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Downloaded fromPENTOBARBITAL AND GLUCOSE UTILIZATION/Crane et al. 13permits the calculation of the rate of brain regional glucose utilization from individual animals, independent of measurements of blood flow and blood brain barrier transport. The additional measurement of brain pentobarbital levels by gas chromatography (GC) has enabled us to report the correlation between brain level of pentobarbital and the rate of glucose utilization. Methods Injection of Barbiturate and Radiolabeled 2-DOGWistar rats (200-250 g), of either sex, were injected intraperitoneally with 0.5 ml of Ringer's solution containing variable doses of sodium pentobarbital. After 10 min, the rats were placed in a restraining device (Gerling-Moore, Palo Alto) and their tails warmed for 30 sec in warm water. A 23 gauge needle was inserted into the dilated tail vein and ten fid of ( 3 H(G)) 2-DOG (sp. act., 10 Ci/mmole) in 0.25 ml of Ringers solution injected, followed by a 0.1 ml wash of Ringers solution. Two minutes later, 2 j*Ci of 2-(l-14 C)-DOG (sp. act., 54 mCi/mmole) in 0.25 ml of Ringers solution were injected through the same needle, followed by 0.1 ml of Ringers solution. Two minutes after the 14 C injection, the rats were killed by intense microwave irradiation.9 Cardiac blood was collected into a heparinized syringe for liquid scintillation counting. The time course of 2-DOG uptake and phosphorylation was determined by single ( 3 H) 2-DOG injections, followed by microwaving at varying times after injection. The brains were excised and stored at -20°C for subsequent analysis. Biochemical And Radiochemical AnalysisThe brains were thawed and the cerebral cortices were weighed and placed in Ten Broeck homogenizers with a measured amount of distilled water (=1.6 ml water -cortex wet wt X 0.8) and 50 #g of sodium secobarbital in 4 ml of methanol to serve as an internal standard in GC. Using a modified procedure of Bligh and Dyer, 10 the cortices were homogenized, 2 ml of chloroform were added, and the tissues were rehomogenized. The homogenates were centrifuged, the supernates saved, and the pellets rehomogenized in 7 ml of chloroform-methanol-water (1:2:0.8) and centrifuged. The supernates were combined, 5 ml of chloroform and 5 ml of water added and the aqueous and organic phases separated after centrifugation. The interfaces were washed three times with methanol-water (1:1), and the washes combined with the aqueous phases.The aqueous phases were further fractionated into neutral (containing glucose and 2-DOG) and anionic (containing 2-deoxy-D-glucose-6-phosphate (2-DOGP)) fractions by ion exchange chromatography on DEAE Sephadex A25 columns (1 ml bed volume). The neutral fractions were recovered unbound from the column, while the anionic fractions were eluted with 0.5M pyridine acetate, pH 5.0. Aliquots were removed from both fractions for simultaneous 3 H and 14 C liquid scintillation counting. The neutral fractions were dried by rotary evaporation and taken up in a 0.1M Tris-HCl (buffered to pH 7.5) and 0.02% sodium azide solution f...

This study investigated the mechanism of myocardial retention of technetium-99m-sestamibi. 99mTc-sestamibi was injected intravenously into guinea pigs, and the myocardium was homogenized and fractionated by differential centrifugation. More than 90% of myocardial 99mTc-sestamibi was localized within the mitochondrial fraction. Calcium was found to release 99mTc-sestamibi from the mitochondrial fraction, with an IC50 of 2.54 +/- 0.98 mM. This effect was potentiated by NaCl, and inhibited by the mitochondrial calcium channel blocker ruthenium red. In vitro uptake of 99mTc-sestamibi was found to increase from 10.5% +/- 3.0% to 61.2% +/- 0.2% with the addition of 10 mM succinate, indicating that respiration is involved. Since irreversible ischemia results in cellular and mitochondrial calcium "overload" and loss of mitochondrial metabolic function, 99mTc-sestamibi should not be retained in necrotic or irreversibly ischemic myocardium, and could potentially act as a sensitive indicator of myocardial cell viability.

Blood-brain barrier penetration of leucine-enkephalin, methionine-enkephalin, and other peptide-like compounds was measured after intracarotid injection of three isotopes and was found to be non-saturable over the nanomolar range of concentrations tested. No significant differences in brain regional extraction of leucine enkephalin (or morphine or heroin) were observed. In contrast to previous reports, the brain extraction of enkephalins was minimally low (E = 2-3%) and about the same order of magnitude as other putative neurotransmitters. Brain extractions of other peptide-like compounds were similarly small: TRH, E = 1%; glutathione, E = 0.5%; beta-alanyl histidine, E = 1%; and thioacetyl coenzyme A, E = 2%. Extraction of the non-diffusible reference dextran was determined to be 1%, suggesting that the blood brain barrier tends to restrict peptide penetration.