normal conscious rats with arterial plasma glucose concentrations varying from normoglycemia (i.e., 8 mM) to hyperglycemia (i.e., 31 mM). The lumped constant for brain was found to decrease very gradually with increasing arterial plasma glucose concentration from a value of approximately 0.45 in the midnormoglycemic range (i.e., 7-8 mM) to approximately 0.38 at 28-31 mM. 3-O-[14C]Methylglucose was used to assess the distribution of glucose within the brain structures in hyperglycemia; the results indicated that the glucose concentration, and therefore also the values for the lumped constant, remain relatively uniform in hyperglycemia with arterial plasma glucose concentrations as high as 34 mM. The values for the lumped constant for rat brain determined in the present studies were combined with those previously determined in this laboratory for hypoglycemia and normoglycemia by the same method to provide a single source for the values for the lumped constant to be used over the full range of arterial plasma glucose concentrations. In several rats the lumped constant for cephalic extracerebral tissues was also evaluated in parallel with those for the brain. The lumped constant for the cephalic extracerebral tissues was found to be about twice that for brain and to be unaffected by changes in arterial plasma glucose levels.

The lumped constant of the deoxyglucose method in hypoglycemia: effects of moderate hypoglycemia on local cerebral glucose utilization in the rat.

The applicability of the [14C]deoxyglucose method for measuring local cerebral glucose utilization (lCMRglc) has been extended for use in hypoglycemia by determination of the values of the lumped constant to be used in rats with plasma glucose concentrations ranging from approximately 2 to 6 mM. Lumped constant values were higher in hypoglycemia and declined from a value of 1.2 at the lowest arterial plasma glucose level (1.9 mM) to about 0.48 in normoglycemia. The distribution of glucose, and therefore also of the lumped constant, was found to remain relatively uniform throughout the brain at the lowest plasma glucose levels studied. lCMRglc in moderate, insulin-induced hypoglycemia (mean arterial plasma glucose concentration +/- SD of 2.4 +/- 0.3 mM) was determined with the appropriate lumped constant corresponding to the animal's plasma glucose concentration and compared with the results obtained in six normoglycemic rats. The weighted average rate of glucose utilization for the brain as a whole was significantly depressed by 14% in the hypoglycemic animals, i.e., 61 mumols/100 g/min in hypoglycemia compared to 71 mumols/100 g/min in the normoglycemic controls (p less than 0.05). lCMRglc was lower in 47 of 49 structures examined but statistically significantly below the rate in normoglycemic rats in only six structures (p less than 0.05) by multiple comparison statistics. Regions within the brainstem were most prominently affected. The greatest reductions, statistically significant or not, occurred in structures in which glucose utilization is normally high, suggesting that glucose delivery and transport to the tissue became rate-limiting first in those structures with the greatest metabolic demands for glucose.

Effects of insulin on local cerebral glucose utilization in the rat.

The effects of hyperinsulinemia on local cerebral glucose utilization were studied by the quantitative autoradiographic 2-[14C]deoxyglucose method in normal conscious rats under steady-state normoglycemic conditions. Hyperinsulinemia and a steady state of normoglycemia were achieved and maintained during the experimental period by a continuous intravenous (i.v.) infusion of insulin given simultaneously with a programmed i.v. infusion of D-glucose. Hyperinsulinemia under normoglycemic conditions did not change the average rate of glucose utilization in the brain as a whole, but significant increases in local glucose utilization were found selectively in the ventromedial, dorsomedial, and anterior hypothalamic nuclei. The results suggest that a known anatomical pathway linking the dorsomedial and anterior nuclei with the ventromedial nucleus of the hypothalamus may be physiologically activated in response to hyperinsulinemia.

Estimation of component and parameter distributions in spectral analysis.

A method is presented for estimating the distributions of the components and parameters determined with spectral analysis when it is applied to a single data set. The method uses bootstrap resampling to simulate the effect of noise on the computed spectrum and to correct for possible bias in the estimates. A number of bootstrap procedures are reviewed, and one is selected for application to the kinetic analysis of positron emission tomography dynamic studies. The technique is shown to require minimal assumptions about noise in the measurements, and its small sample properties are established through Monte-Carlo simulations. The advantages and limitations of spectral analysis with bootstrap resampling for deriving inferences for tracer kinetic modeling are illustrated through sample analyses of time-activity curves for [18F]fluorodeoxyglucose and [15O]-labeled water.

The use of spectral analysis to determine regional cerebral glucose utilization with positron emission tomography and [18F]fluorodeoxyglucose: theory, implementation, and optimization procedures.

A method for kinetic analysis of dynamic positron emission tomography (PET) data by linear programming that allows identification of the components of a measured PET signal without predefining a compartmental model has recently been proposed by Cunningham and co-workers. The method identifies a small subset of functions from a large input set of feasible functions that best fits the time course of total radioactivity measured by PET. To investigate in detail the properties of this technique, we applied it to PET studies with [18F]fluorodeoxyglucose, a tracer with well-characterized kinetic properties. We examined dynamically acquired data over various time intervals in many brain regions and found that the number of components identified by the method is stable and consistent with the presence of kinetic heterogeneity in every region. We optimized the method for determination of regional rates of glucose utilization; calculated rates were found to be somewhat dependent upon the treatment of noise in the measured tissue data and upon the time interval in which the data were collected. The application of a numerical filter to remove noise in the data resulted in values for regional cerebral glucose utilization that were stable with time and consistent with rates determined by the other established techniques. Based on the results of the current study, we expect that the spectral analysis technique will prove to be a highly flexible tool for kinetic analysis of other tracer compounds; it is capable of producing low-variance, time-stable estimates of physiological parameters when optimized for time interval of application, input spectrum of components, and processing of noise in the data.

Refinement of the kinetic model of the 2-[14C]deoxyglucose method to incorporate effects of intracellular compartmentation in brain.

A translocase to transport hexose phosphate formed in the cytosol into the cisterns of the endoplasmic reticulum, where the phosphatase resides, is absent in brain (Fishman and Karnovsky, 1986). 2-Deoxyglucose-6-phosphate (DG-6-P) may therefore have limited access to glucose-6-phosphatase (G-6-Pase), and transport of the DG-6-P across the endoplasmic reticular membrane may be rate limiting to its dephosphorylation. To take this compartmentation into account, a five-rate constant (5K) model was developed to describe the kinetic behavior of 2-deoxyglucose (DG) and its phosphorylated product in brain. Loss of DG-6-P was modeled as a two-step process: (a) transfer of DG-6-P from the cytosol into the cisterns of the endoplasmic reticulum; (b) hydrolysis of DG-6-P by G-6-Pase and subsequent return of the free DG to the precursor pool. Local CMRglc (LCMRglc) was calculated in the rat on the basis of this model and compared with values calculated on the basis of the three-rate constant (3K) and the four-rate constant (4K) models of the DG method. The results show that under normal physiological conditions all three models yield values of LCMRglc that are essentially equivalent for experimental periods between 25 and 45 min. Therefore, the simplest model, the 3K model, is sufficient. For experimental periods from 60 to 120 min, the 4K and 5K models do not correct completely for loss of product, but the 5K model does yield estimates of LCMRglc that are closer to the values at 45 min than those obtained with the 3K and 4K models.