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Journal of Cerebral Blood Flow & Metabolism logoLink to Journal of Cerebral Blood Flow & Metabolism
. 2014 Jan 8;34(3):514–521. doi: 10.1038/jcbfm.2013.229

Propofol compared with isoflurane inhibits mitochondrial metabolism in immature swine cerebral cortex

Masaki Kajimoto 1, Douglas B Atkinson 1, Dolena R Ledee 1, Ernst-Bernhard Kayser 1, Phil G Morgan 1, Margaret M Sedensky 1, Nancy G Isern 2, Christine Des Rosiers 3, Michael A Portman 1,4,*
PMCID: PMC3948133  PMID: 24398942

Abstract

Anesthetics used in infants and children are implicated in the development of neurocognitive disorders. Although propofol induces neuroapoptosis in developing brain, the underlying mechanisms require elucidation and may have an energetic basis. We studied substrate utilization in immature swine anesthetized with either propofol or isoflurane for 4 hours. Piglets were infused with 13-Carbon-labeled glucose and leucine in the common carotid artery to assess citric acid cycle (CAC) metabolism in the parietal cortex. The anesthetics produced similar systemic hemodynamics and cerebral oxygen saturation by near-infrared spectroscopy. Compared with isoflurane, propofol depleted ATP and glycogen stores. Propofol decreased pools of the CAC intermediates, citrate, and α-ketoglutarate, while markedly increasing succinate along with decreasing mitochondrial complex II activity. Propofol also inhibited acetyl-CoA entry into the CAC through pyruvate dehydrogenase, while promoting glycolytic flux with marked lactate accumulation. Although oxygen supply appeared similar between the anesthetic groups, propofol yielded a metabolic phenotype that resembled a hypoxic state. Propofol impairs substrate flux through the CAC in the immature cerebral cortex. These impairments occurred without systemic metabolic perturbations that typically accompany propofol infusion syndrome. These metabolic abnormalities may have a role in the neurotoxity observed with propofol in the vulnerable immature brain.

Keywords: anesthesia, energy metabolism, glucose, mitochondria, MR spectroscopy

Introduction

Recent studies in both rodents and primates have demonstrated alarming widespread neuronal degeneration after conventional doses of common anesthetic medicines such as isoflurane and propofol during a vulnerable window in early development.1, 2, 3 This has been associated with permanent behavioral deficits in these animals. At the same time, learning and behavioral abnormalities have been documented in children exposed as infants to various anesthetics.4 The triggering mechanisms for neuronal degeneration including apoptosis by anesthetics have not been clearly defined. Experimental evidence shows that immature neurons, which exhibit enhanced excitability compared with adults, are particularly vulnerable to caspase activation and hence apoptosis by anesthetics including propofol.1, 5 Disturbances in Ca++ homeostasis can lead to neurodegeneration in the developing brain,6 which shows different mechanisms for depolarization than the adult.5 Anesthetics used in pediatrics, and in particular propofol, can increase Ca++ influx.7 Thus, enhanced caspase activation by Ca++ has been implicated as the mechanism for neuronal death.7 Although, experiments performed in vitro show that the rapid rise in Ca++ induced by propofol can occur before neuronal apoptosis,5 this change in flux does not always precede and therefore does not fully explain the anesthetic-induced neurotoxicity.

Data obtained principally from isolated rodent mitochondria or perfused organs demonstrate that propofol inhibits mitochondrial respiratory function and shifts substrate metabolism toward glycolysis.8, 9 Thus, modifications in energy metabolism resulting in ATP depletion might explain propofol induction of degeneration in immature neurons. Positron emission tomography of adult human brains corroborated that propofol shifted central nervous system metabolism toward glycolysis.10 However, those studies used 2-fluorodeoxyglucose uptake, and did not determine the actual fate of glucose. Similarly, other studies have used metabolic profiling by 1H-nuclear magnetic resonance (NMR) to demonstrate differences between volatile anesthetics and propofol.11 The 1H-NMR method resolves only those metabolites occurring at relatively high concentrations and does not include analyses of citric acid cycle (CAC) intermediates. No detailed studies of anesthetic modulation of mitochondrial function and substrate flux have been performed in the immature brain in the intact animal.

We therefore tested the hypothesis that the volatile anesthetic isoflurane (a substituted ether) and the intravenous anesthetic propofol (a substituted phenol) modify substrate metabolism and CAC flux differently. We performed isotopic labeling experiments to determine the ultimate fate of glucose within the cerebral cortex of immature swine under isoflurane and propofol anesthesia. We analyzed rates of substrate oxidative flux relative to CAC flux using both gas chromatography–mass spectroscopy (GC–MS) and NMR techniques, while also assaying ATP. Previous investigations in various organs have demonstrated that a significant portion of acetyl-CoA to the mitochondria is provided through leucine oxidation. Furthermore, some studies suggest that leucine not only contributes more substantially to oxidative metabolism in the developing brain than in the adult, but also has a regulatory role in overall substrate metabolism.12, 13 The NMR technique allows simultaneous analyses of fractional contributions to the CAC for up to three 13-Carbon (13C)-labeled substrates. We used this technique to advantage and analyzed fractional contributions of both glucose and leucine to the CAC during these anesthetic protocols.

Materials and Methods

Animals

All experimental procedures were approved by Seattle Children's Institutional Animal Use and in accordance with the ARRIVE guidelines. Fourteen male Yorkshire piglets (body weight 7.8 to 14.5 kg, 27 to 41 days of age) were fasted over night with free access to water. They were premedicated with an intramuscular injection of ketamine (33 mg/kg) and xylazine (2 mg/kg) and were placed on a circulating warming blanket. Monitors were placed for electrocardiogram, pulse oximetry (Radical SET, Masimo, Irvine, CA, USA), and rectal temperature. Functional near-infrared spectroscopy (INVOS 5100C Somanetics, Troy, MI, USA) was used to measure regional cerebral oxygen saturation (rSO2). A single pediatric SomaSensor (Somanetics) was placed on the forehead according to the manufacturer's guidelines. The common carotid artery was cannulated for continuous blood pressure monitoring and blood sampling, and the internal jugular vein was cannulated for infusion of substrates (see below). After intubation through surgical tracheostomy, the piglets were mechanically ventilated with 40% to 60% inhaled oxygen. They were then divided into 2 groups, and anesthetized for 4 hours with either inhaled isoflurane or intravenous propofol (see below). An arterial pCO2 of 35 to 45 mm Hg was maintained by adjusting minute ventilation.

Anesthesia Treatment and Infusion of 13-Carbon-Labeled Substrates

Inhaled isoflurane (Forane, Baxter Healthcare Corporation, Deerfield, IL, USA) was maintained at 1% to 3% (group ISO, n=7), and intravenous propofol (Diprivan, AstraZeneca, Cambridge, UK) was infused via the internal jugular vein with a loading dose of 1 to 2 mg/kg followed by an infusion rate of 15 mg/kg per hour for the first 15 minutes to achieve plasma equilibrium concentration, and then decreased infusion (12 mg/kg per hour from 15 to 60 minutes, 9 mg/kg per hour from 1 to 2 hours and 7.5 mg/kg per hour from 2 to 4 hours) as tissue saturation occurred (group PROP, n=7). These anesthetic agents were continuously used for 4 hours. For metabolic analysis, 87 mg/kg of body weight [1-13C]-glucose and 32 mg/kg of body weight [13C6, 15N]-L-leucine (all 99% enriched, Sigma-Aldrich, St Louis, MO, USA), were delivered as tracers of glucose and amino-acid oxidation, respectively into the common carotid artery for the final 60 minutes of the protocol. Immediately upon completion of the labeled infusion, the parietal cortex of the brain was excised through craniotomy and rapidly stored under liquid nitrogen for later extraction.

Metabolic Analyses by Nuclear Magnetic Resonance

13-Carbon nuclear magnetic resonance was performed on the brain cortex for determination of specific carbon glutamate labeling by steady-state isotopomer analysis from Malloy et al14 as previously described.15, 16, 17 Glutamate labeling data by 13C-NMR provides the fractional contributions of substrates to acetyl-CoA entering the CAC. Frozen brain cortex tissues were ground into fine powder under liquid nitrogen and extracted with methanol/chloroform. The final supernatant was lyophilized over night at −50 °C. Lyophilized brain extracts were dissolved in 99.8% 2H2O for NMR spectral acquisition. 13C-NMR spectra were acquired on a Varian Direct Drive (VNMRS) 600 MHz spectrometer (Agilent Technologies, Santa Clara, CA, USA) equipped with a Dell Precision T3500 Linux workstation running VNMRJ 3.2. The number of scans was 8,000 on average for ∼9.5 hours per spectrum. The NMR spectra were analyzed in spectra processed with 0.5 Hz line broadening, based on individual brain. The spectrometer system was outfitted with a Varian triple resonance salt-tolerant cold probe with a cold carbon preamplifier. Protons were decoupled with a Waltz decoupling scheme. Final spectra were obtained using a 45° excitation pulse (7.05 μs at 58 dB), with an acquisition time of 1.3 seconds, a recycle delay of 3 seconds, and a spectral width of 224.1 p.p.m. Fourier-transformed spectra were fitted with commercial software (NUTS, Acorn NMR, Livermore, CA, USA). The labeled carbon resonances (C3–C5) of glutamate were integrated using commercial software (NUTS; Acorn NMR) and the relative multiplet peak areas were input into tcaCALC (kindly provided by the Advanced Imaging Research Center at the University of Texas Southwestern). Our metabolic flux study using 13C-labeled substrates and NMR is based on the formation of different isotopomers of acetyl-CoA, citrate and glutamate resulting from metabolism of [1-13C]-glucose and [13C6, 15N]-L-leucine at the first turn of the CAC. [1-13C]-glucose is converted to [3-13C]-pyruvate and unlabeled pyruvate. These two isotopomers of pyruvate will be decarboxylated leading to the formation of [2-13C]acetyl-CoA and unlabeled acetyl-CoA in the proportion of 1:1, whereas [13C6, 15N]-L-leucine will lead to the formation of [1,2-13C]acetyl-CoA. Subsequently [2-13C]acetyl-CoA will label the C4 of glutamate, whereas [1,2-13C]acetyl-CoA will result in labeling at the C4 and C5 positions of glutamate. This allowed determination of fractional contributions to glutamate labeling via acetyl-CoA between exogenously administrated glucose and leucine.

Metabolic Analyses by Gas Chromatography–Mass Spectrometry

GC–MS (Agilent 6890N gas chromatograph equipped with a HP-5 column coupled to a 5973N mass spectrometer; Agilent Technologies) was performed to measure the 13C-enrichment and concentrations of CAC intermediates (citrate, α-ketoglutarate (α-KG), succinate, fumarate, and malate), lactate, pyruvate, and amino acids as described elsewhere.15, 17, 18 Gas Chromatography–Mass Spectrometry data are reported as the 13C-molar percent enrichment (MPE) and absolute concentration. Mass isotopomers of metabolites containing 1 to n 13C-labeled atoms were identified as Mi, with i=1, 2, … n. The total and absolute MPE of individual 13C-labeled mass isotopomers of a given metabolite was calculated as follows,

graphic file with name jcbfm2013229e1.jpg
graphic file with name jcbfm2013229e2.jpg

where AM and AMi represent the peak areas from ion chromatograms corrected for natural abundance, corresponding to the unlabeled (M) and 13C-labeled mass isotopomers, respectively. Citric acid cycle intermediate enrichment was evaluated via the total MPE of several intermediates in the CAC from the ground tissue samples. The pathway from pyruvate entering into the CAC via acetyl-CoA is called pyruvate decarboxylation (PDC). Pyruvate decarboxylation flux rate relative to citrate synthesis (CIT) was determined using the following formula:

graphic file with name jcbfm2013229e3.jpg

This ratio represents flux through PDC leading to CIT arising from all tissue pyruvate sources.18

Glycogen and ATP Analysis

Glycogen, which can be made by glycogenesis within the brain, was measured using commercial kits (Cayman, Ann Arbor, MI, USA). Intracellular ATP level was measured using luminescence ATP detection assay (ATPlite PerkinElmer, Waltham, MA, USA).

Immunoblot Analysis

The parietal cortex of the brain was pulverized under liquid nitrogen and lysed in radioimmunoprecipitation assay buffer (1X phosphate-buffered saline, 0.5% deoxycholate, 0.1% SDS, 1%NP-40), containing protease/phosphatase inhibitor (Thermo Fisher Scientific, Waltham, MA, USA). For western blot analysis, 20 μg of total protein underwent electrophoresis using a 10% polyacrylamide gel (Mini Protean 3 cell, Bio-Rad, Carlsbad, CA, USA), and transferred to a PDVF plus membrane. The polyvinylidene difluoride membrane was probed using primary antibodies directed against phosphopyruvate dehydrogenase (PDH) at serine 293 (ABS204, EMD Millipore, MA, USA) and total-PDH (Cell Signaling Technology, Danvers, MA, USA). Western blots were developed using a chemiluminescence detection system (SC-2048, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and analyzed using NIH Image J. Membranes were stripped by washing for 2 × 15 minutes with 100 mM dithiothreitol, 2% (w/v) SDS, 62.5 mM Tris-HCl, pH 6.7, at 60°C, followed by three ten-minute washes with Tris-buffered saline. After stripping, the membrane was blocked and incubated with the appropriate secondary antibody to confirm stripping efficiency.

The Electron Transport Chain Assays

Spectrophotometric electron transport chain assays were performed as previously described.19 Frozen tissue preparations were solubilized with cholate, and zero order rates were determined spectrophotometrically for the following enzyme activities: citrate synthase, rotenone-sensitive NADH cytochrome C reductase, antimycin A-sensitive succinate cytochrome C reductase, antimycin A sensitive decylubiquinol cytochrome C reductase, NADH ferricyanide reductase, rotenone-sensitive NADH decylubiquinone reductase, succinate dichlorophenolindophenol (DCPIP) reductase, and duroquinone-stimulated thenoyltrifluoracetone sensitive succinate dichlorophenolindophenol reductase. The first order rate constant was determined for cytochrome C oxidase. Assays and calculations were performed as described by Hoppel et al.20

Blood Analysis

Arterial and venous blood samples were collected and were immediately centrifuged and aliquots of plasma were stored at −80 °C. Blood pH, pCO2, pO2, SO2 and hemoglobin were measured at regular intervals by a Radiometer ABL 800 (Radiometer America, Westlake, OH, USA). Blood glucose was measured at regular intervals using a Bayer Contour point-of-care glucometer (Bayer HealthCare, Tarrytown, NY, USA). Plasma lactate concentration was measured using commercial kits (BioVision, Mountain View, CA, USA).

Statistical Analyses

Reported values are means±standard error (s.e.) in figures, text, and tables. All groups were compared by Mann–Whitney U test. Criterion for significance was P<0.05 for all comparisons.

Results

Hemodynamics and Blood Parameters

The data shown in Table 1 illustrate the hemodynamic stability of this experimental model during a long period (4 hours) of general anesthesia. This stability is essential to define metabolic perturbations and their direct relationship to the anesthetic, as opposed to changes caused indirectly by alterations in perfusion or oxygen supply. Most importantly, we show that rSO2 was not changed during the course of the protocol for either propofol or isoflurane-anesthetized animals. Furthermore, these saturations did not differ between the two protocols, implying that oxygen delivery/consumption ratio was similar. Blood gas values, pO2, and hemoglobin were also similar between the two protocols further supporting the contention that oxygen-carrying capacity and delivery were not different. We performed isotopic-labeled substrate infusion to define flux toward and within the CAC. The substrate, infused over the final hour of the protocol, did not perturb hemodynamic data or oxygen delivery, indexed by rSO2. Also, the concentration of substrates delivered into the carotid arteries did not alter levels for glucose or lactate in the systemic circulation (Table 1).

Table 1. Hemodynamic and blood gas data.

  Start
 End
  ISO PROP ISO PROP
HR (b.p.m.) 95±4 103±3 99±5 106±4
SBP (mm Hg) 90±2 88±4 80±2 84±2
MBP (mm Hg) 72±2 69±3 60±1 64±2
rSO2 (%) 51±2 49±4 59±3 53±3
Hemoglobin (g/dL) 9.4±0.4 9.2±0.2 9.1±0.4 8.9±0.2
Glucose (mg/dL) 96±6 90±9 125±7 111±5
Lactate (mmol/L) 1.9±0.2 1.3±0.2 1.1±0.1 1.1±0.1
         
Arterial blood gas
 pH 7.45±0.02 7.46±0.02 7.46±0.02 7.45±0.01
 PCO2 (mm Hg) 40±3 40±3 38±1 40±1
 PO2 (mm Hg) 330±11 354±10 327±5 334±18
         
Jugular venous blood gas
 pH 7.38±0.02 7.37±0.02 7.38±0.02 7.36±0.01
 PCO2 (mm Hg) 46±4 52±2 49±1 53±1
 PO2 (mm Hg) 43±3 37±1 43±3 45±2
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HR, heart rate; MBP, mean systemic blood pressure; rSO2, cerebral regional oxygen saturation; SBP, systolic systemic blood pressure.

There were no significant differences among groups. Values are means±s.e.; n=7 per group.

Propofol Induces the Depletion of ATP and Glycogen Stores

Despite the stability and the similarity between the anesthetic conditions for oxygen delivery during the protocol, we identified metabolic disturbances caused by propofol. Substantial ATP depletion (∼70%) occurred in the cortex of animals exposed to propofol (Figure 1A). Glycogen stores were also reduced by ∼70% in the propofol group compared with isoflurane (Figure 1B). The substantial depletion of these metabolite pools occurred coincident with stable oxygen supply. Therefore, these data suggested existence of a ‘pseudohypoxic' state consistent caused by metabolic inhibition or substrate supply limitation to the mitochondrial respiratory apparatus.

Figure 1.

Figure 1

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ATP (A) and glycogen (B) in brain cortex. ATP and glycogen stores were significantly higher in ISO group (P=0.00003 and 0.0015). Values are mean±s.e.; n=7 per group. †P<0.01.

Propofol Increases Lactate Production from Glycolysis

In order to further examine oxidative substrate perturbations, which could contribute to these metabolic differences, we examined concentrations for CAC intermediates and their labeling by 13C-glucose and 13C-leucine. As this study was performed in the intact animal, both 13C-labeled and unlabeled glucose contribute to the pyruvate pool and then to lactate derived from pyruvate via lactate dehydrogenase. Ambient glucose and/or glycogen provide unlabeled pyruvate in these experiments. Under propofol anesthetic, we noted a marked decrease in the absolute (pyruvate)/(lactate) ratio compared with isoflurane, caused by lactate accumulation rather than a decrease in pyruvate pool (Figure 2). The infusion of labeled substrates occurred over the final hour of the protocol. However, as glycogen stores were depleted, we noted by the end of the propofol infusion an increase in relative 13C labeling (MPE) for both lactate and pyruvate. Thus, the data overall show an increase in glycolysis and production of lactate. This flux toward lactate was supported principally by exogenous-labeled glucose rather than glucose-6-phosphate derived from glycogen near the end of the 4-hour period.

Figure 2.

Figure 2

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Pyruvate and lactate metabolism. Absolute quantity and 13C-molar percent enrichment (MPE) of pyruvate (A, B), lactate (C, D) in brain by gas chromatography–mass spectroscopy. (A, C) Absolute lactate concentration in PROP was significantly higher compared with ISO (P=0.0003), whereas pyruvate concentration was similar between the two groups. (B) 13C-molar percent enrichment of pyruvate in PROP group was significantly higher (P=0.017). (E) Absolute (pyruvate)/(lactate) in PROP group was also significantly lower (P=0.0002). Values are mean±s.e.; n=7 per group. *P<0.05, †P<0.01.

Propofol Promotes Citric Acid Cycle Perturbations

The propofol shift toward anaerobic glycolysis together with the low ATP pool in this group suggested that mitochondrial energy metabolism was impaired. Thus, we examined concentrations for CAC intermediates to identify potential disturbances in oxidative substrate flux using GC–MS (Figure 3A). Propofol significantly decreased concentrations for intermediates within the first span of the CAC that extends from citrate to α-KG. Propofol diminished the citrate pool by ∼50% and α-KG by 30%. In contrast, propofol increased succinate near threefold, while slightly increasing fumarate, and maintaining malate, all considered within the distal CAC span. Therefore, the marked differences in these concentration profiles between the two anesthetics confirmed propofol-related impairments in flux through the CAC. Succinate represents a critical juncture between the first and distal span of the CAC. As succinate concentration depends partially on flux through mitochondrial complex II, which includes succinate dehydrogenase, we also assayed activity for the mitochondrial complexes. Absolute values for mitochondrial complex activities are shown in Table 2. Propofol did not affect citrate synthase activity but suppressed complex II dependent activities: Complex II (succinate: decylubiquinone oxidoreductase with dichlorophenolindophenol as terminal electron acceptor) and Complex II–III (succinate: cytochrome C oxidoreductase).

Figure 3.

Figure 3

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Absolute quantity of citric acid cycle (CAC) intermediates (A) and amino acids (B–E) in brain by gas chromatography–mass spectroscopy. Isoflurane increased citrate and α-ketoglutarate (α-KG) (P=0.0001 and 0.013), whereas it decreased succinate and fumarate (P=0.0004 and 0.006). Isoflurane significantly elevated aspartate (P=0.003) and glutamate (P=0.021), whereas isoflurane reduced alanine (P=0.004) when compared with propofol. Values are mean±s.e.; n=7 per group. *P<0.05, †P<0.01.

Table 2. Electron transport chain analysis (in nmol/minute per mg protein).

  ISO PROP P
Complex I 45.8±2.8 45.3±11.0 NS
Complex I–III 8.4±0.4 5.8±1.3 NS
Complex II 32.3±2.6 21.9±3.8 0.048
Complex II–III 11.1±1.9 6.2±1.1 0.037
Complex III 69.1±11.8 63.2±18.1 NS
Complex IV 62.2±12.3 48.6±7.2 NS
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Values are mean±s.e.; n=7 per group.

We also evaluated concentrations for amino acids that might be affected by pyruvate flux and CAC flux. We noted that propofol produced significantly higher concentrations for alanine, but statistically lower concentrations for glutamate, aspartate, and a trend toward lower glutamine (Figures 3B–3E).

Propofol Inhibits Pyruvate Decarboxylation Flux

The 13C-labeling protocol provides information regarding relative substrate oxidative flux rates. The cerebral cortex normally oxidizes glucose as the principal substrate to regenerate ATP from ADP. In the mitochondria, PDC produces acetyl-CoA that combines with oxaloacetate to form citrate. Thus, we infused 13C-glucose, but also included labeled leucine, a branched chain amino acid, as a reference source of acetyl-CoA. Representative 13C-NMR spectra are shown in Supplementary Figure S1. The NMR analyses showed that the labeled leucine provided only a small fraction of the total acetyl-CoA supplied to the CAC, regardless of the anesthetic used (Figure 4A). As noted, propofol relative to isoflurane elevated the concentration of 13C-pyruvate relative to pyruvate supplied by other unlabeled glycolytic sources. Consistent with that observation, the NMR data showed that labeled glucose fractional acetyl-CoA contribution increased slightly although not significantly with propofol. We further used the GC–MS data to estimate total (PDC)/(CIT) flux ratio. The (PDC)/(CIT) flux ratio with isoflurane was ∼1.0, indicating that citrate formation depended entirely on acetyl-CoA derived from pyruvate (Figure 4B). With propofol, the (PDC)/(CIT) flux ratio was substantially less than isoflurane (∼40%) implying a marked impairment in PDC. Enzyme activity for the PDH complex, responsible for PDC, is inhibited in part by phosphorylation at serine 293. However, we found paradoxically that propofol reduced phosphorylation status for this protein compared with isoflurane (Figure 4C).

Figure 4.

Figure 4

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Pyruvate decarboxylation. (A) Fractional substrate contribution (FC) to citric acid cycle (CAC) via acetyl-CoA by 13C-nuclear magnetic resonance (13C-NMR). Fractional contribution of glucose and leucine were not significantly different between two groups. (B) Pyruvate decarboxylation (PDC)-to-citrate synthesis (CIT) flux ratio in brain by gas chromatography–mass spectroscopy. Propofol significantly decreased PDC/CIT (P=0.048). (C) Phospho- and total pyruvate dehydrogenase (PDH) levels. Western blot analysis confirmed increased phosphorylation of PDH in ISO. (D) Total anaplerotic component relative to CAC flux by 13C-NMR. The anaplerotic component was not different between the groups. Values are mean±s.e.; n=7 per group. *P<0.05, †P<0.01.

The 13C-MPE generally provides information reflecting flux through the CAC. However, in these experiments the labeling strategy and carbon contributions from unlabeled substrates produced relatively low MPEs for all the intermediates. MPE of pyruvate was higher for propofol than for isoflurane (PROP: 0.098, ISO: 0.048, P=0.017), but this was not reflected similarly in MPE for citrate and the other intermediates. Molar percent enrichments for the intermediates were not statistically different between the two anesthetic conditions (Supplementary Figure S2). Lack of MPE difference for these intermediates between the two groups may be due a combination of factors, including our limited resolution for detecting MPE differences when 13C-MPE is low in both experimental groups.

The tcaCALC program also provides an algorithm for estimation of total anaplerosis from the NMR data. Total anaplerotic component to CAC flux was fairly high in the brain but not different between the anesthetics (Figure 4D). The algorithms for both NMR and GC–MS data could be influenced by recycling of M1-labeled intermediates. The conversion of labeled oxaloacetate (or equivalently malate) back to pyruvate and into acetyl-CoA would cause some scrambling of labeled carbons and result in labeling of the C2-pyruvate, and hence C2-lactate. We identified a very small C2-lactate peak in the NMR spectrum confirming recycling (Supplementary Figure S1). However, this peak was tiny compared with C3-lactate indicating that recycling may be present but is negligible. The small peak could also represent only natural 13C abundance.

Discussion

This study demonstrates that substantial differences exist in cerebral cortex substrate metabolism between anesthetic regimens, isoflurane, and propofol. Potential sites of anesthetic action on substrate metabolism are shown in Figure 5. Overall, propofol infusion results in greater glycogen and ATP depletion in immature swine brain. In this experimental model, we noted no differences between the two anesthetics in systemic hemodynamics or rSO2. We recognize that rSO2 does not provide accurate assessment of brain oxygen extraction or consumption. However, we did observe rSO2 stability in single animals as well as the lack of difference between the two groups. Thus, direct anesthetic effects on mitochondrial function and/or metabolism, rather than modulation of hemodynamics and oxygen supply, are likely responsible for these metabolic differences between the two groups. Furthermore, despite apparently undisturbed oxygen supply, the propofol-exposed cerebral cortex displays multiple metabolic impairments, resembling those commonly observed during hypoxia.

Figure 5.

Figure 5

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A summary schema of the proposed metabolic influences of propofol compared with isoflurane. Bold arrow; the pathways activated by propofol. I–IV: electron transport chain complexes. The schema does not include lactate shuttle from astrocytes to neurons. KG, ketoglutarate; OAA, oxaloacetate.

Glucose is the major metabolic fuel of the brain under the basal aerobic state. Under certain conditions, the brain extracts alternate substrates from the blood for oxidation. For instance, during starvation the brain utilizes acetoacetate and hydroxybutyrate.21, 22 Our GC–MS experimental methods evaluated the oxidative contribution for glucose relative to total CAC flux. Our study shows that propofol compared with isoflurane shifts the oxidative preference from glucose to an alternate, unlabeled substrate. Multiple studies performed in cultured neuronal cells or in brain slices suggested that leucine is also a significant contributor to oxidative metabolism in immature brain and might serve as the alternate substrate for glucose.12, 13 Leucine circumvents the pyruvate oxidative pathway and enters the CAC as acetyl-CoA in a manner analogous to ketone body oxidation. Therefore, we considered leucine as a potential alternate oxidative substrate to glucose. However, our studies performed in the intact pig brain suggest otherwise, as leucine contributed a minor acetyl-CoA fraction that does not change with anesthetic regimen.

We also noted that glycolytic flux to lactate increases with propofol. The net sum of glycolytic and CAC ATP generation with propofol was inadequate to prevent depletion of the ATP pool during 4 hours of anesthesia. We could not directly measure absolute oxidative flux rates for glucose or the CAC in these experiments. The ATP depletion suggests that propofol inhibits overall CAC flux. The GC–MS data showed that propofol also caused marked impairment in PDC via PDH. The precise mechanism for propofol inhibition of PDH flux remains unclear. Activity for this enzyme is regulated through inhibitory phosphorylation by PDH kinases.23 However, we noted a seemingly paradoxical decrease in PDH phosphorylation with propofol. This finding eliminates PDH inhibition by phosphorylation as a cause of the reduced (PDC)/(CIT) flux ratio. The cerebral cortex contains two distinct cell populations, astrocytes and neurons, which display different metabolic phenotypes.24 Substantial experimental evidence supports the lactate-shuttle hypothesis, whereby astrocytes and glial cells produce lactate through aerobic glycolysis, which then undergoes release and then uptake by neurons with subsequent oxidation via the CAC.25 We employed a 13C-labeling strategy that does not differentiate between pyruvate resulting either from astrocyte glycolysis or that produced in neurons via lactate oxidation. Also, our experimental methods do not determine whether lactate accumulated in the intracellular or in the extracellular compartment. Computer modeling based on data, obtained predominantly in vitro, suggests that regulation of aerobic glycolysis in astrocytes and PDC in neurons depend in part on the capacity for lactate transport between these cells.26 Our results showing propofol-induced PDC impairment, accompanied by lactate accumulation, could be explained by a reduction in lactate shuttling capacity into the neurons by the monocarboxylate transporters. These transporters are susceptible to pharmacological inhibition, which is known to suppress pyruvate fueled mitochondrial respiration.27 Thus, the monocarboxylate transporters represent a candidate target for propofol inhibition of PDC.

Lactate oxidation through lactate dehydrogenase requires conversion to pyruvate with production of NADH/H+ from NAD. As lactate is the primary oxidative substrate used by neurons, PDC also depends on recycling of NADH to NAD+.28 The inner mitochondrial membrane is impermeable to NADH and NAD+, and therefore requires shuttles, such as the malate–aspartate shuttle, to transfer reducing equivalents from NADH in the cytosol to the mitochondria. Acceleration of glycolysis and lactate production during ischemia or hypoxia is mediated in part by reduced malate–aspartate shuttle flux along with redistribution of shuttle-associated metabolites.29, 30 In the current study, we did not perform compartmental analyses for these metabolites. However, we did observe anesthetic-related differences in overall concentration for amino acids, aspartate, and glutamate, which participate in NADH/NAD+ shuttling. This may represent a subtle sign that propofol alters shuttling similar to hypoxia, and thereby alters PDH flux, but this possibility requires further investigation.

In addition to anesthetic-related differences in oxidative substrate utilization, we observed discrepancies in handling of the CAC intermediates. The propofol-induced reductions in concentrations for the citrate and α-KG are consistent with a potential lowering of CIT flux, suggested by the ATP depletion. However, the marked increase in succinate with propofol over 4 hours represents the major discrepancy with isoflurane noted within the CAC intermediate pool. Succinate is a recognized marker of anaerobic metabolism in mammals.31 Several metabolic pathways lead to succinate formation. Therefore, the mechanisms for succinate accumulation caused by propofol still require clarification in our model. In fully aerobic cells, including astrocytes and neurons, α-KG oxidation by α-KG dehydrogenase represents the dominant pathway leading to succinate. The forward succinate dehydrogenase reaction then catalyzes succinate to fumarate. This enzyme also catalyzes the reverse reductive reaction, which results in succinate formation from fumarate. This latter reaction generates ATP, but during normoxia operates at a rate ∼1000 times slower than the oxidative flux through the CAC.32 Additional carbons can be supplied for this reaction via pyruvate carboxylation to oxaloacetate, a major mode of pyruvate entry into the CAC in astrocytes.33 Some evidence, although still controversial, exists for pyruvate carboxylation via malic enzyme in neurons that lack pyruvate carboxylase.34 Hypoxia alters the relative succinate dehydrogenase flux rates in favor of fumarate reduction. Our enzyme activity measurements implicate a propofol-induced deficit in forward flux through mitochondrial complex II (succinate dehydrogenase) as a contributor to this imbalance. There are technical and theoretical limitations to this interpretation. Owing to volatility for isoflurane, we cannot offer assurance that concentration in vivo corresponds precisely to the ambient concentration in the mitochondria after isolation. Thus, we could have overestimated the difference between the anesthetics with regard to complex II inactivation. However, this difference is only seen for complex II-related activities; neither complex I nor downstream electron transport chain activities are affected. Second, the succinate dependent complex II assay only measures activity in the forward direction. Accordingly, the data do not include assessment of activity in the reverse direction. Nor do we provide data regarding succinate derived from alternative pathways that remain undefined but contribute substantially to succinate during hypoxia.32

Limitations and Pitfalls

As the techniques used in this study require rapid tissue extraction and freeze clamping, we were able to sample from only one parietal cortex location; therefore, we cannot be certain that these findings occur throughout the brain. However, most previous studies examining CAC metabolism in brain slices indicate uniformity with metabolic perturbations.35 A prior study evaluated the metabolomic profile with 1H-NMR in children (age 2 to 7 years) and found that sevoflurane anesthesia elevated parietal cortex glucose and lactate more than propofol did over 1 hour.36 Unlike our study, that investigation did not use tracers o highly sensitive GC–MS to determine the source of lactate, and those authors acknowledged the technical limitations in accurately assessing the metabolites using 1H-NMR. The discrepancy for results between the two studies could be explained by developmental or species differences. We used piglets just slightly older than neonates in this study. Although, difficult to make neurodevelopmental comparisons between species, several studies show that brain growth rates are similar between humans and pigs during the first year after birth. Accordingly, on the developmental scale, these piglets should approximate human infants between 1 and 6 months of age substantially younger than the children evaluated in that prior study.37 The differences in results between the two studies might also be explained by anesthetic duration and pharmacodynamics of sevoflurane versus isoflurane. In addition, interaction with ketamine, used as a preanesthetic in this study, might differ between propofol and isoflurane. Metabolism was studied more than 4 hours after ketamine, and therefore we believe it is an unlikely contributor to the differences we noted between the two groups.

Clinical Implications

Brain energy metabolite depletion may occur in anesthetized young infants and children with no apparent early clinical signs. One clinical trial used propofol as an anesthetic bridge to extubation in children after surgery for congenital heart disease.38 Duration of propofol in that study was as long as 36 hours. Our study results indicate that a 4-hour propofol infusion may have unrecognized detrimental impact on the brain. These brain metabolic abnormalities occurred without typical systemic metabolic acidosis that occurs in classic propofol infusion syndrome. Patients already at risk for brain injury during cardiopulmonary bypass procedures or those with other metabolic or mitochondrial defects may be particularly vulnerable to propofol. There is currently reluctance among some anesthesiologists to use propofol on children with mitochondrial defects.39, 40 Our data may provide insights into its unique, and potentially toxic, metabolic profile in this patient population.

The authors declare no conflict of interest.

Footnotes

Supplementary Information accompanies the paper on the Journal of Cerebral Blood Flow & Metabolism website (http://www.nature.com/jcbfm)

This work was supported by the National Institutes of Health R01HL60666 to MA Portman. A portion of the research was performed using Environmental Molecular Sciences Laboratory (EMSL), a national scientific user facility sponsored by the Department of Energy's Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory.

Supplementary Material

Supplementary Figure
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Supplementary Figure Legend
Click here for additional data file. (23KB, doc)

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Supplementary Materials

Supplementary Figure
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