Ketone bodies are selectively used by individual brain regions.

Close study of 3-hydroxybutyrate uptake by brain suggests that its metabolism is limited by permeability. Furthermore, the permeability characteristics vary from region to region; areas known to have no blood-brain barrier show the highest rate of utilization. The results imply that rather than substitute fuels, ketone bodies should be considered supplements which partially supply specific areas but are incapable of supporting the entire energy requirement of all brain regions.

Phenylketonuria: phenylalanine inhibits brain pyruvate kinase in vivo.

The hypothesis that brain damage in phenylketonuria is related to inhibition of pyruvate kinase by phenylalanine was examined in rat brain in vivo. One hour after a single injection of phenylalanine into the rat, the brains were removed and completely frozen in less than a second. The concentration of phenylalanine in the brain was comparable to that found in phenylketonuric patients. Changes in brain glycolytic intermediates were consistent with inhibition of pyruvate kinase in vivo. The inhibition of pyruvate kinase was apparently compensated for by an increase in phosphoenolpyruvate; no decrease in adenosine triphosphate or creatine phosphate was found.

Three-dimensional representation and analysis of brain energy metabolism.

Quantitative autoradiography of brain glucose metabolism has been combined with digital image processing to represent the brain as a three-dimensional (3-D) reconstruction of brain energy use. Autoradiographs contain enormous amounts of potentially useful data, but conventional analyses, based on tedious manual methods, can sample and analyze only a small portion of this information. Computer 3-D reconstruction provides a mechanism for observing and analyzing all the data; therefore, a system of computer programs was developed for this purpose. The programs use digital imaging methods for image registration, superimpose whole brain data sets, and allow resampling of the 3-D data in arbitrary planes for pixel-by-pixel comparisons among multiple 3-D sets. These programs operate on the mathematical properties of the images alone, obviating the need for manual image alignment. Various statistical analyses can be applied to the data directly to study the patterns of metabolic changes in different experiments. The system is applied to data from experiments on the influence of injectable anesthetics on cerebral glucose metabolism.

Significance of aromatase activity in human breast cancer.

The significance of in vitro aromatization of [7 alpha-3H]testosterone to estradiol by human breast cancer has been investigated by correlation with (a) estrogen receptor activity and (b) clinical response to endocrine therapy. Evidence for estradiol synthesis was obtained in 66 and estrogen receptor activity in 81 of 110 tumors. Whereas most estrogen receptor-positive tumors synthesized estradiol, the majority of the receptor-negative tumors did not. This tendency for aromatization to be associated with estrogen receptor-positive tumors was statistically significant (p less than 0.005). Mean level of receptor was also significantly higher in tumors with aromatization than in tumors without estradiol synthesis (p less than 0.001). Forty patients with advanced breast cancer have been treated by endocrine therapy. There was a significant trend for tumors with aromatization to be associated with response to treatment (p less than 0.05), but the correlation was not absolute and may simply reflect the association between aromatase activity and estrogen receptors. Within the small subgroup of patients treated with aminoglutethimide or adrenalectomy, tumors with high aromatase activity responded whereas those without aromatization did not. Tumor estrogen biosynthesis may therefore be of clinical significance in selecting patients for treatments which remove sources of precursor for aromatization of inhibit aromatase activity itself.

Expression of the heat shock protein HSP27 in human ovarian cancer.

The relationship of the heat shock protein HSP27 in ovarian cancer to several biological and clinical parameters was investigated in a series of primary tumors and cell lines. Analysis of 72 primary tumors (54 malignant, 5 borderline, and 13 benign neoplasms) indicated that malignant tumors expressed higher HSP27 concentrations than benign tumors (median values, 0.56 versus 0.25 ng/microgram cytosolic protein; P = 0.032). Tumors from patients with advanced stage (stages II, III, or IV) disease contained significantly higher HSP27 concentrations than tumors from stage I patients (P = 0.018), and an HSP27 content >2.0 ng/microgram cytosolic protein was associated with reduced survival (P = 0.03). Tumors that had demonstrated progressive growth after chemotherapy had a significantly higher HSP27 content than tumors that were static or responsive (P = 0.022). These data indicate that HSP27 is associated with more aggressive malignant ovarian disease and with inherent resistance to chemotherapy. Concentrations of HSP27 were also correlated with indicators of estrogen sensitivity. Therefore, the HSP27 concentration correlated with the estrogen receptor (all tumors, P = 0.0014; malignant tumors only, P = 0.047) but not with the progesterone receptor concentration. Analysis of ovarian cancer cell lines in vitro and in vivo indicated that the HSP27 content was higher in cell lines that were estrogen receptor rich and whose growth was modulated by estrogen as compared with those that were not. Additionally, two estrogen receptor-rich ovarian carcinoma lines demonstrated a small but significant decrease in HSP27 levels in response to 17beta-estradiol in culture. These results suggest that HSP27 may help identify tumors responsive to estrogens.

Functionality of the progesterone receptor in ovarian cancer and its regulation by estrogen.

Here, we sought to obtain evidence that the progesterone receptor (PR) may be functional in ovarian cancer and regulated by estrogen. Megestrol acetate inhibited growth of the PR-positive PE04 ovarian carcinoma xenograft but not the PR-negative HOX 60 xenograft. PR concentration was higher in early-stage (I/II) tumors than in advanced-stage (III/IV) tumors (P = 0.007) and in tumors of endometrioid histology compared to other carcinoma subtypes (P = 0.009). Patients with a tumor PR concentration of >40 fmol/mg protein had significantly improved survival over those patients whose tumors contained <40 fmol/mg (P = 0.0007; log-rank). Evidence of PR regulation by estrogen was obtained by endocrine manipulation of the PE04 xenograft. PR content of PE04 xenografts fell from 145 to 7 fmol/mg protein in ovariectomized mice and was 2 fmol/mg in male mice. Administration of 17-beta-estradiol increased PR content to 745 fmol/mg. In primary ovarian carcinomas, PR was significantly associated with ER concentrations (P < 0.0001), suggesting regulation of PR levels by estrogen. This association was present for tumors of endometrioid histology (P < 0.0001) but not for those with serous histology (P = 0.31). These data point to the regulation of PR levels by estrogen in ovarian cancer and to a mediatory role for PR in the inhibition of growth induced by progestin.

Tumor receptor imaging: proceedings of the National Cancer Institute workshop, review of current work, and prospective for further investigations.

In February 1994, the National Cancer Institute held a workshop to evaluate the current and future role of emission tomographic imaging methods, positron emission tomography and single-photon emission computed tomography, in improving the accuracy of cancer diagnosis and the effectiveness of treatment and in elucidating basic aspects of human cancer biology. Reviews covered many of the receptor and transport systems for hormones and growth factors, as well as metabolic changes important in human cancer, and topical presentations reviewed the current status of receptor-based imaging in the most well-characterized systems: somatostatin receptor imaging of neuroendocrine tumors, estrogen receptor imaging of breast cancer, and epidermal growth factor receptor and tumor metabolic imaging. A critical analysis was made of the current research and of new directions for the future development and use of receptor-imaging methods in oncology. In each area, recommendations were made for further investigation, where emerging understanding of tumor cell biology and defined molecular targets might be combined with the methods of radiopharmaceutical design and evaluation, to develop new approaches to critical issues in the diagnosis, staging, and treatment of cancer through tumor receptor imaging.

Loss of heterozygosity at 11q22 correlates with low progesterone receptor content in epithelial ovarian cancer.

Forty-seven epithelial ovarian cancers were analyzed for loss of heterozygosity (LOH) at D11S35 (11q22), close to the progesterone receptor (PR) gene, and for tumoral estrogen receptor (ER) and PR content. Thirty-eight of 47 tumors were informative, and, of these, 14 exhibited LOH. There was a significant association (P = 0.014) between D11S35 LOH and low tumoral PR content. For all informative tumors, there was no correlation between ER and PR; however, exclusion of tumors with LOH from the informative series revealed a linear correlation between tumoral ER and PR (P = 0.013), and established ER (P = 0.025) and PR (P = 0.05) content as significant factors in relation to patient survival. Patients with ER-rich tumors with D11S35 LOH had particularly poor survival compared with ER-rich, D11S35 heterozygous, no loss patients (P = 0.014). Analysis of the same tumors using two other microsatellites, D11S935 (11p13) and NM23 (17q22), showed no statistically significant relationships, although there were nonsignificant trends for the correlation of ER and PR expression in informative tumors without allele loss at these loci. We propose that genomic structural alteration at or close to the PR gene locus has biological and clinical sequelae in ovarian cancer.

Bone metabolic activity measured with positron emission tomography and [18F]fluoride ion in renal osteodystrophy: correlation with bone histomorphometry.

We evaluated the bone metabolic activity in patients with renal osteodystrophy using positron emission tomography and [18F]fluoride ion. Eight patients had secondary hyperparathyroidism (HPT), and three had low-turnover bone disease. Eleven normal subjects were also studied, and three of the eight HPT patients were reevaluated after therapy. A rate constant (K) describing the net transport of [18F] fluoride ion into a bound compartment in bone was calculated using both a three-compartment model and Patlak graphical analysis. Values of K were compared with biochemical data and with histomorphometric indices. The results indicate that K is significantly higher (P < 0.01) in HPT patients than in normal subjects and patients with low-turnover bone disease. Values of K correlated with serum alkaline phosphatase (r = 0.81) and PTH (r = 0.93) levels and with histomorphometric indices of bone formation rate (r = 0.84, P < 0.01) and eroded perimeter (r = 0.77, P < 0.05). Values of K decreased by 40 and 30%, respectively, in two patients who underwent parathyroidectomy and medical therapy. Positron emission tomography studies of bone using [18F]fluoride ion can differentiate low turnover from high turnover lesions of renal osteodystrophy and provide quantitative estimates of bone cell activity that correlate with histomorphometric data.

Effects of temporal sampling, glucose metabolic rates, and disruptions of the blood-brain barrier on the FDG model with and without a vascular compartment: studies in human brain tumors with PET.

The addition of a cerebral blood volume (CBV) compartment in the [18F]2-fluoro-2-deoxy-D-glucose (FDG) model produces estimates of local CBV simultaneously with glucose metabolic rates when kinetic FDG studies are performed. We investigated the influence of this term upon CMRglc values in a series of brain tumor patients and found that significant overestimations of CMRglc are possible if the effect of CBV upon the model is ignored. The magnitude of this potential overestimation is directly related to the absolute value of CBV locally and inversely related to the CMRglc value. The kinetic estimates also permitted an evaluation of the FDG model in an environment with a variable disruption of the blood-brain barrier. Incorporating the vascular compartment in the FDG model also frequently improved the statistical accuracy of model fits to tissue kinetic data. The sampling requirements for this model configuration were also investigated in a series of computer simulations.

Single-scan Bayes estimation of cerebral glucose metabolic rate: comparison with non-Bayes single-scan methods using FDG PET scans in stroke.

Three single-scan (SS) methods are currently available for estimating the local cerebral metabolic rate of glucose (LCMRG) from F-18 deoxyglucose (FDG) positron emission tomography (PET) scan data: SS(SPH), named for Sokoloff, Phelps, and Huang; SS(B), named for Brooks; and SS(H), named for Hutchins and Holden et al. All three of these SS methods make use of prior information in the form of mean values of rate constants from the normal population. We have developed a Bayes estimation (BE) method that uses prior information in the form of rate constant means, variances, and correlations in both the normal and ischemic tissue populations. The BE method selects, based only on the data, whether the LCMRG estimate should be computed using prior information from normal or ischemic tissue. The ability of BE to make this selection gives it an advantage over the other methods. The BE method can be used as a SS method or can use any number of PET scans. We conducted Monte Carlo studies comparing BE as a SS method with the other SS methods, all using a single scan at 60 min. We found SS(H) to be strongly superior to SS(SPH) and SS(B), and we found BE to be definitely superior to SS(H).

Effect of ischemia on quantification of local cerebral glucose metabolic rate in man.

The model for quantifying local cerebral glucose metabolic rates originally developed by Sokoloff et al. and modified by Phelps. Huang and co-workers was applied to humans with cerebral ischemia (i.e., stroke). Rate constants for fluorodeoxyglucose were measured in ischemic and nonischemic regions with position computed tomography. Using measured rate constants for ischemia, the model generate more accurate estimates of local cerebral glucose metabolism as compared to the use constants from normal young adults, because the local metabolic rate is significantly underestimated, and temporal instability of the model is observed when normal values are applied to ischemic regions. A method was also developed to test the stability of the local lumped constant. The estimates of the lumped constant showed no or only small variations between ischemic and nonischemic types. Thus, errors introduced in the calculated local cerebral glucose metabolism by inappropriate rate constants appear to be more significant than those caused by any potential change in the lumped constant in ischemia.

A study of the single compartment tracer kinetic model for the measurement of local cerebral blood flow using 15O-water and positron emission tomography.

The effects of varying the data collection time on the calculation of cerebral blood flow and distribution volume via the integrated projection technique were studied in four human subjects. The significance of these results in terms of the limitations of the single compartment model for 15O-water was explored using computer simulations. The simulations helped to account for causes for the variations seen in blood flow and distribution volume as a function of the data collection time. Two different compartmental models were explored for better quantitation of blood flow and distribution volume.

In vivo cerebral protein synthesis rates with leucyl-transfer RNA used as a precursor pool: determination of biochemical parameters to structure tracer kinetic models for positron emission tomography.

Leucine oxidation and incorporation into proteins were examined in the in vivo rat brain to determine rates and compartmentation of these processes for the purpose of structuring mathematical compartmental models for the noninvasive estimation of in vivo human cerebral protein synthesis rates (CPSR) using positron emission tomography (PET). Leucine specific activity (SA) in arterial plasma and intracellular free amino acids, leucyl-tRNA, alpha-ketoisocaproic acid (KIC), and protein were determined in whole brain of the adult rat during the first 35 min after intravenous bolus injection of L-[1-14C]leucine. Incorporation of leucine into proteins accounted for 90% of total brain radioactivity at 35 min. The lack of [14C]KIC buildup indicates that leucine oxidation in brain is transaminase limited. Characteristic specific activities were maximal between 0 to 2 min after bolus injection with subsequent decline following the pattern: plasma leucine greater than or equal to leucyl-tRNA approximately KIC greater than intracellular leucine. The time integral of leucine SA in plasma was about four times that of tissue leucine and twice those of leucyl-tRNA and KIC, indicating the existence of free leucine, leucyl-tRNA, and KIC tissue compartments, communicating directly with plasma, and separate secondary free leucine, leucyl-tRNA, and KIC tissue compartments originating in unlabeled leucine from proteolysis. Therefore, a relatively simple model configuration based on the key assumptions that (a) protein incorporation and catabolism proceed from a precursor pool communicating with the plasma space, and (b) leucine catabolism is transaminase limited is justified for the in vivo assessment of CPSR from exogenous leucine sources using PET in humans.

Cerebral glucose metabolism as a function of age in man: influence of the rate constants in the fluorodeoxyglucose method.

Measurement of the local cerebral metabolic rate of glucose (LCMRGlc) with the fluorodeoxyglucose (FDG) method requires the utilization of appropriate values for the rate constants of the transport and phosphorylation processes. We measured these rate constants as a function of age to determine whether a decline in LCMRGlc as a function of age, in prior studies with the FDG method, actually represents changes in the rate constants. We found that measurements of LCMRGlc are not significantly affected by changes in rate constants as a function of aging, and that LCMRGlc did not change significantly with age.

Estimation of local cerebral protein synthesis rates with L-[1-11C]leucine and PET: methods, model, and results in animals and humans.

We have estimated the cerebral protein synthesis rates (CPSR) in a series of normal human volunteers and monkeys using L-[1-11C]leucine and positron emission tomography (PET) using a three-compartment model. The model structure, consisting of a tissue precursor, metabolite, and protein compartment, was validated with biochemical assay data obtained in rat studies. The CPSR values estimated in human hemispheres of about 0.5 nmol/min/g agree well with hemispheric estimates in monkeys. The sampling requirements (input function and scanning sequence) for accurate estimates of model parameters were investigated in a series of computer simulation studies.

Models for in vivo kinetic interactions of dopamine D2-neuroreceptors and 3-(2'-[18F]fluoroethyl)spiperone examined with positron emission tomography.

The in vivo tracer kinetics of 3-(2'-[18F]fluoroethyl)spiperone (FESP) in the caudate/striatum and cerebellar regions of the human and monkey brain were studied with positron emission tomography (PET). The minimal model configuration that can describe the kinetics was determined statistically. Three two-compartment model configurations were found to be suitable for describing the kinetics in caudate/striatum and cerebellum: (1) a nonlinear model (five parameters) applicable to studies using nontracer (partially saturating) quantities of FESP in monkey striatum, (2) a linear four-parameter model applicable to the caudate/striatal and cerebellar kinetics in human and monkey studies with tracer quantities of FESP, and (3) a linear three-parameter model derived from the four-parameter model by assuming irreversible binding applicable to tracer studies of the human caudate. In the human studies, when the caudate kinetics (n = 4) were fit by model 2 (with four parameters), the value of the in vivo ligand dissociation constant kd was found to be 0.0015 +/- 0.0032/min. The three-parameter model (model 3) was found to fit the data equally well: this model is equivalent to model 2 with kd set to zero. In the monkey studies, it was found that for short (90 min) studies using tracer quantities of FESP, model 2 fit the striatal kinetics better than model 3. The parameters estimated using model 2 (four parameters) were in better agreement with those estimated by the nonlinear model (model 1) than those estimated using model 3 (three parameters). The use of a graphical approach gives estimates of the plasma-tissue fractional transport rate constant K1 and the net uptake constant K3 comparable to estimates using model 3 for both human and monkey studies.

A double-injection technique for in vivo measurement of dopamine D2-receptor density in monkeys with 3-(2'-[18F]fluoroethyl)spiperone and dynamic positron emission tomography.

Dopamine D2-receptor density in striatum of monkey was measured with 3-(2'-[18F]fluoroethyl)spiperone (FESP) and dynamic positron emission tomography (PET), using a double-injection technique. A first bolus of high specific activity (SA) FESP (5 mCi; approximately equal to 1 Ci/mumol) was injected i.v.; 90 min later, a second bolus of lower SA FESP (5 mCi; approximately equal to 0.04 Ci/mumol) was injected. A dynamic PET study was performed to measure the kinetics of FESP in striatum over 180 min, and the metabolite-corrected concentration of FESP in plasma as a function of time was obtained from arterial blood samples. A nonlinear compartmental model that took into account the saturability of the receptor binding was used to describe the kinetics of FESP in striatum. Model parameters were estimated by regression with a constraint based on information about the equilibrium dissociation constant of the ligand-receptor binding. Dopamine D2-receptor density in striatum was estimated to be 25.9 +/- 12.7 pmol/g in seven Macaca nemestrina monkeys. The method does not require the use of cerebellum as a reference tissue region and an estimate of dopamine D2-receptor density can be obtained from a single study.

A kinetic evaluation of blood-brain barrier permeability in human brain tumors with [68Ga]EDTA and positron computed tomography.

Twelve patients with primary and metastatic brain tumors were evaluated with [68Ga]ethylenediaminetetraacetate (EDTA) and positron computed tomography. Using a two-compartment tracer kinetic model, forward (K1) and reverse (k2) rate constants for molecular diffusion across the blood-brain barrier (BBB) were obtained and averaged 0.0029 +/- 0.0016 (mean +/- SD) ml/min/g for K1 and 0.0310 +/- 0.0156 min-1 for k2. Most tracer kinetic models are based on the assumption that tissue radioactivity contains no vascular component or require independent measures of cerebral blood volume (CBV) which are then subtracted from the measure tissue activity. The model in this work differs from that approach by assuming a vascular compartment in the tissue kinetic data. This vascular parameter is estimated from sequential measurements of activity concentrations in regions with an intact BBB or from measurements of 68Ga concentrations in the plasma (the input function). Thus, this approach does not require the assumption of a zero vascular contribution, does not require a separate measurement of CBV, and uses the criteria of constrained estimation to provide estimates of the local CBV and molecular diffusion through the BBB. Estimates of the relative CBV of the lesions in four studies (three subjects) with [68Ga]EDTA correlated well with those obtained with the C15O hemoglobin technique (correlation coefficient of 0.97).

Regional brain glucose metabolism is altered during rapid eye movement sleep in the cat: a preliminary study.

Glucose utilization was measured in 74 brain regions of the cat during states of wakefulness or rapid eye movement (REM) sleep. These data were obtained from intact, unanesthetized animals which were instrumented for objectively measuring states of consciousness. Through a chronically implanted intravenous catheter, the cats received 250 microCi of magnitude of 6-14C glucose during REM sleep (N = 3) or during wakefulness (N = 3). After spending approximately 8 min in REM sleep or in quiet wakefulness, the cats were administered a lethal dose of barbiturate and the brains were removed and processed for autoradiography. The results revealed site-specific changes in glucose metabolism during REM sleep. Significant alterations in glucose use occurred in the thalamus, the limbic system, and specific regions of the pontine reticular formation. These data demonstrate for the first time that during states comprised entirely of REM sleep there are anatomically specific changes in cerebral glucose metabolism. The majority of brain regions exhibiting REM sleep-dependent changes in glucose metabolism either overlapped with regions known to contain cholinergic cell bodies, or with areas that receive prominent cholinergic input.