Metabolism of acetyl-L-carnitine for energy and neurotransmitter synthesis in the immature rat brain.

Acetyl-L-carnitine (ALCAR) is an endogenous metabolic intermediate that facilitates the influx and efflux of acetyl groups across the mitochondrial inner membrane. Exogenously administered ALCAR has been used as a nutritional supplement and also as an experimental drug with reported neuroprotective properties and effects on brain metabolism. The aim of this study was to determine oxidative metabolism of ALCAR in the immature rat forebrain. Metabolism was studied in 21-22 day-old rat brain at 15, 60 and 120 min after an intraperitoneal injection of [2-(13)C]acetyl-L-carnitine. The amount, pattern, and fractional enrichment of (13)C-labeled metabolites were determined by ex vivo(13)C-NMR spectroscopy. Metabolism of the acetyl moiety from [2-(13)C]ALCAR via the tricarboxylic acid cycle led to incorporation of label into the C4, C3 and C2 positions of glutamate (GLU), glutamine (GLN) and GABA. Labeling patterns indicated that [2-(13)C]ALCAR was metabolized by both neurons and glia; however, the percent enrichment was higher in GLN and GABA than in GLU, demonstrating high metabolism in astrocytes and GABAergic neurons. Incorporation of label into the C3 position of alanine, both C3 and C2 positions of lactate, and the C1 and C5 positions of glutamate and glutamine demonstrated that [2-(13)C]ALCAR was actively metabolized via the pyruvate recycling pathway. The enrichment of metabolites with (13)C from metabolism of ALCAR was highest in alanine C3 (11%) and lactate C3 (10%), with considerable enrichment in GABA C4 (8%), GLN C3 (approximately 4%) and GLN C5 (5%). Overall, our (13)C-NMR studies reveal that the acetyl moiety of ALCAR is metabolized for energy in both astrocytes and neurons and the label incorporated into the neurotransmitters glutamate and GABA. Cycling ratios showed prolonged cycling of carbon from the acetyl moiety of ALCAR in the tricarboxylic acid cycle. Labeling of compounds formed from metabolism of [2-(13)C]ALCAR via the pyruvate recycling pathway was higher than values reported for other precursors and may reflect high activity of this pathway in the developing brain. This is, to our knowledge, the first study to determine the extent and pathways of ALCAR metabolism for energy and neurotransmitter biosynthesis in the brain.

Delayed cerebral oxidative glucose metabolism after traumatic brain injury in young rats.

Traumatic brain injury (TBI) results in a cerebral metabolic crisis that contributes to poor neurologic outcome. The aim of this study was to characterize changes in oxidative glucose metabolism in early periods after injury in the brains of immature animals. At 5 h after controlled cortical impact TBI or sham surgery to the left cortex, 21-22 day old rats were injected intraperitoneally with [1,6-13C]glucose and brains removed 15, 30 and 60 min later and studied by ex vivo 13C-NMR spectroscopy. Oxidative metabolism, determined by incorporation of 13C into glutamate, glutamine and GABA over 15-60 min, was significantly delayed in both hemispheres of brain from TBI rats. The most striking delay was in labeling of the C4 position of glutamate from neuronal metabolism of glucose in the injured, ipsilateral hemisphere which peaked at 60 min, compared with the contralateral and sham-operated brains, where metabolism peaked at 30 and 15 min, respectively. Our findings indicate that (i) neuronal-specific oxidative metabolism of glucose at 5-6 h after TBI is delayed in both injured and contralateral sides compared with sham brain; (ii) labeling from metabolism of glucose via the pyruvate carboxylase pathway in astrocytes was also initially delayed in both sides of TBI brain compared with sham brain; (iii) despite this delayed incorporation, at 6 h after TBI, both sides of the brain showed apparent increased neuronal and glial metabolism, reflecting accumulation of labeled metabolites, suggesting impaired malate aspartate shuttle activity. The presence of delayed metabolism, followed by accumulation of labeled compounds is evidence of severe alterations in homeostasis that could impair mitochondrial metabolism in both ipsilateral and contralateral sides of brain after TBI. However, ongoing oxidative metabolism in mitochondria in injured brain suggests that there is a window of opportunity for therapeutic intervention up to at least 6 h after injury.

Hyperoxic reperfusion after global ischemia decreases hippocampal energy metabolism.

BACKGROUND AND PURPOSE: Previous reports indicate that compared with normoxia, 100% ventilatory O(2) during early reperfusion after global cerebral ischemia decreases hippocampal pyruvate dehydrogenase activity and increases neuronal death. However, current standards of care after cardiac arrest encourage the use of 100% O(2) during resuscitation and for an undefined period thereafter. Using a clinically relevant canine cardiac arrest model, in this study we tested the hypothesis that hyperoxic reperfusion decreases hippocampal glucose metabolism and glutamate synthesis. METHODS: After 10 minutes of cardiac arrest, animals were resuscitated and ventilated for 1 hour with 100% O(2) (hyperoxic) or 21% to 30% O(2) (normoxic). At 30 minutes reperfusion, [1-(13)C]glucose was infused, and at 2 hours, brains were rapidly removed and frozen. Extracted metabolites were analyzed by (13)C nuclear magnetic resonance spectroscopy. RESULTS: Compared with nonischemic controls, the hippocampi from hyperoxic animals had elevated levels of unmetabolized (13)C-glucose and decreased incorporation of (13)C into all isotope isomers of glutamate. These findings indicate impaired neuronal metabolism via the pyruvate dehydrogenase pathway for carbon entry into the tricarboxylic acid cycle and impaired glucose metabolism via the astrocytic pyruvate carboxylase pathway. No differences were observed in the cortex, indicating that the hippocampus is more vulnerable to metabolic changes induced by hyperoxic reperfusion. CONCLUSIONS: These results represent the first direct evidence that hyperoxia after cardiac arrest impairs hippocampal oxidative energy metabolism in the brain and challenge the rationale for using excessively high resuscitative ventilatory O(2).

Aspartate aminotransferase in synaptic and nonsynaptic mitochondria: differential effect of compounds that influence transient hetero-enzyme complex (metabolon) formation.

The enzyme aspartate aminotransferase (AAT) has a number of key roles in astrocytes and neurons in brain. An understanding of the regulation of AAT is important since AAT is involved in many aspects of glutamate metabolism including the synthesis of neurotransmitter glutamate. Mitochondrial AAT binds to a protein and lipids on the inner mitochondrial membrane and also forms a number of transient hetero-enzyme complexes with other enzymes. These complexes serve to facilitate metabolism by essentially channeling substrates and cofactors to other enzymes within the complex. The association and dissociation of transiently formed hetero-enzyme complexes may modulate enzyme activity in "real time" since these complexes are dynamically influenced by changes in the concentration of a number of key metabolites. The influence of several effectors that modulate AAT activity, either directly, or by altering the binding of AAT to mitochondrial lipids, or the association/dissociation into transient hetero-enzyme complexes was determined. The addition of palmitate, malate, citrate, glutamate, bovine serum albumin and Mg(2+) modulated AAT activity differently in synaptic and nonsynaptic mitochondria from brain. These findings suggest that AAT activity and also glutamate metabolism, may be regulated in part, by metabolites that influence binding of the enzyme to lipids or proteins in the inner mitochondrial membrane and/or the association/dissociation of transient hetero-enzyme complexes. This may have a role in the compartmentation of glutamate metabolism in brain.

Isolation of mitochondria with high respiratory control from primary cultures of neurons and astrocytes using nitrogen cavitation.

To study neurons or glia-specific mitochondria one needs to isolate these organelles from primary neuronal or astrocytic cell culture. This work provides novel method for isolation of functional and morphologically intact mitochondria from neurons and astrocytes in cell cultures. In the first step, mitochondria are released from cells by disruption of cell membranes using a nitrogen cavitation technique. This technique is based on rapid decompression of a cell suspension from within a pressure vessel. Mitochondria released from cell bodies are then separated from the rest of cell homogenate by Percoll gradient centrifugation. This is a relatively rapid technique that yields to very well coupled mitochondria that exhibited functional and morphological characteristics comparable to mitochondria isolated from brain tissue using common techniques. This technique thus will allow examination of mitochondria that are exclusively cell specific in origin.

Prevention of Traumatic Brain Injury in Youth and Adolescents.

The goal of this project was to promote bicycle helmet use via an inpatient educational program. We hypothesized that this program would increase bicycle helmet use. One hundred twenty inpatients with history of regular (>1 time per week) bicycle riding (mean age 10.0 ± 3.6 years; 67 males, 53 females; 57 whites, 59 blacks, 4 other) were randomized to treatment (n = 58) or control (n = 62) groups. All participants received a bicycle helmet. At 1 month, 50 (92.6%) of the intervention group and 48 (82.8%) of the control group wore a helmet every bike ride (P < .07). At 3 months, 50 (96.2%) of the intervention group and 44 (80%) of the controls wore a helmet with every bike ride (P < .03). The study proved feasible, requiring trained personnel to deliver the intervention. Providing a helmet without the intervention was effective in 80% to 83% of cases with respect to parental report of helmet wearing compliance.

Postischemic oxidative stress promotes mitochondrial metabolic failure in neurons and astrocytes.

Oxidative stress and mitochondrial dysfunction have been closely associated in many subcellular, cellular, animal, and human studies of both acute brain injury and neurodegenerative diseases. Our animal models of brain injury caused by cardiac arrest illustrate this relationship and demonstrate that both oxidative molecular modifications and mitochondrial metabolic impairment are exacerbated by reoxygenation of the brain using 100% ventilatory O(2) compared to lower levels that maintain normoxemia. Numerous molecular mechanisms may be responsible for mitochondrial dysfunction caused by oxidative stress, including oxidation and inactivation of mitochondrial proteins, promotion of the mitochondrial membrane permeability transition, and consumption of metabolic cofactors and intermediates, for example, NAD(H). Moreover, the relative contribution of these mechanisms to cell injury and death is likely different among different types of brain cells, for example, neurons and astrocytes. In order to better understand these oxidative stress mechanisms and their relevance to neurologic disorders, we have undertaken studies with primary cultures of astrocytes and neurons exposed to O(2) and glucose deprivation and reoxygenation and compared the results of these studies to those using a rat model of neonatal asphyxic brain injury. These results support the hypothesis that release and or consumption of mitochondrial NAD(H) is at least partially responsible for respiratory inhibition, particularly in neurons.

Kinetic parameters and lactate dehydrogenase isozyme activities support possible lactate utilization by neurons.

Lactate is potentially a major energy source in brain, particularly following hypoxia/ischemia; however, the regulation of brain lactate metabolism is not well understood. Lactate dehydrogenase (LDH) isozymes in cytosol from primary cultures of neurons and astrocytes, and freshly isolated synaptic terminals (synaptosomes) from adult rat brain were separated by electrophoresis, visualized with an activity-based stain, and quantified. The activity and kinetics of LDH were determined in the same preparations. In synaptosomes, the forward reaction (pyruvate + NADH + H(+ )--> lactate + NAD(+)), which had a V (max) of 1,163 micromol/min/mg protein was 62% of the rate in astrocyte cytoplasm. In contrast, the reverse reaction (lactate + NAD(+ )--> pyruvate + NADH + H(+)), which had a V (max) of 268 micromol/min/mg protein was 237% of the rate in astrocytes. Although the relative distribution was different, all five isozymes of LDH were present in synaptosomes and primary cultures of cortical neurons and astrocytes from rat brain. LDH1 was 14.1% of the isozyme in synaptic terminals, but only 2.6% and 2.4% in neurons and astrocytes, respectively. LDH5 was considerably lower in synaptic terminals than in neurons and astrocytes, representing 20.4%, 37.3% and 34.8% of the isozyme in these preparations, respectively. The distribution of LDH isozymes in primary cultures of cortical neurons does not directly reflect the kinetics of LDH and the capacity for lactate oxidation. However, the kinetics of LDH in brain are consistent with the possible release of lactate by astrocytes and oxidative use of lactate for energy in synaptic terminals.