No abstract

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 day old rat brain at 15, 60 and 120 minutes after an intraperitoneal injection of [2-13C]acetyl-L-carnitine. The amount, pattern, and fractional enrichment of 13C-labeled metabolites were determined by ex vivo 13C-NMR spectroscopy. Metabolism of the acetyl moiety from [2-13C]ALCAR via the tricarboxylic acid (TCA) cycle led to incorporation of label into the C4, C3 and C2 positions of glutamate (GLU), glutamine (GLN) and GABA. Labeling patterns indicated that [2-13C]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 of lactate, and the C1 and C5 positions of glutamate and glutamine demonstrated that [2-13C]ALCAR was actively metabolized via the pyruvate recycling pathway. The enrichment of metabolites with 13C from metabolism of ALCAR was highest in alanine C3 (10%) and lactate C3 (9%), with considerable enrichment in GABA C4 (8%), GLN C3 (~4%) and GLN C5 (5%). Overall, our 13C-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 TCA cycle. Labeling of compounds formed from metabolism of [2-13C]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.

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 .

It is well documented that the brain preferentially utilizes alternative substrates for energy during brain development; however, less is known about the use of these substrates by synaptic terminals. The present study compared the rates of ¹⁴CO₂ production from 1 mM D-[6-¹⁴C]glucose, L-[U-¹⁴C]glutamine, D-3-hydroxy[3-¹⁴C]butyrate, L-[U-¹⁴C]lactate and L-[U-¹⁴C]malate by synaptic terminals isolated from 17- to 18-day-old and 7- to 8-week-old rat brain. The rates of ¹⁴CO₂ production from glucose, glutamine, 3-hydroxybutyrate, lactate and malate were 8.55 ± 0.78, 25.90 ± 4.58, 42.28 ± 3.54, 48.42 ± 2.09, and 9.31 ± 1.61 nmol/h/mg protein (mean ± SEM), respectively, in synaptic terminals isolated from 17- to 18-day-old rat brain and 12.95 ± 1.64, 30.62 ± 4.19, 16.09 ± 2.62,40.33 ± 6.77, and 8.25 ± 1.69 nmol/ h/mg protein (mean ± SEM), respectively, in synaptic terminals isolated from 7- to 8-week-old rat brain. In competition studies using unlabelled added substrates, the addition of 3-hydroxybutyrate, lactate or glutamine greatly decreased the rate of ¹⁴CO₂ production from labelled glucose. Added unlabelled glucose increased the rate of ¹⁴CO₂ production from 3-hydroxybutyrate in synaptic terminals from 7- to 8-week-old rat brain, but had no effect on ¹⁴CO₂ production from any other substrates. Lactate also increased ¹⁴CO₂ production from 3-hydroxybutyrate at 7–8 weeks, whereas the addition of 3-hydroxybutyrate decreased ¹⁴CO₂ production from lactate only in synaptic terminals from 17- to 18-day-old rat brain. None of the added substrates altered the rate of ¹⁴CO₂ production from labelled glutamine or malate suggesting that these substrates are metabolized in relatively distinct compartments within synaptic terminals. Overall the data demonstrate that synaptic terminals from both weanling and adult rat brain can utilize a variety of substrates for energy. In addition, the competition studies demonstrate that the interactions of substrates change with age and suggest that there are multiple compartments of energy metabolism (or tricarboxylic acid cycle activity) in isolated synaptic terminals.

Abbreviations used: ASP C3, [3][4][5][6][7][8][9][10][11][12][13] C]aspartate; PC, pyruvate carboxylase; PDHC, pyruvate dehydrogenase complex; TBI, traumatic brain injury; TCA, tricarboxylic acid. AbstractTraumatic 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][7][8][9][10][11][12][13] C]glucose and brains removed 15, 30 and 60 min later and studied by ex vivo 13 C-NMR spectroscopy. Oxidative metabolism, determined by incorporation of 13 C 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. Keywords:13 C-NMR spectroscopy, GABA, glucose, gluta- et al. 2005) reported significant increases in brain lactate and glutamine production as early as 3.5 h after TBI in immobilized adult rats. Investigations on the effects of TBI on energy metabolism of the immature brain are limited, despite the prevalence of TBI in children. Nevertheless, brain mitochondrial respiration and pyruvate dehydrogenase complex activity levels are reduced within 4 h after TBI in immature rats (Robertson et al. 2007). A recent 1 H-NMR study reported increased lactate at 4 h, and decreased N-acetylaspartate at 24 h and 7 days after TBI providing further evidence of mitochondrial dysfunction after TBI in immature brain (Casey et al. 2008).The aim of this study was to determine glycolytic and oxidative glucose metabolism and neurotransmitter synthesis using 13 C-NMR spec...