How to Personalize General Anesthesia-A Prospective Theoretical Approach to Conformational Changes of Halogenated Anesthetics in Fire Smoke Poisoning.

Smoke intoxication is a central event in mass burn incidents, and toxic smoke acts at different levels of the body, blocking breathing and oxygenation. The majority of these patients require early induction of anesthesia to preserve vital functions. We studied the influence of hemoglobin (HMG) and myoglobin (MGB) blockade by hydrochloric acid (HCl) in an interaction model with gaseous anesthetics using molecular docking techniques. In the next part of the study, molecular dynamics (MD) simulations were performed on the top-scoring ligand-receptor complexes to investigate the stability of the ligand-receptor complexes and the interactions between ligands and receptors in more detail. Through docking analysis, we observed that hemoglobin creates more stable complexes with anesthetic gases than myoglobin. Intoxication with gaseous hydrochloric acid produces conformational and binding energy changes of anesthetic gases to the substrate (both the pathway and the binding site), the most significant being recorded in the case of desflurane and sevoflurane, while for halothane and isoflurane, they remain unchanged. According to our theoretical model, the selection of anesthetic agents for patients affected by fire smoke containing hydrochloric acid is critical to ensure optimal anesthetic effects. In this regard, our model suggests that halothane and isoflurane are the most suitable choices for predicting the anesthetic effects in such patients when compared to sevoflurane and desflurane.

Scientific Accuracy Matters.

The Solubility of Halothane in Blood and Tissue Homogenates. By Larson CP, Eger EI, Severinghaus JW. Anesthesiology 1962; 23:349-55. Measured samples of human and bovine blood, human hemoglobin, and tissue homogenates from human fat and both human and bovine liver, kidney, muscle, whole brain, and separated gray and white cortex were added to stoppered 2,000-ml Erlenmeyer flasks. To each flask, 0.1 ml of liquid halothane was added under negative pressure using a calibrated micropipette. After the flask was agitated for 2 to 4 h to achieve equilibrium between the gas and blood or tissue contents, a calibrated infrared halothane analyzer was used to measure the concentration of halothane vapor. Calculated partition coefficients ranged from 0.7 for water to 2.3 for blood and from 3.5 for human or bovine kidney to 6 for human whole brain or liver and 8 for human muscle. Human peritoneal fat had a value of 138. The human blood-gas partition coefficient of 2.3 as determined by this equilibration method was well below the previously published value of 3.6.

Molecular Bases for Anesthetic Agents: Halothane as a Halogen- and Hydrogen-Bond Donor.

Although instrumental for optimizing their pharmacological activity, a molecular understanding of the preferential interactions given by volatile anesthetics is quite poor. This paper confirms the ability of halothane to work as a hydrogen-bond (HB) donor and gives the first experimental proof that halothane also works as a halogen-bond (HaB) donor in the solid state and in solution. A halothane/hexamethylphosphortriamide co-crystal is described and its single-crystal X-ray structure shows short HaBs between bromine, or chlorine, and the phosphoryl oxygen. New UV/Vis absorption bands appear upon addition of diazabicyclooctane and tetra(n-butyl)ammonium iodide to halothane solutions, indicating that nitrogen atoms and anions may mediate the HaB-driven binding processes involving halothane as well. The ability of halothane to work as a bidentate/tridentate tecton by acting as a HaB and HB donor gives an atomic rationale for the eudismic ratio shown by this agent.

Molecular dynamics simulation study of the effect of halothane on mixed DPPC/DPPE phospholipid membranes.

We report results of a molecular dynamics simulation study of the effect of one general anesthetic, halothane, on some properties of mixed DPPC/DPPE phospholipid membranes. This is a suitable model for the study of simple, two-phospholipid membrane systems. From the simulation runs, we determined several membrane properties for five different molecular proportions of DPPC/DPPE. The effect of halothane on the studied membrane properties (area per lipid molecule, density of membrane, order parameter, etc.) was rather small. The distribution of halothane is not uniform through the bilayer thickness. Instead, there is a maximum of anesthetic concentration around 1.2 nm from the center of the membrane. The anesthetic molecule is located close to the phospholipid headgroups. The position of the halothane density maximum depends slightly on the DPPC/DPPE molar proportion. Snapshots taken over the plane of the membrane, as well as calculated two-dimensional radial distribution functions show that the anesthetic has no preference for either phospholipid (DPPC or DPPE). Our results indicate that this anesthetic molecule has only small effects on DPPC/DPPE mixed membranes. In addition, halothane displays no preferential location around DPPC or DPPE. This is probably due to the hydrophobic nature of halothane and to the fact that the chosen phospholipids have the same hydrophobic tails.

Investigation of Anesthetic-Protein Interactions by a Thermodynamic Approach.

Anesthetics can interact with a wide variety of proteins in the body, including ion channels and alter their activity, but little is known about the molecular mechanisms of the interactions responsible for the functional activity. Characterization of the nature of anesthetic-protein interactions therefore is important and requires the complete analysis of the binding energetics. Isothermal titration calorimetry (ITC) is the only technique that allows quantitative determination of all thermodynamic parameters, including the equilibrium binding constant (KB), the standard Gibbs free energy change (ΔG), the enthalpy change (ΔH), the entropy change (ΔS), heat capacity change (ΔCp), and stoichiometry (n) of the reaction. ITC does not require any labeling or modification of the interacting partners analyzed and can be performed in solution with small amounts of reagents. In this chapter we describe the general properties of the ITC method, highlighting some critical aspects of experimental planning and data analysis, with practical application to anesthetic-protein interactions.

Solution NMR Studies of Anesthetic Interactions with Ion Channels.

NMR spectroscopy is one of the major tools to provide atomic resolution protein structural information. It has been used to elucidate the molecular details of interactions between anesthetics and ion channels, to identify anesthetic binding sites, and to characterize channel dynamics and changes introduced by anesthetics. In this chapter, we present solution NMR methods essential for investigating interactions between ion channels and general anesthetics, including both volatile and intravenous anesthetics. Case studies are provided with a focus on pentameric ligand-gated ion channels and the voltage-gated sodium channel NaChBac.

Ligand partitioning into lipid bilayer membranes under high pressure: Implication of variation in phase-transition temperatures.

The variation in phase-transition temperatures of dipalmitoylphosphatidylcholine (DPPC) bilayer membrane by adding two membrane-active ligands, a long-chain fatty acid (palmitic acid (PA)) and an inhalation anesthetic (halothane (HAL)), was investigated by light-transmittance measurements and fluorometry. By assuming the thermodynamic colligative property for the bilayer membrane at low ligand concentrations, the partitioning behavior of these ligands into the DPPC bilayer membrane was considered. It was proved from the differential partition coefficients between two phases that PA has strong affinity with the gel (lamellar gel) phase in a micro-molal concentration range and makes the bilayer membrane more ordered, while HAL has strong affinity with the liquid crystalline phase in a milli-molal concentration range and does the bilayer membrane more disordered. The transfer volumes of both ligands from the aqueous solution to each phase of the DPPC bilayer membrane showed that the preferential partitioning of the PA molecule into the gel (lamellar gel) produces about 20% decrease in transfer volume as compared with the liquid crystalline phase, whereas that of the HAL molecule into the liquid crystalline phase does about twice increase in transfer volume as compared with the gel (ripple gel) phase. Furthermore, changes in thermotropic and barotropic phase behavior of the DPPC bilayer membrane by adding the ligand was discussed from the viewpoint of the ligand partitioning. Reflecting the contrastive partitioning of PA and HAL into the pressure-induced interdigitated gel phase among the gel phases, it was revealed that PA suppresses the formation of the interdigitated gel phase under high pressure while HAL promotes it. These results clearly indicate that each phase of the DPPC bilayer membrane has a potential to recognize various ligand molecules.

Lateral Pressure Profile and Free Volume Properties in Phospholipid Membranes Containing Anesthetics.

The effect of four general anesthetics, namely chloroform, halothane, diethyl ether, and enflurane on the free volume fraction and lateral pressure profiles in a fully hydrated dipalmitoylphosphatidylcholime (DPPC) membrane is investigated by means of computer simulation. In order to find changes that can be related to the molecular mechanism of anesthesia as well as its pressure reversal, the simulations are performed both at atmospheric and high (1000 bar) pressures. The obtained results show that the additional free volume occurring in the membrane is localized around the anesthetic molecules themselves. Correspondingly, the fraction of the free volume is increased in the outer of the two membrane regions (i.e., at the outer edge of the hydrocarbon phase) where anesthetic molecules prefer to stay in every case. As a consequence, the presence of anesthetics decreases the lateral pressure in the nearby region of the lipid chain ester groups, in which the anesthetic molecules themselves do not penetrate. Both of these changes, occurring upon introducing anesthetics in the membrane, are clearly reverted by the increase of the global pressure. These findings are in accordance both with the more than 60 years old "critical volume hypothesis" of Mullins, and with the more recent "lateral pressure hypothesis" of Cantor. Our results suggest that if anesthesia is indeed caused by conformational changes of certain membrane-bound proteins, induced by changes in the lateral pressure profile, as proposed by Cantor, the relevant conformational changes are expected to occur in the membrane region where the ester groups are located.

Hydrostatic Pressure Promotes Domain Formation in Model Lipid Raft Membranes.

Neutron diffraction measurements demonstrate that hydrostatic pressure promotes liquid-ordered (Lo) domain formation in lipid membranes prepared as both oriented multilayers and unilamellar vesicles made of a canonical ternary lipid mixture for which demixing transitions have been extensively studied. The results demonstrate an unusually large dependence of the mixing transition on hydrostatic pressure. Additionally, data at 28 °C show that the magnitude of increase in Lo caused by 10 MPa pressure is much the same as the decrease in Lo produced by twice minimum alveolar concentrations (MAC) of general anesthetics such as halothane, nitrous oxide, and xenon. Therefore, the results may provide a plausible explanation for the reversal of general anesthesia by hydrostatic pressure.

Emulsified halothane produces long-term epidural anesthetic effect: a study in rabbits.

Previous studies have demonstrated that volatile anesthetics could produce local anesthesia. Emulsified isoflurane at 8% has been reported to produce epidural anesthetic effect in rabbits. This study was designed to investigate the long-term epidural anesthetic effect of emulsified halothane in rabbits. In this study, 40 healthy adult rabbits (weighting 2.0-2.5 kg) with an epidural catheter were randomly divided into 4 groups (n=10/group), receiving epidural administration of 1% lidocaine (lido group), 8% emulsified isoflurane 1ml (8% E-iso group), 8% emulsified halothane (8% E-Halo group) and 12% emulsified halothane (12% E-Halo group). After administration, sensory and motor functions as well as consciousness state were assessed until 60 minutes after sensory and motor function returned to its baseline or at least for 180 min. After epidural anesthesia, all the rabbits were continuously observed for 7 days and sacrificed for pathological evaluations. As a result, all the four study solutions produced typical epidural anesthesia. Onset times of sensory and motor function blockade were similar among the four groups (P>0.05). Duration of sensory blockade in 12% E-Halo group (83±13 min) was significantly longer than other groups: 51±12 min in 8% E-Halo group (P<0.01), 57±8 min in 8% E-iso group (P<0.01) and 47±9 min in lido group (P<0.01). Duration of sensory blockade in 8% E-iso group is longer than lido group (P<0.05). Duration of motor blockade in 12% E-Halo group (81±12 min) was also significantly longer than other groups: 40±8 min in 8% E-Halo group (P<0.01), 37±3 min in 8% E-iso group (P<0.01), 37±6 min in lido group (P<0.01). Normal consciousness was found in the rabbits from 8% E-Halo, 8% E-iso and lido groups while there were four rabbits in 12% E-Halo group (4/10) showed a light sedation. For all the rabbits, no pathological injury was found. The present study demonstrates that emulsified halothane produces reversible concentration-dependent epidural anesthesia and at 12% (v/v), emulsified halothane could produce long-term anesthesia without pathological injury.

Electronic State Spectroscopy of Halothane As Studied by ab Initio Calculations, Vacuum Ultraviolet Synchrotron Radiation, and Electron Scattering Methods.

We present the first set of ab initio calculations (vertical energies and oscillator strengths) of the valence and Rydberg transitions of the anaesthetic compound halothane (CF3CHBrCl). These results are complemented by high-resolution vacuum ultraviolet photoabsorption measurements over the wavelength range 115-310 nm (10.8-4.0 eV). The spectrum reveals several new features that were not previously reported in the literature. Spin-orbit effects have been considered in the calculations for the lowest-lying states, allowing us to explain the broad nature of the 6.1 and 7.5 eV absorption bands assigned to σ*(C-Br) ← nBr and σ*(C-Cl) ← n(Cl) transitions. Novel absolute photoabsorption cross sections from electron scattering data were derived in the 4.0-40.0 eV range. The measured absolute photoabsorption cross sections have been used to calculate the photolysis lifetime of halothane in the upper stratosphere (20-50 km).

Calculation of volatile anaesthetics consumption from agent concentration and fresh gas flow.

BACKGROUND: The assessment of volatile agents' consumption can be performed by weighing vapourisers before and after use. This method is technically demanding and unavailable for retrospective analysis of anaesthesia records. Therefore, a method based on calculations from fresh gas flow and agent concentration is presented here. METHODS: The presented calculation method herein enables a precise estimation of volatile agent consumption when average fresh gas flows and volatile agent concentrations are known. A pre-condition for these calculations is the knowledge of the vapour amount deriving from 1 ml fluid volatile agent. The necessary formulas for these calculations and an example for a sevoflurane anaesthesia are presented. RESULTS: The amount of volatile agent vapour deriving from 1 ml of fluid agent are for halothane 229 ml, isoflurane 195 ml, sevoflurane 184 m, and desflurane 210 ml. The constant for sevoflurane is used in a fictitious clinical case to exemplify the calculation of its consumption in daily routine resulting in a total expenditure of 23.6 ml liquid agent. CONCLUSIONS: By application of the presented specific volatile agent constants and equations, it becomes easy to calculate volatile agent consumption if the fresh gas flows and the resulting inhaled concentration of the volatile agent are known. By this method, it is possible to extract data about volatile agent consumption both ways: (1) retrospectively from sufficiently detailed and accurate anaesthesia recordings, as well as (2) by application of this method in a prospective setting. Therefore, this method is a valuable contribution to perform pharmacoeconomical surveys.

NMR structures of the human α7 nAChR transmembrane domain and associated anesthetic binding sites.

The α7 nicotinic acetylcholine receptor (nAChR), assembled as homomeric pentameric ligand-gated ion channels, is one of the most abundant nAChR subtypes in the brain. Despite its importance in memory, learning and cognition, no structure has been determined for the α7 nAChR TM domain, a target for allosteric modulators. Using solution state NMR, we determined the structure of the human α7 nAChR TM domain (PDB ID: 2MAW) and demonstrated that the α7 TM domain formed functional channels in Xenopus oocytes. We identified the associated binding sites for the anesthetics halothane and ketamine; the former cannot sensitively inhibit α7 function, but the latter can. The α7 TM domain folds into the expected four-helical bundle motif, but the intra-subunit cavity at the extracellular end of the α7 TM domain is smaller than the equivalent cavity in the α4ß2 nAChRs (PDB IDs: 2LLY; 2LM2). Neither drug binds to the extracellular end of the α7 TM domain, but two halothane molecules or one ketamine molecule binds to the intracellular end of the α7 TM domain. Halothane and ketamine binding sites are partially overlapped. Ketamine, but not halothane, perturbed the α7 channel-gate residue L9'. Furthermore, halothane did not induce profound dynamics changes in the α7 channel as observed in α4ß2. The study offers a novel high-resolution structure for the human α7 nAChR TM domain that is invaluable for developing α7-specific therapeutics. It also provides evidence to support the hypothesis: only when anesthetic binding perturbs the channel pore or alters the channel motion, can binding generate functional consequences.

DNA testing for malignant hyperthermia: the reality and the dream.

The advent of the polymerase chain reaction and the availability of data from various global human genome projects should make it possible, using a DNA sample isolated from white blood cells, to diagnose rapidly and accurately almost any monogenic condition resulting from single nucleotide changes. DNA-based diagnosis for malignant hyperthermia (MH) is an attractive proposition, because it could replace the invasive and morbid caffeine-halothane/in vitro contracture tests of skeletal muscle biopsy tissue. Moreover, MH is preventable if an accurate diagnosis of susceptibility can be made before general anesthesia, the most common trigger of an MH episode. Diagnosis of MH using DNA was suggested as early as 1990 when the skeletal muscle ryanodine receptor gene (RYR1), and a single point mutation therein, was linked to MH susceptibility. In 1994, a single point mutation in the α 1 subunit of the dihydropyridine receptor gene (CACNA1S) was identified and also subsequently shown to be causative of MH. In the succeeding years, the number of identified mutations in RYR1 has grown, as has the number of potential susceptibility loci, although no other gene has yet been definitively associated with MH. In addition, it has become clear that MH is associated with either of these 2 genes (RYR1 and CACNA1S) in only 50% to 70% of affected families. While DNA testing for MH susceptibility has now become widespread, it still does not replace the in vitro contracture tests. Whole exome sequence analysis makes it potentially possible to identify all variants within human coding regions, but the complexity of the genome, the heterogeneity of MH, the limitations of bioinformatic tools, and the lack of precise genotype/phenotype correlations are all confounding factors. In addition, the requirement for demonstration of causality, by in vitro functional analysis, of any familial mutation currently precludes DNA-based diagnosis as the sole test for MH susceptibility. Nevertheless, familial DNA testing for MH susceptibility is now widespread although limited to a positive diagnosis and to those few mutations that have been functionally characterized. Identification of new susceptibility genes remains elusive. When new genes are identified, it will be the role of the biochemists, physiologists, and biophysicists to devise functional assays in appropriate systems. This will remain the bottleneck unless high throughput platforms can be designed for functional work. Analysis of entire genomes from several individuals simultaneously is a reality. DNA testing for MH, based on current criteria, remains the dream.

NMR resolved multiple anesthetic binding sites in the TM domains of the α4ß2 nAChR.

The α4ß2 nicotinic acetylcholine receptor (nAChR) has significant roles in nervous system function and disease. It is also a molecular target of general anesthetics. Anesthetics inhibit the α4ß2 nAChR at clinically relevant concentrations, but their binding sites in α4ß2 remain unclear. The recently determined NMR structures of the α4ß2 nAChR transmembrane (TM) domains provide valuable frameworks for identifying the binding sites. In this study, we performed solution NMR experiments on the α4ß2 TM domains in the absence and presence of halothane and ketamine. Both anesthetics were found in an intra-subunit cavity near the extracellular end of the ß2 transmembrane helices, homologous to a common anesthetic binding site observed in X-ray structures of anesthetic-bound GLIC (Nury et al., [32]). Halothane, but not ketamine, was also found in cavities adjacent to the common anesthetic site at the interface of α4 and ß2. In addition, both anesthetics bound to cavities near the ion selectivity filter at the intracellular end of the TM domains. Anesthetic binding induced profound changes in protein conformational exchanges. A number of residues, close to or remote from the binding sites, showed resonance signal splitting from single to double peaks, signifying that anesthetics decreased conformation exchange rates. It was also evident that anesthetics shifted population of two conformations. Altogether, the study comprehensively resolved anesthetic binding sites in the α4ß2 nAChR. Furthermore, the study provided compelling experimental evidence of anesthetic-induced changes in protein dynamics, especially near regions of the hydrophobic gate and ion selectivity filter that directly regulate channel functions.

Anesthetic molecules embedded in a lipid membrane: a computer simulation study.

The effect of four general anesthetic molecules, i.e., chloroform, halothane, diethyl ether and enflurane, on the properties of a fully hydrated dipalmitoylphosphatidylcholine (DPPC) membrane is studied in detail by long molecular dynamics simulations. Furthermore, to address the problem of pressure reversal, the effect of pressure on the anesthetic containing membranes is also investigated. In order to ensure sufficient equilibration and adequate sampling, the simulations performed have been at least an order of magnitude longer than the studies reported previously in the literature on general anesthetics. The results obtained can help in resolving several long-standing contradictions concerning the effect of anesthetics, some of which were the consequence of too short simulation time used in several previous studies. More importantly, a number of seeming contradictions are found to originate from the fact that different anesthetic molecules affect the membrane structure differently in several respects. In particular, halothane, being able to weakly hydrogen bound to the ester group of the lipid tails, is found to behave in a markedly different way than the other three molecules considered. Besides, we also found that two changes, namely lateral expansion of the membrane and increasing local disorder in the lipid tails next to the anesthetic molecules, are clearly induced by all four anesthetic molecules tested here in the same way, and both of these effects are reverted by the increase in pressure.

Effects of isoflurane, halothane, and chloroform on the interactions and lateral organization of lipids in the liquid-ordered phase.

The first quantitative insight has been obtained into the effects that volatile anesthetics have on the interactions and lateral organization of lipids in model membranes that mimic "lipid rafts". Specifically, nearest-neighbor recogntion measurements, in combination with Monte Carlo simulations, have been used to investigate the action of isoflurane, halothane, and chloroform on the compactness and lateral organization of cholesterol-rich bilayers of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in the liquid-ordered (l(o)) phase. All three anesthetics induce a similar weakening of sterol-phospholipid association, corresponding to ca. 30 cal/mol of lipid at clinically relevant concentrations. Monte Carlo lattice simulations show that the lateral organization of the l(o) phase, under such conditions, remains virtually unchanged. In sharp contrast to their action on the l(o) phase, these anesthetics have been found to have a similar strengthening effect on sterol-phospholipid association in the liquid-disordered (l(d)) phase. The possibility of discrete complexes being formed between DPPC and these anesthetics and the biological relevance of these findings are discussed.

Halogen bonded complexes between volatile anaesthetics (chloroform, halothane, enflurane, isoflurane) and formaldehyde: a theoretical study.

The structures and intermolecular interactions in the halogen bonded complexes of anaesthetics (chloroform, halothane, enflurane and isoflurane) with formaldehyde were studied by ab initio MP2 and CCSD(T) methods. The CCSD(T)/CBS calculated binding energies of these complexes are between -2.83 and -4.21 kcal mol(-1). The largest stabilization energy has been found for the C-Br···O bonded halothane···OCH(2) complex. In all complexes the C-X bond length (where X = Cl, Br) is slightly shortened, in comparison to a free compound, and an increase of the C-X stretching frequency is observed. The electrostatic interaction was excluded as being responsible for the C-X bond contraction. It is suggested that contraction of the C-X bond length can be explained in terms of the Pauli repulsion (the exchange overlap) between the electron pairs of oxygen and halogen atoms in the investigated complexes. This is supported by the DFT-SAPT results, which indicate that the repulsive exchange energy overcompensates the electrostatic one. Moreover, the dispersion and electrostatic contributions cover about 95% of the total attraction forces, in these complexes.

G protein {beta}{gamma} gating confers volatile anesthetic inhibition to Kir3 channels.

G protein-activated inwardly rectifying potassium (GIRK or Kir3) channels are directly gated by the ßγ subunits of G proteins and contribute to inhibitory neurotransmitter signaling pathways. Paradoxically, volatile anesthetics such as halothane inhibit these channels. We find that neuronal Kir3 currents are highly sensitive to inhibition by halothane. Given that Kir3 currents result from increased Gßγ available to the channels, we asked whether reducing available Gßγ to the channel would adversely affect halothane inhibition. Remarkably, scavenging Gßγ using the C-terminal domain of ß-adrenergic receptor kinase (cßARK) resulted in channel activation by halothane. Consistent with this effect, channel mutants that impair Gßγ activation were also activated by halothane. A single residue, phenylalanine 192, occupies the putative Gßγ gate of neuronal Kir3.2 channels. Mutation of Phe-192 at the gate to other residues rendered the channel non-responsive, either activated or inhibited by halothane. These data indicated that halothane predominantly interferes with Gßγ-mediated Kir3 currents, such as those functioning during inhibitory synaptic activity. Our report identifies the molecular correlate for anesthetic inhibition of Kir3 channels and highlights the significance of these effects in modulating neurotransmitter-mediated inhibitory signaling.