Feasibility of imaging pentose cycle glucose metabolism in gliomas with PET: studies in rat brain tumor models.

UNLABELLED: The feasibility of imaging pentose cycle (PC) glucose utilization in human gliomas with PET was explored in two rat glioma models by means of glucose radiolabeled in either the carbon-1 (C-1) or carbon-6 (C-6) position. METHODS: In vitro, monolayers of T-36B-10 glioma, tissue slices of intracerebral glioma grafts or slices of normal brain were fed [1-14C]glucose or [6-14C]glucose, and the generated [14C]CO2 was trapped to quantitate the ratio of [14C]CO2 from 14C-1 versus 14C-6. In vivo, rats bearing grafts of either T-36B-10 or T-C6 rat gliomas at six subcutaneous sites received simultaneous intravenous injections of either [1-11C]glucose and [6-14C]glucose, or [1-14C]glucose and [6-11C]glucose. Tumors were excised between 5 and 55 min postinjection to quantify tracer uptake while arterial plasma was collected to derive time-activity input curves. RESULTS: In vitro, the C-1/C-6 ratio for CO2 production from T-36B-10 monolayers was 8.8 +/- 0.4 (s.d.), in glioma slices it was 6.1 +/- 2.1 and in normal brain slices it was 1.1 +/- 0.7. PC metabolism in T-36B-10 was 1.8% +/- 0.5 of total glucose utilization. In vivo, tumor radioactivity levels normalized by plasma isotopic glucose levels showed that retained C-1 relative to C-6 radiolabeled glucose was significantly lower in both gliomas, 4.9% lower in T-36B-10 (p < 0.01) and 4.7% lower in T-C6 (p < 0.01). In an additional group of rats bearing T-36B-10 gliomas and exposed to 10 Gy of 137Cs irradiation 4 hr before isotope injection, the C-1 level was 5.6% lower than that for C-6 (p < 0.05). These results were analyzed with a model of glucose metabolism that simultaneously optimized parameters for C-1 and C-6 glucose kinetics by simulating the C-1 and C-6 tumor time-activity curves. The rate constant for loss of radiolabeled carbon from the tumors, k4, was higher for C-1 than for C-6 in all groups of rats (19% higher for T-36B-10 unirradiated, 32% for T-36B-10 irradiated and 32% for T-C6 unirradiated). CONCLUSION: Mathematical modeling, Monte Carlo simulations and construction of receiver-operator-characteristic curves show that if human gliomas have a similar fractional use of the PC, it should be measurable with PET using sequential studies with [1-11C]glucose and [6-11C]glucose.

Deoxyglucose lumped constant estimated in a transplanted rat astrocytic glioma by the hexose utilization index.

The lumped constant (LC) for calculating the regional glucose (glc) metabolic rate by the deoxyglucose (DG) method was estimated in a transplanted rat glioma and normal rat brain. First, the hexose utilization index (HUI) was measured at 1.5, 3.0, and 4.5 min in right hemisphere glioma implants and uninvolved contralateral hemisphere following bolus intravenous injections of [3H]DG and [14C]glucose. At these times, the glioma HUI values were 0.639, 0.732, and 0.712, respectively, and the coordinate left hemisphere values were 0.432, 0.449, and 0.418. Second, the volumes of distribution of DG and glucose were determined to be 0.436 and 0.235 in glioma implants and 0.402 and 0.237 in left hemisphere, respectively. Third, following simultaneous intracarotid injections of [3H]DG and [14C]glucose, the ratio K1/K1 was 1.1 in glioma grafts and 1.3 in left hemisphere. With these values for HUI, volume of distribution, and K1 ratio, the LC in this rat glioma was estimated to be 2.1 times higher than the left hemisphere LC (p less than 0.02). These results suggest that measurement of brain tumor CMRglc using a normal brain LC may significantly overestimate the true tumor CMRglc.

Determination of the deoxyglucose and glucose phosphorylation ratio and the lumped constant in rat brain and a transplantable rat glioma.

Mitochondrially bound hexokinase (ATP-D-hexose-6-phosphotransferase; EC 2.7.1.1) was dissociatively extracted from normal rat brains and intracerebral and subcutaneous implants of the 36B-10 glioma. At least 70% of the total hexokinase enzyme activity in normal and glioma tissue was associated with the mitochondrial fraction. Purification of the crude tissue extracts by ion-exchange and affinity chromatography followed by analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a successive purification of the enzyme to homogeneity with a molecular size of 98 kilodaltons. Enzyme kinetics with glucose or 2-deoxyglucose (2-DG) as the substrate were measured spectrophotometrically by coupling the appropriate reactions to either NADPH or NAD+ formation. The Km of hexokinase with glucose as the substrate in the intracerebral glioma (0.138 mM) and subcutaneous glioma (0.183 mM) tissues was 2.1-2.7-fold higher than that observed in normal brain tissue (0.067 mM) (p less than 0.001). No significant differences were observed in the Km for hexokinase with 2-DG as the substrate in the glioma and normal brain tissue. The phosphorylation ratio for normal brain was 0.320 and was increased in the intracerebral glioma to 0.694 and in the subcutaneous glioma to 0.519. The ratios of deoxyglucose and glucose volumes of distribution in normal brain and intracerebral glioma tissues were 1.70 and 1.85, respectively. The lumped constants calculated directly from the phosphorylation ratios and the volumes of distribution of deoxyglucose and glucose were 0.517 in normal brain and 1.168 in intracerebral glioma. Our results indicate the lumped constant is increased 2.26-fold in intracerebral glioma compared with normal brain.

Deoxyglucose kinetics in a rat brain tumor.

Accurate quantitation of local glucose metabolic rates (LMRglc) of abnormal tissues such as brain tumors with the 2-deoxyglucose (DG) method requires knowledge of the tissue rate constants and lumped constant. The deoxyglucose rate constants were measured in an experimental intracerebral glioma in 24 awake rats with a dual tracer [(3H)-DG and (14C)-DG] method. Tissue time points were obtained at 2, 5, 10, 18, 30, 60, 90, and 180 min after injection by decapitation and liquid scintillation counting. Blood samples were obtained at 1 min intervals initially and at longer intervals later. The rate constants were estimated with parameter estimation. LMRglc was calculated from the rate constants, assuming a lumped constant of 0.5. K1 for normal cerebrum was found to be 0.258 ml/g/min, and k2-k4 were 0.406, 0.075, and 0.0103 min-1; LMRglc = 65.1 mumol/100 g/min. The corresponding values for the glioma were 0.108, 0.126, 0.040, and 0.0019 with LMRglc = 41.7. The considerably lower k4 in the glioma was reflected in persistent higher activity in the glioma at longer times. Thus, tissue activity alone cannot be used to assess relative glucose metabolic rates in abnormal tissues such as gliomas, particularly at late times after injection.

Blood flow changes following 137Cs irradiation in a rat glioma model.

Blood flow changes in response to 20 Gy 137Cs whole brain irradiation were measured with quantitative autoradiography of [14C]iodoantipyrine (IAP) in intracerebral grafts of the 36B-10 rat glioma, the brain around tumor (BAT), the contralateral corpus callosum, and the contralateral cerebral cortex. Irradiations were delivered on Day 14 post-transplantation, and measurements of flow (F) were performed with IAP on Day 15 or Day 16. Mean values of F were determined in individual tumors and in treatment groups. In 15- and 16-day-old unirradiated control tumors, the group mean F was 0.31 ml.g-1.min-1. In both 15- and 16-day-old tumor groups irradiated on Day 14 (Day 1 and 2 postirradiation tumors) the mean F for each day's group was 0.52 ml.g-1.min-1, 68% higher than the control (P less than 0.01). Flow in the BAT and the contralateral corpus callosum similarly was increased at these times (P less than 0.01). Flow in the contralateral cerebral cortex was 1.1, 1.5, and 1.3 ml.g-1.min-1 in the control, 1 day postirradiated, and 2 day postirradiated groups, respectively, but these increases were not significantly different from the control. These data indicate that flow increases in the intracerebral gliomas as well as in normal brain regions during the 2 days following 20 Gy irradiation. Changes such as these following radiotherapy may have important effects on the bioavailability of chemotherapeutic drugs.

Regional blood-to-tissue transport in an irradiated rat glioma model.

To assess vascular permeability in intracerebral grafts of the 36B-10, F-344 rat glioma following 20 Gy 137Cs whole brain irradiation, the blood-to-tissue transport constant, K, of [14C]-alpha-aminoisobutyric acid (AIB) was measured with quantitative autoradiography. Mean, 90th percentile, and 95th percentile values of K were determined in individual tumors and in treatment groups. In 15-day-old unirradiated control tumors, mean, 90th percentile, and 95th percentile values of K were, respectively, 11.3, 18.4, and 20.8 ml kg-1 min-1. In 15-day-old tumors irradiated on Day 14 (Day 1 postirradiation tumors) the K values were 5.9, 9.4, and 10.4, all of which were significantly less than the respective control values (P less than 0.01). In 16-day-old tumors irradiated on Day 14 (Day 2 postirradiation tumors), the K values were 10.8, 15.0, and 16.0, respectively, none of which was significantly different from control tumors. Mean K values for Day 2 vs Day 1 postirradiation tumors (10.8 vs 5.9) yielded P less than 0.05, but the 90th percentile and 95th percentile values for Day 2 vs Day 1 yielded 0.05 less than P less than 0.10. Separate experiments measured AIB and 86RbCl uptake in 36B-10 cells in vitro 1 and 2 days following 20 Gy irradiation to assess whether this radiation dose reduced the capacity of tumor cells to trap AIB or Rb+. Irradiation did not reduce the accumulation of either tracer, but rather was associated with an increased accumulation of AIB. Therefore, the AIB transport data suggest that vascular permeability and/or surface area decreases significantly in the day following 20 Gy irradiation and that this decrease reverses by the second day following irradiation.

Blood flow in an experimental rat brain tumor by tissue equilibration and indicator fractionation.

The tissue equilibration technique (Kety) was compared with the indicator fractionation technique for the measurement of blood flow to normal brain and an experimental brain tumor in the rat. The tumor was a cloned astrocytic glioma implanted in the cerebral hemisphere of F-344 rats. I-125 Iodoantipyrine, using a rising infusion for one minute, was used for the tissue equilibration technique. C-14 butanol, injected as a bolus 8 seconds before sacrifice, was used for the indicator fractionation technique. Samples were assayed using liquid scintillation counting and the iodoantipyrine results were regressed against the butanol results. For normal tissue R = 0.832, SEE = 0.115 ml/g/min, and Slope = 0.626. For tumor R = 0.796, SEE = 0.070 ml/g/min, and Slope = 0.441. The iodoantipyrine tissue/blood partition coefficient for normal hemisphere (gray and white matter) was 0.861 +/-0.037 (SD) and for tumor was 0.876 +/-0.042. The indicator fractionation technique with C-14 butanol underestimated blood flow in a consistent manner, probably because of incomplete extraction, early washout of activity from tissue and from evaporation of butanol during processing. Our experiments revealed no differences between tumor and normal brain tissue that might invalidate the comparison of iodoantipyrine blood flow results in brain tumors and surrounding normal brain.

High-speed bubble-segmented blood sampler for the rat.

An arterial blood sampler for use in the conscious rat is described. With this apparatus it is possible to obtain small (10 microliter) whole-blood or plasma samples as frequently as 1/s and to derive accurate arterial time-concentration curves in the first 60-120 s after compounds are injected for regional blood flow or pharmacokinetic measurements. The blood is withdrawn from an implanted arterial catheter into polyethylene tubing at a constant rate while bubbles are introduced at regular intervals via a side channel into the column of blood. Although some dispersion of blood samples occurs as the tubing is traversed, this can be mathematically corrected. However, correction is unnecessary if the information of interest is the area under the time-concentration curve.