Abstract
Cerebral micro-dialysis allows continuous sampling of extracellular metabolites, including glucose, lactate and pyruvate. Transient ischemic events cause a rapid drop in glucose and a rise in lactate levels. Following such events, the lactate/pyruvate (L/P) ratio may remain elevated for a prolonged period of time. In neurointensive care clinics, this ratio is considered a metabolic marker of ischemia and/or mitochondrial dysfunction. Here we propose a novel, sensitive microdialysis liquid chromatography-mass spectrometry (LC-MS) approach to monitor mitochondrial dysfunction in living brain using perfusion with 13C-labeled succinate and analysis of 13C-labeled tricarboxylic acid cycle (TCA) intermediates. This approach was evaluated in rat brain using malonate-perfusion (10–50 mM) and endothelin-1 (ET-1)-induced transient cerebral ischemia. In the malonate model, the expected changes upon inhibition of succinate dehydrogenase (SDH) were observed, i.e., an increase in endogenous succinate and decreases in fumaric acid and malic acid. The inhibition was further elaborated by incorporation of 13C into specific TCA intermediates from 13C-labeled succinate. In the ET-1 model, increases in non-labeled TCA metabolites (reflecting release of intracellular compounds) and decreases in 13C-labeled TCA metabolites (reflecting inhibition of de novo synthesis) were observed. The analysis of 13C incorporation provides further layers of information to identify metabolic disturbances in experimental models and neuro-intensive care patients.
Highlights
In neuro-intensive care units, cerebral microdialysis is routinely used to monitor interstitial levels of glucose, lactate and pyruvate, the main energy substrates of the brain
Elevated L/P ratios with normal or increased levels of pyruvate after an ischemic insult are considered a metabolic marker of mitochondrial dysfunction, which has been associated with delayed neurological deterioration (DND) [3,4]
In the ET-1-induced cerebral ischemia-reperfusion model we observed a glucose drop below 50%
Summary
In neuro-intensive care units, cerebral microdialysis is routinely used to monitor interstitial levels of glucose, lactate and pyruvate, the main energy substrates of the brain. Elevated L/P ratios with normal or increased levels of pyruvate after an ischemic insult are considered a metabolic marker of mitochondrial dysfunction, which has been associated with delayed neurological deterioration (DND) [3,4]. Detection and monitoring of mitochondrial dysfunction, as well as understanding the underlying mechanisms of disturbed energy metabolism post-injury are essential to improve treatments and outcome in neuro-intensive care patients. Brain energy metabolism has been studied in vitro and in vivo by perfusion with 14 C-labeled (radioactive) energy substrates, including glucose, lactate, pyruvate, glutamate and glutamine, and monitoring 14 CO2 production under various experimental conditions. Studies using fluorocitrate, which at lower concentrations inhibits glial, but not neuronal TCA cycle activity, combined with
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