Randy E. Keen

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.

Retention and clearance of C-11 palmitic acid in ischemic and reperfused canine myocardium

Free fatty acids are the major energy source for cardiac muscle. Oxidation of fatty acid decreases or even ceases during ischemia. Its recovery after transient ischemia remains largely unexplored. Using intracoronary carbon-11 palmitic acid as a tracer of myocardial fatty acid metabolism in an open chest dog model, retention and clearance of tracer in myocardium were evaluated at control, during ischemia and after reperfusion following a 20 minute occlusion of the left anterior descending coronary artery. Myocardial C-11 time-activity curves were analyzed with biexponential curve-fitting routines yielding fractional distribution and clearance half-times of C-11 palmitic acid in myocardial tissue. In animals with permanent occlusion and intracoronary injection of C-11 palmitic acid distal to the occlusion site, the relative size and half-time of the early clearance curve component differed markedly from control values and did not change with ongoing ischemia. Conversely, in animals with only 20 minutes of coronary occlusion, the relative size of the early C-11 clearance phase was still significantly depressed at 20 and 90 minutes of reperfusion but returned to control level at 180 minutes. Tissue C-11 clearance half-times remained significantly prolonged throughout the reperfusion period. Regional function in reperfused myocardium monitored with ultrasonic crystals recovered slowly and was still less than control after 3 hours of reperfusion. The data indicate that after transient ischemia, myocardial fatty acid metabolism fails to recover immediately. Because the metabolic recovery occurs in parallel with recovery of regional function, C-11 palmitic acid in conjunction with positron tomography may be useful for studying regional fatty acid metabolism noninvasively after an ischemic injury, and may be helpful in identifying reversible tissue injury.

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, α-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 ≥ leucyl-tRNA ≈ KIC > 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.

Effects of anoxia on kinetics of [13N] glutamate and 13NH3 metabolism in rabbit myocardium.

Positron emission tomography is a unique noninvasive imaging technique that provides cross-sectional images of radiotracer concentrations in myocardium and permits measurement of blood flow as well as metabolism. Ammonia and glutamate have been labeled with the positron-emitter 13N (half-life 10 minutes) for use with positron emission tomography as tracers of flow and metabolism, respectively. In order to characterize the fate of these 13N-labelled compounds in myocardium, isolated rabbit interventricular septa were used to study the kinetics of [13N] glutamate ([13N]glu) and 13NH3 under aerobic and anoxic conditions. Tissue analyses 6 minutes after injection of a [13N]glu bolus into myocardium revealed that 70% of the 13N-label was present in [13N]glu 12%, 11%, and 4% in [13N]alanine ([13N]ala), [13N]aspartate ([13N]asp), and [13N]glutamine ([13N]gln), respectively. The corresponding relative specific activities were 1.0:0.4:0.5:0.01. Anoxia resulted in a significant increase in [13N]ala with a reduction in [13N]glu. This was consistent with increased pyruvate production due to increased anaerobic glycolysis and transamination of pyruvate with [13N]glu to yield [13N]ala. In support of this, addition of 2 mM pyruvate to the perfusate under control conditions produced a tissue distribution of 13N similar to that with anoxia. Six minutes after a bolus of 13NH3 during both control and anoxic conditions, 60% of the tissue 13N-label was in [13N]gln with no detectable amounts in other amino acids. The rest of the 13N-label was in 13NH3. Time-activity curve analyses demonstrated that anoxia significantly reduced the tissue retention of 13N-label from 13NH3 but not from [13N]glu. Thus, 13N from 13NH3 and [13N]glu was retained in tissue by different mechanisms involving glutamate, which were affected differentially by anoxia. These results suggest that positron emission tomography imaging with 13NH3 and [13N]glu in combination may be useful in identifying ischemic myocardium.

The elusive 8-fluoroadenosine

Abstract The synthesis of 8-fluoroadenosine has been accomplished for the first time. The kinetics of deamination of 8-fluoroadenosine with the enzyme adenosine deaminase has also been measured.

Concepts and Techniques Used in Metabolic Tracer Studies

Studies of ammonia, amino acid, fatty acid, and glucose metabolism have relied primarily on the use of 15N, 14C, 13C, and 3H-labeled compounds. However, tissue sampling and analysis of enriched metabolites, i.e., 15N and 13C by mass spectrometry [1] and NMR [2–4], and 14C and 3H by liquid scintillation counting, limit the application of these techniques for the noninvasive determination of in vivo tissue tracer kinetics.

Nitrogen-13 flux from L-[13N]glutamate in the isolated rabbit heart: effect of substrates and transminase inhibition

Kinetic and biochemical parameters of nitrogen-13 flux from L-[13N]glutamate in myocardium were examined. Tissue radioactivity kinetics and chemical analyses were determined after bolus injection of L-[13N]glutamate into isolated arterially perfused interventricular septa under various metabolic states, which included addition of lactate, pyruvate, aminooxyacetate (a transaminase inhibitor), or a combination of aminooxyacetate and pyruvate to the standard perfusate containing insulin and glucose. Chemical analysis of tissue and effluent at 6 min allowed determination of the composition of the slow third kinetic component of the time-activity curves. 13N-labeled aspartate, alanine and glutamate accounted for more than 80% of the tissue nitrogen-13 under the experimental conditions used. Specific activities for these amino acids were constant, but not identical to each other, from 6 through 15 min after administration of L-[13N]glutamate. Little labeled ammonia (1.9%) and glutamine (4.7%) were produced, indicating limited accessibility of exogenous glutamate to catabolic mitochondrial glutamate dehydrogenase and glutamine synthetase, under control conditions. Lactate and pyruvate additions did not affect tissue amino acid specific activities. Aminooxyacetate suppressed formation of 13N-labeled alanine and aspartate and increased production of L-[13N]glutamine and [13N]ammonia. Formation of [13N]ammonia was, however, substantially decreased when aminooxyacetate was used in the presence of exogenous pyruvate. The data support a model for glutamate compartmentation in myocardium not affected by increasing the velocity of enzymatic reactions through increased substrate (i.e., lactate or pyruvate) concentrations but which can be altered by competitive inhibition of transaminases (via aminooxyacetate) making exogenous glutamate more available to other compartments.