Paul D. Crane

Dose dependent reduction of glucose utilization by pentobarbital in rat brain.

A new method of determining the rate of glucose utilization in brain regions of individual rats has been used to measure the dose dependency of the reduction of the metabolic activity of the cerebral cortex by pentobarbital. Cerebral cortical glucose utilization is depressed to a basal level of 44% of the control rate when cerebral pentobarbital levels exceed 50 μg per g of tissue. The major portion of this effect occurs between the cerebral pentobarbital range of 10-20 μg per g, which can be achieved by 1/5 to 1/10 the normal anesthetic intraperitoneal dosage. If a depression of brain metabolism is responsible for the previously reported protection of the brain from ischemic damage, these data suggest a substantial reduction of brain metabolic rate is achieved in the rat at a barbiturate dosage which may be therapeutically relevant in the human after acute brain ischemia.

Kinetics of transport and phosphorylation of 2-fluoro-2-deoxy-D-glucose in rat brain.

Abstract: The kinetics of transport across the blood‐brain barrier and metabolism in brain (hemisphere) of [14C]2‐fluoro‐2‐deoxy‐d‐glucose (FDG) were compared to that of [3H]2‐deoxy‐d‐glucose (DG) and d‐glucose in the pentobarbital‐anesthetized adult rat. Saturation kinetics of transport were measured with the brain uptake index (BUI) method. The BUI for FDG was 54.3 ± 5.6. Nonlinear regression analysis gave a Km of 6.9 ± 1.1 mM and a Vmax of 1.70 ± 0.32 μmol/min/g. The K1 for glucose inhibition of FDG transport was 10.7 ± 4.4 mM. The kinetic constants of influx (k1) and efflux (K2) for FDG were calculated from the Km, Vmax, and glucose concentrations of the hemisphere and plasma (2.3 ± 0.2 μmol/g and 9.9 ± 0.4 mM, respectively). The transport coefficient (k1 FDG/k1glucose) was 1.67 ± 0.07 and the phosphorylation constant was 0.55 ± 0.16. The predicted lumped constant for FDG was 0.89, whereas the measured hexose utilization index for FDG was 0.85 ± 0.16. Conclusion: The value for the lumped constant can be predicted on the basis of the known kinetic constants of FDG and glucose transport and metabolism, as well as brain and plasma glucose levels. Knowledge of the lumped constant is crucial in interpreting data obtained from 18FDG analysis of regional glucose utilization in human brain in pathological states. We propose that the lumped constant will rise to a maximum equal to the transport coefficient for FDG under conditions of transport limitation (hypoglycemia) or elevated glycolysis (ischemia, seizures), and will fall to a minimum equal to the phosphorylation coefficient during phosphorylation limitation (extreme hyperglycemia).

Kinetics of Regional Blood–Brain Barrier Transport and Brain Phosphorylation of Glucose and 2-Deoxyglucose in the Barbiturate–Anesthetized Rat

Abstract: Recent studies indicate the lumped constant (LC), which defines the relative rates of brain utilization of glucose and 2‐deoxyglucose (2‐DG), doubles to values > 1.0 under conditions of hypoglycemia. Since changes in the LC should be predictable given the kinetic parameters of blood‐brain barrier (BBB) transport and brain phosphorylation of glucose and 2‐DG, the present studies were designed to measure the necessary kinetic parameters. The carotid injection technique was used to determine cerebral blood flow and the Km, Vmax, and KD of glucose and 2‐DG transport through the BBB in seven brain regions in rats anesthetized with 50 mg/kg i.p. pentobarbital. Regional glucose transport through the BBB was characterized by an average Km= 6.3 mm, average Vmax= 0.53 μmol min−1g−1, and average KD= 0.022 ml min−1g−1. The nonsaturable route of transport of glucose represented on the average 40% of the total glucose influx into brain regions at an arterial glucose concentration of 10 mm. In addition, the rate constants of phosphorylation of glucose and 2‐DG were measured for each region. Substitutions of the measured kinetic parameters for sugar transport and phosphorylation into equations defining the LC confirm the observation that the LC would be expected to vary under extreme conditions such as hypoglycemia and to exceed values of 1.0 under these conditions.

The Interaction of Transport and Metabolism on Brain Glucose Utilization: A Reevaluation of the Lumped Constant

Abstract: The relative cerebral cortical metabolism of glucose (GLU) and 2‐deoxy‐D‐glucose (DG) was measured in vivo in control and insulin‐treated hypoglycemic rats. The ratio of the utilization rate constants for the two hexoses, i.e., KDG/KCLU is defined as the Hexose Utilization Index (HUI). The HUI was found to be invariant in rats whose cerebral glucose content exceeded 1 μmo1.g−1 wet weight (HUI = 0.48 ± 0.07). Severe hypoglycemia (plasma glucose <2 mM) effected a shift in the HUI to 1.04 ± 0.21. The results are consistent with a model in which the interpretation of the HUI is determined by the rate of transport into brain, or subsequent phosphorylation, as the rate‐limiting step for hexose utilization.

Nomogram for 2-Deoxyglucose Lumped Constant for Rat Brain Cortex

The quantitation of local cerebral metabolic rate of glucose with the 2-deoxyglucose technique of Sokoloff requires the use of a correction factor, or lumped constant. We have shown previously (Pardridge et al., 1982) that a simple model may be formulated to predict changes in the lumped constant that occur due to alterations in the distribution of glucose and 2-deoxyglucose in brain. Given experimentally observed values for brain and plasma glucose concentrations, the 2-deoxyglucose lumped constant may be determined from a nomogram constructed from knowledge of the blood–brain barrier transport constants (KM, Vmax, KD) for glucose and for 2-deoxyglucose. However, the nomogram is constructed from transport constants determined in the barbiturate-anesthetized state. The applicability of the nomogram to other physiologic states was examined in the present studies. Large changes in blood–brain barrier hexose transport constants do not appreciably alter the shape of the nomogram, if the changes in KM or Vmax for glucose or for 2-deoxyglucose are the same. Moreover, glucose and 2-deoxyglucose are both transported by the same hexose carrier, and selective changes in the transport of only one hexose have not been reported. Therefore, it is probable that the nomogram constructed from transport constants measured under barbiturate anesthesia is useful in predicting the lumped constant in a variety of physiologic states.

Cerebral Cortical Glucose Utilization in the Conscious Rat: Evidence for a Circadian Rhythm

Abstract: The presence of a circadian rhythm of glucose utilization was demonstrated in vivo in rat cerebral cortex. The activity pattern of the rats, living in a controlled lighting regimen with lights on from 7 a.m. to 7 p. m., appeared to coincide with the rate of glucose consumption in the brain. The rate of utilization was measured at 3‐h intervals throughout the day and was found to fall from a maximum at 3 a.m. of 0.98 ± 0.13 μmol min−1 g−1 to a minimum of 0.70 ± 0.08 μmol min−1 g−1 at 3 p. m. Brain glucose also varied with time and its fluctuating level weakly correlated with its rate of utilization. Animals entrained on a 5‐h (4: 30‐9: 30 p. m.) feeding schedule had a similar circadian rhythm, with only a slight increase in amplitude. Reversal of the light cycle caused a disruption in the normal rhythm, but utilization still varied significantly with time of day. The results both indicate the potential error that can be encountered in experiments done at different times of the day and stress the need for awareness of time of day as a factor in measurements of alterations of metabolic rate in the brain.

Two-Day Starvation Does Not Alter the Kinetics of Blood-Brain Barrier Transport and Phosphorylation of Glucose in Rat Brain

The blood-brain barrier (BBB) transport and brain phosphorylation of glucose were assessed in conscious rats subjected to 2 days of starvation. Although plasma glucose decreased, no significant changes in brain blood flow, BBB glucose transport, or 2-deoxy-d-glucose phosphorylation were observed. The data suggest that adaptive changes of brain glucose metabolism previously observed in starvation are located beyond the initial steps of brain entry and phosphorylation.

Blood-Brain Barrier Transport of Basic Amino Acids Is Selectively Inhibited at Low pH

Abstract: The transport of amino acids across the blood‐brain barrier was measured with the single‐pass carotid injection method. The pH of the injected bolus varied between 4.5 and 8.5. Arginine and lysine uptakes were inhibited 24% at pH 5.5 and 59% at pH 4.5. The uptakes of 2‐aminobicyclo (2,2,1) heptane‐2‐carboxylic acid and phenylalanine were unaffected at this pH. There were also no changes observed in choline, glucose, or butanol transport. The Ki of arginine transport inhibition by H+ was 2.4 ± 0.5 μM; i.e., pH 5.6 ± 0.1. No change with pH occurred in the Km of arginine transport, while a significant decrease (p < 0.01) was observed in the Vmax (10.2 ± 2.3 nmol min−1 and 5.6 ± 2.3 nmol min−1 g−1 at pH 7.5 and pH 5.5, respectively). This noncompetitive inhibition was found to be transient as arginine uptake at pH 7.5; it was measured by carotid injection 30 sec following a previous bolus which was buffered to pH 4.5, and was not significantly different from the control. This selective inhibition of the blood‐brain barrier basic amino acid carrier demonstrates the advantage of the carotid injection approach in exposing the capillary exchange site to extreme alterations in chemical composition which could not be tolerated systemically.

Rapid, transient drop in brain glucose after intravenous phloretin or 3-0-methyl-D-glucose.

Rats were injected intravenously with either phloretin (100 mg/kg) or 3-0-methyl glucose (2 g/kg) to reduce the carrier-mediated flux of glucose into brain. Plasma glucose and brain free glucose (BFG), lactate, and glycogen were measured over a 16 min time course. Injection of these substances caused a rapid drop in BFG to 60% of control at one minute and a minimum (50% of control values) at 4 min., followed by a gradual rise to control levels at 16 min. While plasma glucose fell, and then increased after injection, brain lactate and glycogen content was unaffected. Repeated injections of phloretin eventually caused a drop in brain glycogen; but with either competitor, BFG never fell below 50% of normal values. The i.v. injection of the glucose analog, 3-0-methyl glucose (the less toxic of the two drugs) is proposed as a possible means of cutting off the potentially hazardous supply of blood glucose to the postischemic brain.

Reduction in Brain Glucose Utilization Rate after Tryptophol (3-Indole Ethanol) Treatment

Abstract: 3‐Indole ethanol has been recently identified as the hypnotic agent in trypanosomal sleeping sickness, and because it is formed in vivo after ethanol or disulfiram treatment, it is also associated with the study of alcoholism. When administered intraperitoneally to rats (250 mg/kg) tryptophol induced a sleep‐like state that lasted less than an hour (no righting reflex was apparent 2 min after injection, but it returned at 11 min in bovine serum albumin solution, and 47 min in 40% ethanol solution). In ethanol solutions, tryptophol reduced brain cortical glucose utilization by 55% to the basal brain metabolic rate, and this effect lasted less than 1 h. Synergistic effects of tryptophol and ethanol were suggested by the observation that in albumin solution, tryptophol reduced brain glucose utilization by 35%, but a normal rate was not observed until 2 h postinjection.