Dr Florian Nachon
email: florian@nachon.net

Eugenie Carletti

Marielle Wandhammer

Yvain Nicolet

Lawrence M Schopfer

Patrick Masson

Oksana Lockridge

X-ray crystallographic snapshots of reaction intermediates in the G117H mutant of human butyrylcholinesterase, a nerve agent target engineered into a catalytic bioscavenger

Keywords: acetylcholinesterase, butyrylcholinesterase, bioscavenger, reactivation, engineering, organophosphorus Biochemical AChE, Acetylcholinesterase, BChE, Butyrylcholinesterase, ChE, Cholinesterase, CHO, Chinese hamster ovary

Introduction

Recent progress in the understanding of enzyme catalysis, through the use of structural biology and molecular modeling has opened a new era in protein engineering. It is now possible to develop rational strategies for modifying enzymes to confer upon them the ability to catalyze reactions for which they were not originally designed [START_REF] Gerlt | Enzyme (re)design: lessons from natural evolution and computation[END_REF]. Human butyrylcholinesterase (BChE; EC 3.1.1.8) is an enzyme for which modifying the catalytic activity has been an ongoing subject of interest. This enzyme is related to acetylcholinesterase (AChE; EC 3.1.1.7), the enzyme that terminates the action of the neurotransmitter acetylcholine at postsynaptic membranes and neuromuscular junctions. BChE differs from AChE by its substrate specificity and inhibitor sensitivity [START_REF] Harel | Conversion of acetylcholinesterase to butyrylcholinesterase: modeling and mutagenesis[END_REF][START_REF] Kaplan | Does "butyrylization" of acetylcholinesterase through substitution of the six divergent aromatic amino acids in the active center gorge generate an enzyme mimic of butyrylcholinesterase?[END_REF]. Although BChE is present in numerous vertebrate tissues, its physiological role remains unclear [START_REF] Li | Abundant tissue butyrylcholinesterase and its possible function in the acetylcholinesterase knockout mouse[END_REF].

Considerable interest has been shown to BChE because it hydrolyzes a wide range of toxic esters, including heroin and cocaine, and because it scavenges toxic organophosphylate pesticides and nerve agents [START_REF] Schopfer | Development of diagnostics in the search for an explanation of toxic airline syndrome[END_REF][START_REF] Lockridge | Pesticides and susceptible populations: people with butyrylcholinesterase genetic variants may be at risk[END_REF][START_REF] Masson | Structure, Activities and Biomedical Applications of Human Butyrylcholinesterase[END_REF]. A large effort has been expended to improve its ability to hydrolyze these compounds. In this regard, it is noteworthy to mention that redesign of the active site improved its (-)-cocaine hydrolase activity by 2000-fold, turning it into a very efficient cocaine detoxifying tool [START_REF] Zheng | Most efficient cocaine hydrolase designed by virtual screening of transition states[END_REF]. This redesign was based on hybrid quantum mechanical/ molecular mechanical (QM/MM) studies aimed at finding ways to stabilize the acylation transition state of the cocaine hydrolysis reaction.

Reaction of cholinesterases with organophosphorus compounds (OPs) is another area in which modification of the native cholinesterase activity has received considerable attention. Interest in this area is a consequence of the irreversible inhibition of AChE (at the neuronal synapses and neuromuscular junctions) by organophosphorus compounds which leads to accumulation of acetylcholine and results in paralysis, seizures, and other symptoms of the cholinergic syndrome. Under extreme conditions, inhibition of AChE can lead to death [START_REF] Heilbronn | Molecular biology of cholinesterases, a background and an introduction[END_REF]. Native plasma BChE works well as a stoichiometric bioscavenger because it reacts very efficiently with organophosphorus compounds, thus trapping them as a one-to-one complex in the blood stream before they can reach synaptic AChE. Injection of wild-type BChE iv or im protects animals against 3 to 5 LD50 doses of the nerve agents soman, VX, and tabun [10]. BChE purified from human plasma (Baxter Healthcare Corporation) or produced in the milk of transgenic goats [START_REF] Huang | Recombinant human butyrylcholinesterase from milk of transgenic animals to protect against organophosphate poisoning[END_REF] has successfully completed clinical trials and is about to reach the market as a bioscavenger for pretreatment against OP intoxication. A BChE dose up to 250 mg / 70 kg is required to achieve efficient protection of humans following a challenge with 1 LD50 of OPs [START_REF] Ashani | Estimation of the upper limit of human butyrylcholinesterase dose required for protection against organophosphates toxicity: a mathematically based toxicokinetic model[END_REF]. Thus, a large amount of enzyme is necessary to get an efficient protection. This is due to the stoichiometric and irreversible nature of the reaction between the OP and butyrylcholinesterase, and to the unfavorable OP/BChE mass ratio. Such large doses are expensive, and will prevent the widespread use of wild-type BChE as a pretreatment. Research efforts now are devoted to circumventing this limitation by developing catalytic bioscavengers able to hydrolyze OPs [START_REF] Lenz | Stoichiometric and catalytic scavengers as protection against nerve agent toxicity: a mini review[END_REF].

Very early in the study of OP inhibition of cholinesterases, it was suggested that OP inhibitors could be considered as hemisubstrates of cholinesterases because formation of the enzyme-inhibitor complex was efficient but the dephosphylation was not. Slow dephosphylation was presumed to be a consequence of there being no amino acid groups at the appropriate positions to catalyze hydrolysis by in-line nucleophilic attack of water [START_REF] Järv | Stereochemical aspects of cholinesterase catalysis[END_REF]. The idea of introducing a residue into the active site, that was capable of catalyzing the dephosphylation step, followed. In principle, after such a modification, OPs would become full substrates for the modified cholinesterase, or in other words, the added residue would turn the cholinesterase into an organophospho-hydrolase, i.e. a catalytic bioscavenger [START_REF] Masson | A collaborative endeavor to design cholinesterase-based catalytic scavengers against toxic organophosphorus esters[END_REF].

This approach proved to be qualitatively successful. BChE was shown to gain OP hydrolase activity when a histidine was substituted for glycine at position 117 [START_REF] Millard | Design and expression of organophosphorus acid anhydride hydrolase activity in human butyrylcholinesterase[END_REF][START_REF] Lockridge | A single amino acid substitution, Gly117His, confers phosphotriesterase (organophosphorus acid anhydride hydrolase) activity on human butyrylcholinesterase[END_REF]. The G117H mutant is remarkably efficient at hydrolyzing echothiophate, having a catalytic rate constant, k cat = 0.75 min -1 in 0.1 M potassium phosphate, pH 7.0, 25°C. G117H can also hydrolyze the nerve agents sarin, VX, and additionally soman if a second mutation (E197Q) is introduced to prevent fast aging of the conjugate [START_REF] Millard | Organophosphorus acid anhydride hydrolase activity in human butyrylcholinesterase: synergy results in a somanase[END_REF]. Dephosphylation remains the rate-limiting step with k cat = 5.2x10 -3 min -1 for sarin, 7.2x10 -3 min -1 for VX, and 77.0x10 -3 min -1 for the most toxic isomer of soman (P S C S ) (0.067 M phosphate buffer, pH 7.5, 25 °C). Furthermore, it has been shown that transgenic mice expressing the G117H mutant are resistant to OP [START_REF] Wang | Resistance to organophosphorus agent toxicity in transgenic mice expressing the G117H mutant of human butyrylcholinesterase[END_REF]. Although G117H/E197Q is generally considered to reactivate too slowly to be of general use as a catalytic bioscavenger for nerve agents, it provides an improvement over the native enzyme. Spontaneous reactivation of soman-, sarin-or VX-inhibited G117H/E197Q replenishes half of the affected enzyme in about 10 minutes [START_REF] Millard | Organophosphorus acid anhydride hydrolase activity in human butyrylcholinesterase: synergy results in a somanase[END_REF]. This could be lifesaving in a scenario where successive exposures to these OPs occur.

To date, more than sixty BChE mutants have been made in an effort to improve the OP hydrolase activity of the original G117H mutant. It was found that G117D, G117E, L286H, and several G117Hbased double and triple mutants were capable of hydrolyzing echothiophate [START_REF] Schopfer | Mutants of human butyrylcholinesterase with organophosphate hydrolase activity; evidence that His117 serves as a general base catalyst[END_REF]. However, G117H remains the most efficient mutant by at least two orders of magnitude. One reason for this relative failure to find an improved mutant is that the dephosphylation mechanism is poorly understood. Knowledge of the structure of the active site of G117H now appears to be critical in order to understand the catalytic mechanism and to design more efficient G117H-based mutants. An indication of the pressing need for an experimentally determined structure of the active site of G117H is provided by two recent publications [21,[START_REF] Vyas | Butyrylcholinesterase and G116H, G116S, G117H, G117N, E197Q and G117H/E197Q mutants: A molecular dynamics study[END_REF] in which the three dimensional structure of the G117H BChE mutant was determined computationally. Though they are interesting, such models cannot replace an experimental structure.

Here we present the X-ray structure of the G117H mutant, the structures of the transient covalent adducts formed upon reaction of G117H with echothiophate and VX (Scheme 1), and the structure of a fluoride complex which was the consequence of an attempt to create a transition-state phosphate analog in the form of MgF 3 -. These crystal structures give a true picture of the active site of G117H under a variety of conditions, and provide the essential information needed to perform QM/MM studies aimed at designing mutants with improved hydrolytic capabilities.

Methods

Production of the recombinant G117H mutant of human BChE, purification and crystallization. The crystallizable, low-glycosylated, monomeric form of human BChE was mutated into G117H [START_REF] Millard | Design and expression of organophosphorus acid anhydride hydrolase activity in human butyrylcholinesterase[END_REF], produced in CHO cells, and purified under conditions similar to those described for the wild-type enzyme [START_REF] Nachon | Engineering of a monomeric and low-glycosylated form of human butyrylcholinesterase: expression, purification, characterization and crystallization[END_REF]. Crystals of G117H were prepared in five ways. First, crystals of G117H were grown in an ammonium sulfate containing mother liquor as previously reported, using the hanging-drop vapor-diffusion method, at room temperature [START_REF] Nachon | Engineering of a monomeric and low-glycosylated form of human butyrylcholinesterase: expression, purification, characterization and crystallization[END_REF]. Second, crystals were grown in the mother liquor plus 5 mM MgCl 2 and 10 mM NaF as additives. The purpose for this crystallization condition was to generate crystals wherein MgF 3 -would appear as a mimic for PO 3 -in the active site of G117H. Third, crystals were grown in the presence of 5 mM Al(NO 3 ) 3 -and 10 mM NaF for the purpose of generating crystals with AlF 4 -in the active site, again as a mimic for PO 3 -. Fourth and fifth, crystals of the VX-G117H and echothiophate-G117H phosphylated enzyme transient adducts were prepared by flash-soaking the G117H crystals for 2 min in 0.1 M MES buffer, pH 6.5, 6% glycerol, 2.1 M ammonium sulfate containing either 1 mM VX or 1 mM echothiophate. The crystals were then washed with a cryoprotectant solution [0.1 M MES buffer pH 6.5 with 2.1 M ammonium sulfate, containing 20% glycerol] and flash-cooled in liquid nitrogen. At this stage, the VX-adduct in the crystals of VX-G117H BChE presents absolutely no danger.

X-ray Data Collection and Structure Solution. Diffraction data for G117H 1) without additives,

Results

Crystallographic structure of G117H without additives. Crystals were grown in the absence of any extra additives, using ammonium sulfate as a precipitant. Data were collected from tetragonal crystals of space group I422 and refined to 2.1 Å. The space group is identical to that formed by the wild-type enzyme. Data and refinement statistics are summarized in Table 1. The mutation does not induce any noticeable conformational change in the enzyme as shown by the marginal all-atoms root mean square deviation of 0.205 Å between G117H BChE and the wild-type BChE (pdb entry 1P0I). In particular, the main chain atoms of Gly117 in the wild type enzyme and of His117 in the mutant enzyme superimpose very well (RMSD=0.225 Å).

Unexpectedly, there is a sulfate ion bound in the active site, opportunely mimicking an organophosphate about to be attacked by the catalytic serine (Fig 1A). The sulfur atom of the sulfate ion is 2.8 Å from Ser198-Oγ; the O1 and S atoms of SO 4

2-and the Ser198-Oγ are collinear (175°); and the distance between O1 and Ser198-Oγ is 4.3 Å which compares favorably to the distance between the oxygens located at the apical vertices of a trigonal-bipyramidal phosphorous transition state (4.1 Å). However, the 2⏐Fo⏐-⏐Fc⏐ map does not reveal any continuity of electron density between S and Ser198-Oγ. Such continuity would have suggested a covalent bond between these atoms. O4 of the SO 4

2-is well stabilized in the oxyanion hole, being at hydrogen-bond distance from the main chain nitrogens of Ala199, Gly116 and His117 (2.9, 3.0 and 2.9 Å, respectively). O3 is stabilized by a strong hydrogen bond with Ser198-Oγ (2.6 Å) and a salt bridge with His117-Nδ1 (2.5 Å). A salt bridge between O2 and His438-Nε2 (2.6 Å) further stabilizes the sulfate ion. His117 adopts a conformation that is not standard (c1=-116°, c2=44°) according to the penultimate rotamer library [START_REF] Lovell | The penultimate rotamer library[END_REF]. This adjustment probably results from the interaction of His117-Nε2 with the main chain carbonyl oxygen of Leu286 (2.8 Å), and from a salt bridge between His117-Nδ1 and the O3 atom of the sulfate (2.5 Å). This conformation is probably not representative of that adopted by His117 in the absence of the sulfate ion, though it could indicate the conformation in the OP complex just prior to phosphorylation of the Ser198.

As is often observed in the crystal structures of wild type BChE [START_REF] Nicolet | Crystal structure of human butyrylcholinesterase and of its complexes with substrate and products[END_REF][START_REF] Nachon | Role of water in aging of human butyrylcholinesterase inhibited by echothiophate: the crystal structure suggests two alternative mechanisms of aging[END_REF][START_REF] Carletti | Aging of cholinesterases phosphylated by tabun proceeds through O-dealkylation[END_REF][START_REF] Carletti | Structure-activity analysis of aging and reactivation of human butyrylcholinesterase inhibited by analogues of tabun[END_REF], there is an unidentified ligand stacked against Trp82 that we chose to model by dummy atoms. This ligand is at hydrogen bond distance to O2 of SO 4

2-(2.9 Å). In addition, O1 is at hydrogen-bond distance (2.5-3.0 Å) to another unidentified ligand, the electron density of which stretches between Gln119, His117 and Thr120. This density was also modeled by dummy atoms. Crystallographic structure of G117H in complex with fluoride. Data were collected from tetragonal crystals grown in the presence of MgCl 2 and NaF. The original rational for using these additives was to trap a transition state analogue in which MgF 3 -would mimic PO 3 - [START_REF] Baxter | A Trojan horse transition state analogue generated by MgF3-formation in an enzyme active site[END_REF]. This structure was refined to 2.4 Å resolution (See statistics in Table 1).

There are two peaks of electron density in the ⏐Fo⏐-⏐Fc⏐ omit maps (green mesh, 4.5 σ). Both are present at the heart of a dense hydrogen bond network involving the oxyanion hole, the catalytic serine (Ser198) and the catalytic histidine (His438) (Fig 1B). One peak likely corresponds to a water molecule which forms one edge of a triangular H-bond network involving Ser198-Oγ and His438-Nε2. The second peak corresponds to an atom that interacts strongly with both the water molecule (2.6 Å) and Ser198-Oγ (2.5 Å), and is at hydrogen bonding distance to the 3 main chain nitrogens of the oxyanion hole residues (Gly116, 3.0 Å; His117, 2.8 Å; Ala199, 2.9 Å). Because the molecule represented by this density was able to displace the sulfate ion that was observed in the absence of NaF, and because there is a three-pronged interaction with the oxyanion hole as expected for a fluoride anion, we modeled this density as a putative fluoride ion.

This interpretation is supported by the fact that the fluoride anion is known to be a reversible inhibitor of BChE [START_REF] Cimasoni | Inhibition of cholinesterases by fluoride in vitro[END_REF][START_REF] Page | Butyrylcholinesterase: inhibition by arsenite, fluoride, and other ligands, cooperativity in binding[END_REF][START_REF] Masson | Recombinant human butyrylcholinesterase G390V, the fluoride-2 variant, expressed in Chinese hamster ovary cells, is a low affinity variant[END_REF]. Though binding sites have not been identified yet, it is reported that fluoride competes with organophosphates and it was proposed that binding involves two or more hydrogen donors necessary for enzyme catalysis [START_REF] Ashani | The effect of fluoride on the scavenging of organophosphates by human butyrylcholinesterase in buffer solutions and human plasma[END_REF]. Alternatively, a water molecule has been observed in a position similar to that of this putative fluoride ion for some BChE structures [41]. But, the interaction of that water with the oxyanion hole was clearly weaker based on the distance of hydrogen donors, suggesting a two-pronged hydrogen bond interaction.

While the general conformation of His117 in this structure is well resolved, the orientation of the imidazole ring is not. This is because there are two peaks of electron density in the ⏐Fo⏐-⏐Fc⏐ maps, one on each side of the imidazole ring suggesting alternate interactions of His117-Nδ1 with 2 water molecules at relatively low occupancy. In the first option, the c1 and c2 angles of His117 are -48° and 103°, respectively (this is the structure shown in Fig 1B) This conformation is reasonably close to the m-70° histidine conformation from the penultimate rotamer library (c1=-65°; c2=-70°) [START_REF] Lovell | The penultimate rotamer library[END_REF]. We favored the first option for the final model of His117 because it better modeled the strongest peak in the ⏐Fo⏐-⏐Fc⏐map and because it kept both water molecules in the structure. The conformation of His117 adopted in Fig 1B is likely to be representative of that found in the mutant structure, in the absence of ligands.

Following the same logic we employed for the MgCl 2 and NaF as additives, we grew crystals in the presence of 5 mM Al(NO 3 ) 3 and 10 mM NaF. The idea was to trap an AlF 4 -based transition state analog of PO 3 -in the active site [START_REF] Xu | AlF3 mimics the transition state of protein phosphorylation in the crystal structure of nucleoside diphosphate kinase and MgADP[END_REF]. The crystal structure was refined to 2.4 Å resolution (data not shown). It yielded a crystal structure that was identical to that found when the crystals were grown in the presence of MgCl 2 and NaF. This confirmed that fluoride was the key additive leading to the observed complex.

Crystallographic structure of echothiophate-G117H transient adduct. Data were collected from a crystal that was flash-soaked for 2 minutes in a solution containing 1 mM echothiophate. We chose to prepare the crystal in this way because dephosphorylation is partly rate-limiting for the hydrolysis of echothiophate (1.2 min -1 ) [START_REF] Lockridge | A single amino acid substitution, Gly117His, confers phosphotriesterase (organophosphorus acid anhydride hydrolase) activity on human butyrylcholinesterase[END_REF]. Therefore, we assumed that the phosphoryl intermediate would likely accumulate in the crystal in the presence of excess echothiophate. The structure was refined to 2.3 Å (See statistics in Table 1).

A strong peak of positive electronic density (11.8 σ), within covalent bond distance of the catalytic serine was seen in the initial⏐Fo⏐-⏐Fc⏐ map (Fig 1C). This is consistent with the presence of a bound diethoxyphosphoryl moiety (DEP). The position of DEP is similar to that which it adopts in wildtype BChE (PDB entry 1XLW) [START_REF] Nachon | Role of water in aging of human butyrylcholinesterase inhibited by echothiophate: the crystal structure suggests two alternative mechanisms of aging[END_REF]. The phosphorus atom is found at a covalent bonding distance of 1.64 Å from the Ser198-Oγ atom. O3, the phosphoryl oxygen, is at hydrogen bonding distance from the main chain amide nitrogens of the oxyanion hole residues with the shortest distance being to His117N (2.7 Å). There is a very dense hydrogen bond network involving His438-Nε2, Ser198-Oγ, DEP-O2, water molecule 101 and Glu197-Oε1. Given the interatomic distances and angles, the strongest H-bonds are between His438-Nε2 and H 2 O-101, and Glu197-Oε1 and H 2 O-101 (3.0 Å each).

The H 2 O-101 is in the same position as the water molecule 101 described in the DEP conjugate with native BChE (PDB entry 1XLW). This water molecule promotes "aging", i.e. dealkylation of the nearby ethoxy group, by attacking DEP-C3 [START_REF] Nachon | Role of water in aging of human butyrylcholinesterase inhibited by echothiophate: the crystal structure suggests two alternative mechanisms of aging[END_REF][START_REF] Li | Aging pathways for organophosphate-inhibited human butyrylcholinesterase, including novel pathways for isomalathion, resolved by mass spectrometry[END_REF]. Aging via dealkylation was proven by mass spectral analysis of the product after aging in the presence of 18 O-water [START_REF] Nachon | Role of water in aging of human butyrylcholinesterase inhibited by echothiophate: the crystal structure suggests two alternative mechanisms of aging[END_REF][START_REF] Li | Aging pathways for organophosphate-inhibited human butyrylcholinesterase, including novel pathways for isomalathion, resolved by mass spectrometry[END_REF].

Comparing DEP-G117H to wild-type DEP-BChE it appears that the His117 side chain induces a concerted shift of DEP-C3 toward H 2 O-101 (by 1.2 Å) and H 2 O-101 toward Trp82 (by 0.7 Å). As a consequence the distance between DEP-C3 and H 2 O-101 is only 2.9 Å in DEP-G117H compared to 3.8 Å in wild-type DEP-BChE. Thus, the imidazole of His117 appears to be an obstacle to the formation of the phosphylated enzyme.

Given the preeminent role of H 2 O-101 in the dealkylation of DEP, it is expected that a shorter distance between DEP-C3 and H 2 O-101 would be more favorable for the aging reaction. This was experimentally verified. The dealkylation rate of DEP-G117H is about 7 times faster than that for wildtype DEP-BChE, under similar conditions [START_REF] Lockridge | A single amino acid substitution, Gly117His, confers phosphotriesterase (organophosphorus acid anhydride hydrolase) activity on human butyrylcholinesterase[END_REF].

His117 also induces a slight rearrangement of the second ethoxy group of DEP and its environment. The imidazole ring prevents DEP-C2 from pointing toward Val288, and thus DEP-C2 is forced toward Leu286. In response, Leu286-Cδ1 and Leu286-Cδ2 shift away by about 1 Å. A similar rearrangement of Leu286 was previously observed in an N-diethylphosphoramidyl conjugate of BChE [START_REF] Carletti | Structure-activity analysis of aging and reactivation of human butyrylcholinesterase inhibited by analogues of tabun[END_REF].

His117 adopts a stable conformation (c1=-62°; c2=-76°) corresponding to m-70° from the penultimate rotamer library. The imidazole ring is lying flat, above the diethoxyphosphoryl moiety. This conformation is stabilized by a hydrogen bond between His117-Nε2 and the Pro285-carbonyl-O, and a hydrogen bond to a water molecule bridging His117-Nδ1, and Thr120-Oγ1.

The distances between His117 and the phosphorus of DEP provide some basis on which to address the mechanism of dephosphorylation for DEP-G117H. His117-Nδ1 is 4.6 Å from DEP-P and 3.9 Å from DEP-C3 (respectively 1.3 and 0.6 Å channel width). These distances are too short to allow a water molecule to fit between His117 and the phosphorus as depicted on scheme 2 (pathway B, "attack is anti"). For this path to be active, a conformation change of His117 would have to occur. Alternatively, because His117-Nδ1, DEP-P and Ser198-Oγ are almost collinear (168°), a simple rotation of the imidazole ring by -60° around Cβ-Cγ would place Nδ1 of His117 3.7 Å from DEP-P with the nitrogen lone pair pointing toward the phosphorus atom. This situation seems plausible for a nucleophilic attack of His117 on the phosphorus, i.e. the "Nucleophile is His117" path as depicted in scheme 2 (pathway A).

Crystallographic structure of VX-G117H transient adduct. Data were collected from a crystal that was flash-soaked for 2 minutes in a solution containing 1 mM VX. The reasons for preparing the crystal in this way are the same as those described in the section on the "Crystallographic structure of echothiophate-G117H transient adduct". Because the dephosphonylation rate of VX-G117H is 200 fold lower than that of diethylphosphoryl-G117H, trapping the hydrolysis intermediate was expected to be less challenging. The structure was refined to 2.7 Å (see statistics in Table 1).

No water molecule equivalent to H 2 O-101 is present in the active site of the VX-adduct structure (Fig 1D ). A peak of electron density more than 4 Å away from His438 and Glu197 was modeled as an ammonium ion likely to be electrostatically interacting with Glu197 and the π system of Trp82 (data not shown).

A peak in the initial⏐Fo⏐-⏐Fc⏐ map (3.7 σ) was found at a covalent bonding distance to Ser198-Oγ (1.7 Å). The absolute configuration of the phosphorous atom is P(S). The methyl group is pointing toward His438 (Fig 1D). The ethoxy group is located in the acyl-binding pocket defined by residues Leu286, Val288 and Trp231. This orientation is the mirror image of that observed in VX-TcAChE [START_REF] Millard | Reaction Products of Acetylcholinesterase and VX Reveal a Mobile Histidine in the Catalytic Triad[END_REF], but identical to that observed in VX-BChE (Wandhammer et al, submitted).

The ethoxy group points toward Val288, and superimposes very well on the equivalent ethoxy in the wild-type DEP-BChE structure. However, this orientation is different from that just described for the DEP-G117H structure where the ethoxy group was pointing toward Leu286. As a consequence, the shift of Leu286 that is observed in the DEP-G117H conjugate does not occur in the VX-G117H conjugate. And, the carbonyl oxygen from Pro285 is not available for interaction with His117-Nε2. This is reflected in two conformations for His117, which differ by the ring orientation: c1=-7° and c2=-124° or c1=-7° and c2=57°. These conformations are far from common histidine rotamers. This unusual conformational arrangement may be a consequence of the asymmetry of the adduct. That is, the His117 imidazole ring is shifted over the methyl substituent and away from the bulkier ethoxy. In this position, it cannot be stabilized by H-bonding because it is too far from the acceptors in the acyl-binding pocket, and too close to Thr120 to let a bridging water molecule slip in. This translates into much higher A c c e p t e d M a n u s c r i p t residual B-factors for the imidazole ring atoms (≈53 Å 3 ) in comparison to main chain atoms (≈38 Å 3 ). The corresponding B-factors are much smaller for DEP-G117H, where they are 16 Å 3 for Ca, and 10 Å 3 for His117-Nε2. The large B-factors for the imidazole of His117 in VX-G117H do not permit the orientation of the imidazole ring to be determined with confidence.

Much like in the DEP-G117H structure, His117-Nδ1 is at 4.5 Å from DEP-P and 3.8 Å from DEP-C3. These short distances would not allow a water molecule to get between the phosphorus and His117 in the absence of a large conformational change. His117-Nδ1, DEP-P and Ser198-Oγ are less collinear (157°) than in the DEP-G117H conjugate. Cβ for His117 in VX-G117H is offset by 0.7 Å from its position in DEP-G117H (Fig 2). Thus, the maximum allowable side chain rotation around Cβ-Cγ is -50°. That would partially orient the lone electron pair of His117-Nδ1 toward the adduct, but any further motion toward the phosphorus atom would lead to a clash between His117-Nδ1/Cε2 and the methyl substituent of the VX. This situation is less favorable for a nucleophilic attack of His117 on the phosphorus than was the case in the DEP-G117H intermediate. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Discussion

Impact of mutations at position 117 on the activity of BChE and related enzymes. When BChE is mutated at position G117 it loses catalytic efficiency for the hydrolysis of thio-and oxo-esters [START_REF] Masson | A collaborative endeavor to design cholinesterase-based catalytic scavengers against toxic organophosphorus esters[END_REF][START_REF] Millard | Design and expression of organophosphorus acid anhydride hydrolase activity in human butyrylcholinesterase[END_REF]. It was hypothesized that this loss in activity was related to a change in the orientation of the H117 nitrogen proton donor of the oxyanion hole, leading to weaker transition state stabilization due to suboptimal H-bonding [START_REF] Masson | Hydrolysis of oxo-and thio-esters by human butyrylcholinesterase[END_REF]. We observe no such change in the X-ray structures of G117H presented here (Figure 2). Though there is some heterogeneity in the positions adopted by His117, depending on the adduct, the positions of the critical oxyanion hole residues are unchanged.

Alternatively, loss of activity toward the normal thio-oxo-esters may be due to the imidazole ring of His117 blocking the approach of substrate and water. This suggestion is based on the fluoride-G117H structure, which we take to be representative of the unliganded, resting enzyme. In this structure, His117 clutters access to the oxyanion hole (Fig 2B ), and is very likely a steric obstruction to the formation of all substrate complexes, including the transition state. A change in the position of His117 would be necessary in order to properly orient a substrate in the oxyanion hole, even then optimal orientation may not be achieved.

Such a conformational change is seen in the sulfate complex. If His117 were to maintain the position observed in the fluoride complex, it would collide with the sulfate oxygen that is opposite to the serine. Consequently, the histidine is forced sideways in the sulfate complex.

Similarly, proper fitting of an OP into the active site of BChE requires a conformational change of His117 (compare Fig 1A to Fig 1C and Fig 1D). Such steric hindrance from His117 could explain the 2-to 4-order of magnitude decrease in phosphylation rates observed for OPs reacting with G117H [START_REF] Broomfield | Protein engineering of a human enzyme that hydrolyzes V and G nerve agents: design, construction and characterization[END_REF].

Loss of sensitivity to organophosphate inhibitors in insects may be attributed to the same phenomenon. For example, a natural mutation in mosquito acetylcholinesterase, G119S -a position equivalent of G117 in BChE -is at the origin of their resistance to organophosphate pesticides [START_REF] Weill | Comparative genomics: Insecticide resistance in mosquito vectors[END_REF]. No OP hydrolase activity was reported for this mutant, but resistance could arise from decreased affinity for OP. It is noteworthy to mention that no OP-hydrolase activity was observed for the equivalent BChE mutant, G117S [START_REF] Schopfer | Mutants of human butyrylcholinesterase with organophosphate hydrolase activity; evidence that His117 serves as a general base catalyst[END_REF]. A second example is the G137D mutant of carboxylesterase from the sheep blowfly [START_REF] Newcomb | A single amino acid substitution converts a carboxylesterase to an organophosphorus hydrolase and confers insecticide resistance on a blowfly[END_REF]. Position 137 is equivalent to position 117 in BChE and substitution of Asp for Gly at this position would be expected to hinder access to the oxyanion hole. The G137D mutation reduces sensitivity to OPs and confers OP-hydrolase activity on the carboxylesterase. OP-hydrolase activity was also found for the equivalent G117D mutation in BChE [START_REF] Schopfer | Mutants of human butyrylcholinesterase with organophosphate hydrolase activity; evidence that His117 serves as a general base catalyst[END_REF]. Finally, a triple mutation in Bungarus fasciatus AChE aimed at introducing a histidine into a position homologous to 117 of BChE reduces sensitivity to OPs and turns this enzyme into an OP hydrolase [START_REF] Poyot | Mutant of Bungarus fasciatus acetylcholinesterase with low affinity and low hydrolase activity toward organophosphorus esters[END_REF].

Candidate mechanisms for catalytic dephosphylation. Four different mechanisms have been proposed by previous investigators to explain why dephosphylation of G117H BChE is faster than dephosphylation of the native enzyme (Scheme 2).

Mechanism A

The first mechanism is based on a nucleophilic attack of His117 on the phosphorus, leading to a phosphylhistidine intermediate that is subsequently hydrolyzed (Scheme 2 pathway A, the "Nucleophile is His117" pathway). This mechanism was originally proposed by Fortier on the basis of molecular mechanics calculations [START_REF] Albaret | Mechanical aspects of the phosphotriesterase activity of human butyrylcholinesterase G117H mutant[END_REF].

One argument against this mechanism is that phosphohistidines have a high-energy phosphoramidate bond and tend to transfer the phosphoryl group to other molecules [START_REF] Stock | Signal transduction in bacteria[END_REF][START_REF] Attwood | Focus on phosphohistidine[END_REF]. Thus it would be expected that a phosphylhistidine would transfer its phosphyl moiety to the serine and not the other way around. Following this reasoning, early investigators proposed that the OP-serine adduct was formed via an intermediate OP-histidine donor rather than through a direct phosphylation by the OP [START_REF] Hobbiger | Effect of nicotinhydroxamic acid methiodide on human plasma cholinesterase inhibited by organophosphates containing a dialkylphosphato group[END_REF]. A c c e p t e d M a n u s c r i p t Support for nucleophilic attack by His117 on the phosphorus comes from the colinearity of His117-Nδ1, the phosphorus atom and Ser198-Oγ in the DEP-G117H conjugate, and to a lesser extent in the VX-G117H conjugate. A 60° rotation of the imidazole ring around the Cβ-Cγ bond would orient the lone pair of electrons on the nitrogen toward the phosphorus for direct nucleophilic attack. Unfortunately, the phosphorus substituents are in close contact with the imidazole ring and these interactions would prevent a simple rotation around Cβ-Cγ bond. Rotation could occur only if His117 moved back from the phosphorus substantially. Even if the simple rotation were to occur, the imidazole would still be 3.7 Å from the phosphorus, too far for covalent interaction. Taken together, the crystal data and the thermodynamics argue that pathway A is unlikely. Proof for this mechanism would require trapping of the phosphylhistidine intermediate.

Mechanism B

The second mechanism is a nucleophilic attack by water on the phosphorus atom, promoted by His117. The oxygen of the attacking water would have to be positioned opposite the serine-phosphorus bond, i.e. in an apical or "anti" position (Scheme 2, pathway B, "attack is anti"). This mechanism formed the basis of the original rationale for the design of the G117H mutant [START_REF] Broomfield | Enzymes of the Cholinesterase Family[END_REF]. Broomfield and Millard reasoned that "it might be possible to introduce a second nucleophilic center into the active site in such a position that it could carry an activated water molecule to the face of the phosphorus moiety opposite the phosphorus-serine bond and thereby reactivate the enzyme" [START_REF] Broomfield | Enzymes of the Cholinesterase Family[END_REF]. Newcomb et al. extended this mechanism to the G137D mutant of the sheep blowfly carboxylesterase, the aspartate acting as a base to orientate a water molecule in the appropriate position for hydrolysis [START_REF] Newcomb | A single amino acid substitution converts a carboxylesterase to an organophosphorus hydrolase and confers insecticide resistance on a blowfly[END_REF]. By analogy, this mechanism was also proposed for the G117D, G117E, and L286H BChE mutants [START_REF] Schopfer | Mutants of human butyrylcholinesterase with organophosphate hydrolase activity; evidence that His117 serves as a general base catalyst[END_REF]. Finally, this mechanism was recently re-proposed by Amitay et al. based on similarities with RNase A [21]. The active site of RNase A contains two histidine residues, His12 and His119, in locations comparable to His117 and His438 of BChE. His12 serves as a base to abstract a proton from the 2'-oxygen of the RNA substrate molecule, thereby activating it for attack on the phosphorus. His119 serves as an acid to protonate the oxygen of the leaving group [START_REF] Raines | Ribonuclease A[END_REF].

The X-ray structures in Figures 1C and1D show that the imidazole of His117 is in close contact with the phosphorus. In this position His117 would not allow a water molecule to approach the phosphorus. A large conformational change of His117 would be required to make room. In addition, if one considers the sulfate-G117H complex to be a structural analog of the dephosphylation transition state for mechanism B, then the formation of the transition state is obstructed by the ethoxy moiety in the acyl-binding pocket as seen by the superimposition of the sulfate, VX and DEP-G117H (Figure 2). Finally, the imidazole of His117 is an obstacle to the formation of the dephosphylation transition state, much like it is an obstacle for the formation of the phosphylation transition state in agreement with kinetic data [START_REF] Millard | Design and expression of organophosphorus acid anhydride hydrolase activity in human butyrylcholinesterase[END_REF]. These observations argue that a conformational change in the active center would be necessary for Mechanism B to occur. A partially rate limiting conformational change in course of forming the transition state for dephosphylation is consistent with the weak pH dependencies observed for dephosphylation [START_REF] Millard | Design and expression of organophosphorus acid anhydride hydrolase activity in human butyrylcholinesterase[END_REF][START_REF] Lockridge | A single amino acid substitution, Gly117His, confers phosphotriesterase (organophosphorus acid anhydride hydrolase) activity on human butyrylcholinesterase[END_REF]. QM/MM calculations based on the structures herein would be needed in order to determine if the change of conformation required corresponds to an energy barrier compatible with the measured dephosphylation rate.

Mechanism C

The third mechanism, which was proposed by Millard et al. [START_REF] Millard | Design and expression of organophosphorus acid anhydride hydrolase activity in human butyrylcholinesterase[END_REF], derives from the suggestion of Kovach that the dephosphylation rate is inversely proportional to the electron density on the phosphorus [START_REF] Kovach | Structure and dynamics of serine hydrolase-organophosphate adducts[END_REF]. Millard proposed that a hydrogen bond between His438 and an alkoxy ligand to the phosphorus stabilized the linkage between the phosphorus and Ser198-Oγ by making His438 unavailable to serve in its traditional role of transferring a proton to Ser198. It was proposed that His117 would compete with His438 by forming a hydrogen bond with the one of the ethoxy groups thus setting His438 free to form a hydrogen bond with a water molecule. Abstraction of a proton from that water molecule, would shows His117 making H-bond contact with water molecules but not with the ethoxy substituent. In the DEP-G117H complex His438 is at an optimal position to transfer a proton between a water molecule and the serine while maintaining a hydrogen bond to an ethoxy group. These observations refute the idea that H-bonding of His438 to an ethoxy group is a critical feature of phosphylated G117H that would prevent it from acting as a base catalyst.

Mechanism D

The structure in Fig 1C supports a mechanism in which a water molecule could be activated by His438 for attack on the phosphorus atom. His438 in DEP-G117H is hydrogen bonded to a water located near the wide-open face of the DEP adduct, adjacent to Ser198 (Scheme 2, pathway C). If His438 is deprotonated then it could abstract a proton from the entering water molecule and subsequently transfer it to the serine. Alternatively, if it is protonated then it could serve as a relay in a concerted proton transfer from the water molecule to the catalytic serine as depicted in scheme 2, pathway C. A similar adjacent nucleophilic substitution mechanism has been proposed in some phosphylation and aging reactions of cholinesterases [START_REF] Kwasnieski | Fixation of the two Tabun isomers in acetylcholinesterase: a QM/MM study[END_REF][START_REF] Nachon | Aging mechanism of butyrylcholinesterase inhibited by an N-methyl analogue of tabun: Implications of the trigonalbipyramidal transition state rearrangement for the phosphylation or reactivation of cholinesterases[END_REF].

H-bonding of the water to Glu197 that is seen in Fig 1C can further stabilize the approaching water molecule. A role for Glu197 in dephosphylation is supported by the observation that there is 40fold decrease in the dephosphorylation rate of echothiophate-G117H/E197Q [START_REF] Schopfer | Mutants of human butyrylcholinesterase with organophosphate hydrolase activity; evidence that His117 serves as a general base catalyst[END_REF], but a 10-fold increase in the dephosphonylation rate for VX-and sarin-G117H/E197Q mutants [START_REF] Millard | Organophosphorus acid anhydride hydrolase activity in human butyrylcholinesterase: synergy results in a somanase[END_REF].

In Mechanism D, His117 could accelerate dephosphylation by destabilizing the transient phosphylserine by steric obstruction. Indeed, the conformation of His117 in the DEP-and methylethoxyphosphonyl-G117H transient conjugates, appears to be strained by comparison to the relaxed conformation observed in the fluoride-G117H complex (Figure 2). The imidazole ring puts a lot of strain on the phosphyl moiety and can be viewed as a molecular analog of a loaded spring. This should result in a high-energy structure, destabilizing the adduct. The destabilizing role of His117 in the transition state would therefore be simply steric exclusion. Given this thesis, a diethylphosphoryl adduct is expected to be under greater strain than a methylethoxyphosphonyl adduct, thus explaining the higher hydrolysis rate for echothiophate than for VX. Additionally, if His117 is charged, it is expected to enhance the electrophily of the phosphorus atom by a local electrostatic effect. These effects would also be consistent with the modest pH dependence on the dephosphylation step [START_REF] Millard | Design and expression of organophosphorus acid anhydride hydrolase activity in human butyrylcholinesterase[END_REF][START_REF] Lockridge | A single amino acid substitution, Gly117His, confers phosphotriesterase (organophosphorus acid anhydride hydrolase) activity on human butyrylcholinesterase[END_REF]. All things considered, mechanism D appears to be most consistent with the X-ray data.

In summary, the structures presented here are not sufficient to definitively identify the molecular mechanism of G117H phosphyl hydrolysis, however they do provide a basis on which to question the currently advanced possibilities. In addition, the X-ray structures of echothiophate-G117H and VX-G117H provide indispensable structural templates for future QM/MM calculations and valuable structural starting points for further explorations into the hydrolysis mechanism. The proposals provided in this discussion also may provide guidance for computational mutagenesis directed at improving the dephosphylation rates for G117H. A mutagenesis strategy based on computational design was successfully implemented to create a mutant of BChE with improved cocaine hydrolase activity [START_REF] Zhan | Fundamental reaction mechanism for cocaine hydrolysis in human butyrylcholinesterase[END_REF]. Such a strategy could be applied to the design of new cholinesterases or other serine hydrolase mutants that can more efficiently reactivate from OP inhibition. Efficient catalytic bioscavengers would provide a great improvement in prophylaxis, decontamination and treatment of organophosphylate poisoning. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 19 Nov 2010 as manuscript BJ20101648 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101648 A c c e p t e d M a n u s c r i p t Experimental Materials VX, O-ethyl-S-[2[bis(1-methyl-ethyl) amino] ethyl] methylphosphonothioate, was obtained from the Centre DGA Maîtrise NRBC (Vert-le-Petit, France). VX is highly toxic. It belongs to schedule 1 Chemicals as defined in the Chemical Weapons Convention. All work with VX is regulated by that convention. Echothiophate, O-diethyl-S-(2-trimethylammonium ethyl) phosphorothioate, was obtained from Biobasal (Basel, Switzerland). The handling of VX and echothiophate is dangerous and requires suitable personal protection, training, and facilities.

  . The water molecule (peak at 4.1 σ) is at hydrogen bond distance from the backbone nitrogen of Thr120 (3.1 Å), and interacts strongly with His117-Nδ1 (2.5 Å). In the alternative refined model (position not shown in Fig 1B), the ring is simply flipped with c1=-46° and c2=-71°. The water molecule (peak at 3.5 σ) is at hydrogen bond distance from the carbonyl oxygen of Leu286 (3.1 Å), and interacts very strongly with His117-Nδ1 (2.4 Å).

Biochemical

  Journal Immediate Publication. Published on 19 Nov 2010 as manuscript BJ20101648 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101648

Biochemical

  Journal Immediate Publication. Published on 19 Nov 2010 as manuscript BJ20101648 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101648

Biochemical

  Journal Immediate Publication. Published on 19 Nov 2010 as manuscript BJ20101648 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101648

Biochemical

  Journal Immediate Publication. Published on 19 Nov 2010 as manuscript BJ20101648 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101648 A c c e p t e d M a n u s c r i p t promote attack of the water on the phosphorus atom. The structure of VX-G117H (Fig 1D) shows no hydrogen bonding interactions for the imidazole of His117 with the ethoxy substituents. The structure of DEP-G117H (Fig 1C)

Biochemical

  Journal Immediate Publication. Published on 19 Nov 2010 as manuscript BJ20101648 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101648 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2010 The Authors Journal compilation © 2010 Portland Press Limited

Figure legends Scheme 1 :Scheme 2 :

 12 Figure legends

Figure 1 :

 1 Figure 1: The active site of G117H BChE: (A) in the presence of sulfate; (B) in the presence of fluoride; (C) with diethylphosphate covalently attached to Ser198, i.e an echothiophate adduct; and (D) with O-ethyl-methylphosphonate attached to Ser198, i.e. a VX adduct. Active site residues are shown in the ball-and-sticks format. Carbon atoms are shown in bright orange (or cyan for key residues), nitrogen atoms in deep blue, the phosphorus atom in orange, and oxygen atoms in red. Hydrogen bonds are represented by dashes. The electron density ⏐Fo⏐-⏐Fc⏐ omit map is represented by a green mesh contoured at 3σ.

Figure 2 :

 2 Figure 2: Two views of the active site of butyryl-BChE (blue, PDB code 1p0i) superimposed on the G117H complex with sulfate (orange), fluoride (cyan), echothiophate (green) and VX (red). Side (A) and front (B)

  Publication. Published on 19 Nov 2010 as manuscript BJ20101648 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101648 A c c e p t e d M a n u s c r i p t

Figure 1 Biochemical

 1 Figure 1

TableTable 1 :

 1 Data Collection and Refinement Statistics R-factor=Σ |Fo -|Fc|| / Σ |Fo|, Fo and Fc are observed and calculated structure factors b R-free set uses about 1000 randomly chosen reflections.

	Ligand	Sulfate	Fluoride	Echothiophate	VX
	PDB entry code	2xmb	2xmc	2xmd	2xmg
	Data
	ESRF Beamline	ID14-2	ID14-1	ID14-2	ID23-2
	space group	I422	I422	I422	I422
	unit cell axes, a=b, c (Å)	154.8, 134.6	155.6, 128.0	154.9, 127.5	156.6, 128.4
	no. of measured reflections	308251	154794	249541	166904
	unique reflections	47525	29897	33216	20940
	resolution (Å)	37.6-2.1(2.4-2.1)	41.1-2.4(2.5-2.4)	28.1-2.3(2.4-2.3)	55.5-2.7(2.8-2.7)
	completeness (%)	99.5(99.0)	96.7(96.2)	95.9(85.4)	94.3(95.0)
	Rsym (%)	6.1(36.4)	5.6(51.0)	4.9(23.9)	7.1(56.0)
	I/σ(I)	21.3(5.7)	28.0(3.6)	28.9(6.0)	23.7(3.8)
	Redundancy	6.4(6.5)	5.1(4.9)	7.5(4.5)	8.0(7.4)
	Refinement Statistics
	R-factor a (R-free b )	16.8(20.8)	19.2(25.2)	17.0(21.4)	17.6(25.0)
	no. of atoms
	protein	4209	4216	4226	4226
	solvent	380	229	285	151
	others	189	171	198	168
	mean B-factor (Å 2 )	41.7	46.08	40.08	56.14
	RMS from ideality
	bond length (Å)	0.024	0.021	0.023	0.018
	angles (deg)	2.182	2.009	2.090	1.933
	chiral (Å 3 )	0.154	0.140	0.157	0.128

a

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2010 The Authors Journal compilation © 2010 Portland Press Limited

© 2010 The Authors Journal compilation © 2010 Portland Press Limited

was supported by ANR-06-BLAN-0163 and DGA/PEA 08co501 to FN.


Angela Woods

Amanda Heslegrave

Phillip J Muckett

Adam Levene

Melanie Clements

Margaret Mobberley

Timothy A Ryder

Shadi Abu-Hayyeh

Catherine Williamson

Robert D Goldin

LKB1 is required for hepatic bile acid transport and canalicular membrane integrity in mice

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

  Figure 1

  Figure 4

  Figure 5

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2010 The Authors Journal compilation © 2010 Portland Press Limited

© 2010 The Authors Journal compilation © 2010 Portland Press Limited


M.D Frank Weber
email: f.weber@erasmusmc.nl

Nadia Kriek

M.D Heleen J Blussé Van Oud-Alblas

State Index in children under propofol-anaesthesia -a prospective randomized clinical trial

Keywords: electroencephalogram, bispectral index, cerebral state index, children, propofol, neuromuscular block electroencephalogram, bispectral index, cerebral state index, children, propofol, neuromuscular block

The effects of mivacurium induced neuromuscular block on Bispectral Index and Cerebral State Index in children under propofol-anaesthesia -a prospective randomized clinical trial

in anaesthetised subjects, whereas absence of EMG activity in awake subjects might result in a falsely low DH-index [START_REF] Jensen | Pitfalls and challenges when assessing the depth of hypnosis during general anaesthesia by clinical signs and electronic indices[END_REF].

Already in 1946 Gray and Halton (2) suggested some kind of synergism between curare and barbiturates and an even more pronounced synergism between this combination and the inhalational anaesthetics ether and cyclopropane. In a recent editorial Bonhomme and Hans (3) gave an overview of the theories that have been published about how neuromuscular blocking agents (NMBA) may affect both the hypnotic and the antinociceptive component of anaesthesia.

The de-afferention theory suggests that prolonged sensory deprivation results in a decrease in power together with a shift of the power-spectrum towards lower frequencies in the human EEG [START_REF] Heron | Effects of prolonged perceptual isolation on the human electroencephalogram[END_REF]. However, with respect to NMBA it is noteworthy to stress that muscle relaxation does not produce total sensory deprivation (3). Schwartz et al. [START_REF] Schwartz | Pancuronium increases the duration of electroencephalogram burst suppression in dogs anesthetized with isoflurane[END_REF] demonstrated that pancuronium increases the duration of isoelectricity during isoflurane EEG burst suppression in dogs and suggested that the effect of isoflurane on the brain is enhanced by nondepolarizing NMBA. Lanier et al. [START_REF] Lanier | The cerebral and systemic effects of movement in response to a noxious stimulus in lightly anesthetized dogs. Possible modulation of cerebral function by muscle afferents[END_REF] suggested that in lightly anaesthetised dogs muscle afferent receptors mediate effects of active muscle movement in response to painful stimulation on the brain in terms of EEG activation, cerebral vasodilatation and increased cerebral blood flow. Pancuronium induced paralysis abolished movement and the afferent response, resulting in attenuated responses of EEG, cerebral blood flow and cerebral vascular resistance following painful stimulation.

Messner et al. [START_REF] Messner | The bispectral index declines during neuromuscular block in fully awake persons[END_REF] reported a significant decrease in Bispectral Index (BIS) values in two fully awake volunteers after succinylcholine administration.

Like the BIS™-monitor (Aspect Medical Systems, Natick, U.S.A.), the Cerebral State Monitor In this prospective randomised trial we tested the hypothesis that mivacurium induced NMB in children anaesthetised with propofol results in a lower level of consciousness as detected by the BIS and the CSI, thus a decrease of both indices.

Materials and methods

After approval of the institutional ethics committee of the Erasmus University Medical Center

Rotterdam, The Netherlands, and written informed consent was obtained from the patients' parents or caregivers, 40 children, aged between 2 and 12 years, ASA physical status I or II, scheduled for elective minor paediatric or urological surgery were studied. Patients were primarily excluded from the study if they suffered from significant cardiovascular, respiratory or neurological disease or if they were taking chronic medication known to affect the central nervous system.

All patients had a standardised anaesthetic. Induction of general anaesthesia was performed as follows: After establishment of intravenous access, intravenous sufentanil 0.3 µg kg -1 was administered. Two minutes later propofol 3 mg kg -1 was injected, if necessary followed by subsequent doses of 0.5 mg kg -1 until loss of responsiveness (LOR). LOR was defined as the moment of loss of eyelash reflex combined with the absence of response to verbal or mild tactile stimulation. At that moment patients were considered to be unconscious. Then a continuous propofol infusion was started, initially at a rate of 10 mg kg -1 h -1 . Immediately after LOR both BIS and CSI monitoring were established. The continuous propofol infusion was then titrated to achieve a stable BIS value of 50±5 for at least 60 seconds. Then, patients were prospectively -5 -randomized to receive in a double blinded fashion either mivacurium 0.25 mg kg -1 (Group Miva), or an equal volume of NaCl 0.9% (0.125 ml/kg) (Group Con). The syringes were labelled as "study medication". Patients were randomised by the sealed envelope method.

Initially patients were either breathing spontaneously via a face mask or received manually assisted ventilation in order to maintain an end-tidal carbon dioxide tension of 35-45 mmHg.

With the onset of NMB following mivacurium administration patients were completely ventilated.

During the whole study period (1 min. before administration of study drugs and 5 min. thereafter) the patients remained untouched except of face mask ventilation, furthermore care was taken to minimize the sound level of the anaesthesia induction room to a minimum.

Five minutes after study drug administration the registration of BIS, and CSI data was stopped.

After a deblinding procedure patients who had been randomised to the Control group were allowed to receive mivacurium and the conduct of anaesthesia was further unrestricted at the discretion of the anaesthesiologist in charge. Assessment of NMB was performed by Train-of-Four (TOF) stimulation of the ulnar nerve 5 minutes after study drug administration. During the study period no TOF stimulations were applied in order to avoid unnecessary artefact contamination of the EEG readings.

Cerebral State Index (CSI) monitoring

After the skin was prepared with alcohol and abraded with gauze, three silver-silver chloride electrodes (Medicotest A/S, Olstykke, Denmark) were positioned at the mid forehead (+), left forehead (reference) and left cheekbone (-). Electrode placement and skin preparation were performed until the electrodes' impedance was less than 1000 Ohms. The algorithm that calculates the CSI is based on several sub parameters within the 6-42.5 Hz frequency band of the EEG and on burst suppression. The CSI is a dimensionless scale, ranging from 100 (fully awake) to 0 (very deep hypnosis). CSI values between 40 and 60 can be considered as the range for surgical anaesthesia [START_REF] Jensen | Pitfalls and challenges when assessing the depth of hypnosis during general anaesthesia by clinical signs and electronic indices[END_REF]. Besides the CSI, the Cerebral State Monitor also gives information about

Bispectral Index (BIS) monitoring

For BIS registration a paediatric four-sensor BIS™ probe (BIS™ Paediatric Sensor; Aspect Medical Systems International BV, De Meern, The Netherlands) was attached to the patient's forehead according to the instructions of the manufacturer. A BIS XP ™ monitor was used. On the BIS XP ™ monitor EMG activity is being displayed as the power in the 70-110 Hz frequency range of the EEG in dB-units [START_REF] Dahaba | The effect of different stages of neuromuscular block on the bispectral index and the bispectral index-XP under remifentanil/propofol anesthesia[END_REF].

All EEG data, incl. EMG in dB, were collected at 5-sec intervals using Rugloop™ (Demed, Temse, Belgium), synchronized with Labgrab™ (Demed), and stored on a personal computer for subsequent analysis.

Statistics:

Individual mean BIS and CSI values of two periods of 60 seconds were calculated for the occasions "Baseline" (after stable BIS values of 50±5 for at least 1 min, prior to study drug administration) and 5 min. after study drug administration. Mean values per patient were subsequently used for statistical analysis. Datasets were furthermore tested for normality by the Kolmogorov-Smirnov test. Intergroup comparison of the change in BIS and CSI values was performed by the Mann-Whitney Rank Sum Test. Intragroup comparison of mean BIS and CSI (baseline versus 5 min. after drug administration) was performed by paired t-test.

In order to reach a power of 0.9 and an alpha level of 0.05 a total of 36 patients would be required to detect a 10 percent difference in BIS values between both study groups. In order to compete for possible drop outs 20 patients per group were included.

Results

Forty patients were enrolled between March and November 2008. Due to synchronisation problems, EEG data acquisition failed in one patient of the control group. Per protocol analysis was performed using the data obtained from 39 patients. Study groups did not differ with respect to age (group Miva 85±49 months, group Con 73±28 months) and body weight (group Miva 24.9±11.3 kg, group Con 22.5±5.8 kg). Propofol consumption was also comparable between groups (Group Miva 10.0±0.6 mg kg -1 h -1 , Group Con 10.0±0.2 mg kg -1 h -1 ; n.s.).

Pooled mean values per patient of BIS and CSI (baseline and 5 minutes after study drug administration) together with the results of intragroup comparison of the change in BIS and CSI are presented in table 1. Both BIS and CSI values were higher 5 min. after study drug as compared to baseline in all participants of the study. With respect to the change in BIS and CSI (baseline vs. +5 min.) no differences were found between groups Miva and Con (see table 2).

The absence of all four twitch responses to TOF stimulation of the ulnar nerve in all patients of group M confirmed complete NMB, whereas TOF stimulation evoked four twitches in all patients allocated to group C. The BIS XP monitor recorded a constant level of EMG activity throughout the study period irrespective of study drug administration (Group Miva 32±1 dB, Group Con 32±1, n.s.). No EMG activity was recorded by the CSM throughout the whole study period. No side effects or adverse events were recorded in any patient.

Discussion

In the present paediatric study mivacurium induced NMB had no effect on both the BIS and the CSI in paediatric patients anaesthetised with propofol. To our best knowledge until now there are speculative to some degree. However, it cannot be ruled out that at a BIS level of 48±4, indicative of surgical anaesthesia and an EMG activity of 32±1 in children under propofol anaesthesia the application of NMB has no impact on both EMG activity and hypnotic depth as measured by the BIS.

In our study propofol was titrated using a conventional infusion pump to achieve a stable BIS of 50±5 prior to the application of the study medication. Once having achieved the desired BIS level, the propofol infusion rate remained unaltered until the end of the study period. It would undoubtedly have been desirable to apply TCI technology but unfortunately we had no devices available which incorporate paediatric pharmacokinetic models validated for use in small children. The design of this study renders thus the possibility that, due to redistribution, a constant infusion rate even over a period as short as 6 minutes, does not necessarily result in a stable DoH.

That might explain the mild increase of BIS and CSI values in both study groups over time, even in the absence of physical or acoustic stimulation, indicating the likelihood of a decrease of hypnotic depth as a result of distribution of propofol among the compartments of the body, which leads to both a lower plasma-and effect-site drug concentration. In the present study recordings of facial EMG via the BIS monitor revealed a constant activity of 32dB in all patients throughout the study period, whereas the Cerebral State Monitor did not detect any EMG activity. TOF stimulation of the ulnar nerve confirmed complete NMB in all patients allocated to group Miva. However, that did not result in any changes of facial EMG activity, as measured by the BIS-monitor. The site of EMG-monitoring might contribute to this finding. Human skeletal muscles consist of two different types of fibres which can be classified on the basis of their metabolic and electrophysiological characteristics. Type I, or slow twitch muscle fibres are the predominant fibres in muscles with an almost exclusive postural function, whereas a high percentage of type II (fast twitch) fibres, can be found in muscles with usually phasic activity. For the adductor pollicis Johnson et al. [START_REF] Johnson | Data on the distribution of fibre types in thirtysix human muscles. An autopsy study[END_REF] reported a percentage of type I fibres as high as 80%, whereas in the orbicularis oculi they found 85% of type II fibres. Secher et al. [START_REF] Secher | Effect of tubocurarine on human soleus and gastrocnemius muscles[END_REF] suggested that tubocurarine affects human muscles in proportion to their type I muscle fibre In their study the application of tubocurarine resulted in a significantly greater reduction of muscle strength of the m. gastrocnemius (54% type I fibres) than in the m. soleus (71% type I fibres). Not surprisingly there is thus significant evidence that upper face muscles are less sensitive to non-depolarizing neuromuscular blockers than hand muscles, at least under anaesthesia conditions [START_REF] Edmonds | Comparison of frontalis and hypothenar EMG responses to vecuronium[END_REF][START_REF] Paloheimo | Comparison of neuromuscular blockade in upper facial and hypothenar muscles[END_REF][START_REF] Pathak | A comparison of the response of hand and facial muscles to non-depolarising relaxants[END_REF].

Given the algorithms implemented in the BIS-monitor and the CSM, EMG contamination of the EEG signal renders the possibility to interfere with the calculation of both the BIS and the CSI.

EMG frequencies overlap the 30-47 Hz range of the EEG (1), from which the 'beta-ratio' of the BIS algorithm is calculated. Thus, theoretically, elimination of EMG activity by NMB should lead to another beta-ratio, resulting in an altered BIS value. In the present study, mivacurium induced NMB had no impact on facial EMG activity as measured by the BIS-monitor. That might explain, why the BIS remained unchanged despite complete NMB, as confirmed by TOF stimulation of the ulnar nerve. 17) applied atracurium 0.4 mg kg -1 to adult patients anaesthetised with propofol/remifentanil after establishing a stable BIS value of 55 using TCI. They reported that NMB had no effect on the BIS. However, the application of neostigmine-glycopyrrolate led to a significant increase BIS of 7.1±7.5. -11 -might be explained by the fact, that the CSM did not detect any EMG activity even prior to mivacurium administration. This was possibly due to the rather narrow EEG band of 6-42.5 Hz used by the CSM for calculation of the CSI. Unfortunately there is no specific information available to the public about the frequency range of the CSM used for measurement of EMG activity. Facial EMG activity peaks a approximately 70Hz, and may even reach 300Hz [START_REF] Jensen | Pitfalls and challenges when assessing the depth of hypnosis during general anaesthesia by clinical signs and electronic indices[END_REF].

Vasella et al. (

As opposed to the BIS little is known about the Cerebral

Assuming that the CSM does not record any EEG/EMG activity outside the 6-42.5 Hz window this might, at least partly, explain why there wasn't any detection of EMG activity at all. The likelihood that mivacurium influences the BIS or the CSI appears to have very much to do with the amount of the facial EMG signal. Our data suggest that, due to the known relative insensitivity of the supraorbital muscles to NMBA (14-16), administration of mivacurium did not result in any changes of the amount of EMG activity as measured by the BIS and the CSM. In addition to that, propofol itself reduces EMG activity [START_REF] Rigouzzo | Effect of Muscle Relaxant on Bispectral Index in Anesthetized Children: Propofol Versus Sevoflurane[END_REF]. Since in the children who participated in our study propofol was titrated to achieve a rather deep level of hypnosis, it is, from a basic science point of view, less likely that mivacurium induced NMB influenced facial EMG activity.

The results of this study should not be extrapolated to other combinations of anaesthetics, muscle relaxants and monitors of hypnotic depth. Even with respect to other levels of hypnotic depth using the same drug-monitor combination it remains speculative whether or not the impact of neuromuscular block might the same as in this trial.

In conclusion in this paediatric study mivacurium induced neuromuscular block did not alter the level of consciousness as measured by either the Bispectral Index or the Cerebral State Index in children anaesthetised with propofol.

7

Outcomes and estimation 17

For each primary and secondary outcome, a summary of results for each group, and the estimated effect size and its precision (e.g., 95% confidence interval).

7, 14, 15

Ancillary analyses 18 Address multiplicity by reporting any other analyses performed, including subgroup analyses and adjusted analyses, indicating those pre-specified and those exploratory.

n.a.

Adverse events 19

All important adverse events or side effects in each intervention group. The CONSORT Statement 2001 checklist is intended to be accompanied with the explanatory document that facilitates its use. For more information, visit www.consort-statement.org.

Introduction

  For the adult patient population there are conflicting data in the literature as to whether or not frontal electromyogram activity (EMG ) affects the reliability of the BIS as a measure of depth of hypnosis (DH). On the other hand it is nowadays widely accepted that high frontal EMG activity might falsely elevate electroencephalogram (EEG) based indices of the depth of hypnosis (DoH)

  Danmeter A/S, Odense, Denmark) performs a computerised analysis of the EEG. CSM EEG processing results in an index indicating the hypnotic component of anaesthesia, the Cerebral State Index (CSI). Until now there are, to our best knowledge, no published data available with respect to the possible effects of neuromuscular block (NMB) on the accuracy of CSI calculation (3).

  activity. Unfortunately there is no information available to the public about the EEG frequency range, which is being used by the Cerebral State Monitor to measure and quantify EMG activity (dB).

  analysis was performed using SigmaStat, version 3.5 (Systat Software Inc., San Jose, CA, USA). For power analysis G*Power, version 3.0.10 was used (9). P values of <0.05 were considered statistically significant. Variables are presented as mean±sd unless otherwise stated.

  published which addressed this issue in the paediatric surgical population. To compare our results with other published data we mainly have to refer to papers dealing with the use of the BIS in adult patients.Rigouzzo et al.[START_REF] Rigouzzo | Effect of Muscle Relaxant on Bispectral Index in Anesthetized Children: Propofol Versus Sevoflurane[END_REF] presented in abstract form paediatric data on the effects of atracurium induced NMB on the BIS in children anaesthetised with propofol and sevoflurane. The authors applied Target Controlled Infusion (TCI) technique and observed a significant decrease of BIS values after injection of atracurium in children with low propofol plasma concentrations (2 and 3 µg ml -1 ), whereas there was no effect of NMB with higher plasma concentrations (4-6 µg ml -1 ) and in children anaesthetised with sevoflurane. These data suggest a direct, dose dependent effect of propofol on the EMG.The results of this study agree with data published by Greif et al.[START_REF] Greif | Muscle relaxation does not alter hypnotic level during propofol anesthesia[END_REF] who investigated the impact of different stages of mivacurium induced neuromuscular block on the BIS and fronto-temporal EMG activity in adult volunteers under propofol anaesthesia. In their study Greif et al. used a TCI system to achieve a constant level of hypnotic depth. A propofol plasma concentration of 3.8±0.4 µg ml -1 resulted in a BIS level of 39±4. Compared to our paediatric study with an overall baseline BIS value of 48±4 this represents a considerably deeper level of hypnosis. Greif et al furthermore measured a constant level of EMG activity of 28±1 dB irrespective of the degree of NMB. They hypothesised that the combination of a deep level of hypnosis and minimal EMG background activity NMB had no further impact on both the BIS and the EMG. Any extrapolation of the conclusions made by Greif et al. to the paediatric patients of our study is certainly

  In a recent study byDahaba et al. (8) the administration of mivacurium 0.15 mg/kg to adult patients under deep propofol/remifentanil anaesthesia resulted in a transient decline of BIS XP values from 41±3 to 35±3 (p = 0.003). TCI was applied to establish stable BIS of approximately 40 which was deemed to reflect a state of deep surgical anaesthesia. Recordings of BIS values were started after verifying the absence of EMG activity.

  State Monitor. There are at least two recent publications indicating that the CSI is capable to detect the graduated levels of propofol anaesthesia both in adults and children. Adult data published by Weber-Jensen et al. (18) showed a prediction probability (PK) value between the CSI and the Observer's Assessment of Alertness and Sedation score of 0.92. For paediatric patients under various levels of propofol sedation Disma et al. (19) reported a strong negative correlation (r = -0.823; P < 0.0001) between the CSI and the University of Michigan Sedation Scale for children.The present study suggests that mivacurium induced neuromuscular block does not alter the level of consciousness as measured by the CSI in children anaesthetised with propofol. This finding

  Denmark). Aspect Medical Systems and Danmeter A/S were not involved in the design, conduct or analysis of this study.

12 From

 12 results, taking into account study hypotheses, sources of potential bias or imprecision and the dangers associated with multiplicity of analyses and outcomes. validity) of the trial findings. 7-12 Overall evidence 22 General interpretation of the results in the context of current evidence. 7-Moher D, Schulz KF, Altman DG. The CONSORT statement: revised recommendations for improving the quality of reports of parallel-group randomised trials. Lancet 2001; 357(9263):1191-1194.

Table 1

 1 Intragroup comparison of EEG parameters BIS and CSI

	Parameter	BIS		CSI
	Group	Miva	Control	Miva	Contral
	Baseline	48.0 ± 4.8	48.7 ± 3.2	54.0 ± 7.7	54.8 ± 6.1
	+ 5 min.	53.0 ± 6.6	52.0 ± 4.0	57.4 ± 7.1	56.8 ± 6.8
	Diff. of means	-5.0 ± 7.1	-3.3 ± 4.5	-3.4 ± 5.3	-2.0 ± 2.5
	95 % CI diff. of means	-8.7 --1.3	-5.4 --1.1	-5.9 --1.0	-3.2 --0.8
	P value	0.01	0.005	0.009	0.002
	Mean values (±sd) of Bispectral Index (BIS) and Cerebral State Index (CSI), at baseline and 5
	minutes after study drug administration (+5 min.), together with the differences in means between
	baseline and +5 min., and 95 percent confidence interval for difference in means. P-values
	represent the results of a paired t-test.

Table 2

 2 Intergroup comparison of differences in BIS and CSI values before and after administration of How participants were allocated to interventions (e.g., "random allocation", "randomized", or "randomly assigned"). Specifically, for each group report the numbers of participants randomly assigned, receiving intended treatment, completing the study protocol, and analyzed for the primary outcome. Describe protocol deviations from study as planned, together with reasons. (denominator) in each group included in each analysis and whether the analysis was by "intention-to-treat". State the results in absolute numbers when feasible (e.g., 10/20, not 50%).

	study drugs
		BIS	CSI
	Group Miva
	x (diff.)	2.8	4.3
	IQR	0.8 -11.1	0.5 -6.4
	Group Control
	x (diff.)	1.9	2.5
	IQR	0.2 -5.8	0.2 -4.0
	P value	0.57 (n.s.)	0.29 (n.s.)
	Median values (x diff.) and interquartile range (IQR) of differences in EEG parameters Bispectral
	Index (BIS) and Cerebral State Index (CSI) between baseline and 5 minutes after study drug
	administration (+5 min.). P values represent the results of a Mann-Whitney Rank Sum Test.

Acknowledgements

The Rugloop™ software was provided by Aspect Medical Systems International B.V. (de Meern, The Netherlands). Labgrab™ software and AEP electrodes were provided by Danmeter A/S

This study was solely supported by departmental funding of the Department of Anaesthesia,


Richard Beringer
email: richard.beringer@uhbristol.nhs.uk

Noami Skeahan

Suzette Sheppard

Philip Ragg

Nick Martin

Ian Mckenzie

Andrew Davidson

Naomi Skeahan

Consultant Anaesthetist

Study to

Keywords: topical anesthesia, pediatric airway, lignocaine, local anaesthetic

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Background: Topical local anaesthesia of the airway of anaesthetised children has many potential benefits. In our institution, lignocaine is topically instilled blindly into the back of the mouth with the expectation that it will come into contact with the larynx. The volume and method of application varies between clinicians. There is no published evidence to support the plausibility of this technique.

Aim: To determine whether this technique of instillation results in the local anaesthetic coming into contact with key laryngeal structures and whether this is influenced by volume or additional physical manoeuvres.

Methods/Materials: Sixty three healthy anaesthetised children between six months and 16 years old had lignocaine stained with methylene blue poured into the back of their mouths. The volume and subsequent physical manoeuvre were determined by randomisation. A blinded observer assessed staining of the vocal cords, epiglottis, vallecula and piriform fossae by direct laryngoscopy. Airway complications were recorded.

Results: Fifty three of the 63 children had complete staining of all four areas. Four children had one area unstained and all others had at least partial staining of all four structures. Nine children coughed following induction of anaesthesia. Coughing was more likely in children with incomplete staining (p=0.03), low volume lignocaine (p=0.003) and following a head lift (p=0.02). Applying local anaesthetics topically to the airway of anaesthetised patients has many potential benefits such as: reducing the incidence of coughing and laryngospasm on induction or emergence from general anaesthesia (1-3), improving conditions for tracheal intubation in the absence of neuromuscular blockade (4), reducing the frequency of postoperative sore throat [START_REF] Tanaka | Lidocaine for preventing postoperative sore throat[END_REF] and attenuation of the sympathetic nervous system response to intubation [START_REF] Denlinger | Effects of intratracheal lidocaine on circulatory responses to tracheal intubation[END_REF](7)(8)(9).

Lignocaine is the most frequently used local anaesthetic agent for this purpose. It has been shown to be beneficial when used topically or systemically, but the evidence suggests a superior effect from topical use, with a lower total dose and lower systemic blood concentration (8).

These advantages must however be balanced by the potential risk to a child with a topically anaesthetised and unprotected airway, for example following extubation. Although there is no published literature to support a significant risk of aspiration in this situation, care must be taken with looking after a child with obtunded protective reflexes.

The most frequently described method for topical application of lignocaine is to spray it onto the laryngeal and tracheal mucosa under direct vision. An alternative technique has evolved in our institution, in which the local anaesthetic is blindly instilled into the back of the mouth, with the expectation that it will anaesthetise the hypopharynx and proximal laryngeal structures. Laryngoscopy is a potent cause of sympathetic stimulation and is also associated with adverse reflexes such as laryngospasm and coughing. It is therefore best performed once the airway has been anaesthetised. Review of the published literature does not reveal any previous studies assessing the use of local anaesthetic in this way. There is therefore some uncertainty if this practice is clinically effective, or if it is even plausible that the local anaesthetic reaches the area of intended action.

This practice has some variations including use of different volumes of local

In this study we added a dye (methylene blue) to the local anaesthetic and observed the degree of staining of the laryngeal structures. The purpose of this study was to gain evidence regarding the plausibility that this technique could anaesthetise the airway. In addition we wished to identify whether factors such as volume of local anaesthetic or additional physical manoeuvres influenced whether the local anaesthetic reached the intended site of action.

By determining if the local anaesthetic does indeed reach the relevant structures, this could inform future practice, and provide the scientific

Sample size and statistics

This was a prospective, randomised, blinded, observational study. We initially intended to recruit a total of 120 patients. This would have resulted in 24

RESULTS

Sixty three children were recruited. More children were recruited from the older age groups, but otherwise the groups were well balanced (Table 1). Age Quantitative analysis of the differences in the degree of staining between the different subgroups using the Chi squared test with Yates correction for continuity revealed no evidence for differences between: the five age groups (p=0.9); the two different volumes (p=0. The standard method of topically anaesthetising the airway is to spray local anaesthetic onto the larynx under direct vision using a laryngoscope. This might result in more frequent complete coverage than was demonstrated by this study, and therefore may be the technique of choice when local anaesthesia is absolutely necessary. However, laryngoscopy is not without risks and is associated with coughing, laryngospasm and regurgitation as well as stimulation of the sympathetic nervous system. A major potential benefit of the technique used in this study is to reduce the complications of laryngoscopy by providing anaesthesia before the laryngoscope comes into contact with the patient. Supplementary spraying of the trachea under direct vision may then be appropriate for some patients, such as those undergoing bronchoscopy. The observation that staining was extensive suggests it is plausible that the technique we use could be as effective as spraying under direct vision.

We wanted to identify whether factors such as volume of local anaesthetic or additional physical manoeuvres influenced whether the drug reached the intended site of action. No significant associations were observed with regards to staining of the larynx. However, the small numbers limit the power of this analysis.

Complications related to airway instrumentation during this study were a secondary outcome measure and relatively few in number, so it is difficult to draw firm conclusions. However, there was evidence for an association between both volume and technique, and coughing on induction. There was also evidence for an association between degree of staining and coughing.

Coughing was more frequent in patients with incomplete coverage of the larynx, only occurred in patients who received the lower volume of lignocaine and was also associated with the use of head lift after administration of lignocaine. These findings would support using the higher volume of local anaesthetic and avoiding head lift in future practice.

The observation that topical lignocaine reduces risk of coughing is consistent with the findings of other investigators who have shown reduced frequency and severity of coughing for up to two hours following application of topical lignocaine [START_REF] Minogue | Laryngotracheal topicalization with lidocaine before intubation decreases the incidence of coughing on emergence from general anesthesia[END_REF]. It seems plausible that by reducing the frequency of coughing, this technique may also reduce the frequency of more severe complications such as laryngospasm. Further study is necessary to determine whether this is the case.

The study has some limitations. The most significant is that staining does not necessarily equate with sufficient local anaesthesia to reduce reflex coughing or other responses to tracheal intubation. Although, as mentioned above we found evidence for some associations between staining and coughing, the anaesthetic conditions such as use of agent and depth of anaesthesia were not controlled at this period. These could influence any observed association.

In order to design a study to assess the effect of topical local anaesthetic on adverse reflexes during anaesthesia, it would be necessary to standardise the anaesthetic technique and also the time between instillation of local anaesthetic and laryngoscopy.

There is a theoretical potential for physicochemical interactions between methylene blue and lignocaine which may have influenced the findings.

Methylene blue is a weak acid (pH 3 to 4.5) and may alter the lipid solubility of lignocaine and the detergent effect of methylene blue may have enhanced the spread of the mixture over mucus membranes.

The main disadvantage to using topical local anaesthetic within the airway is that persisting anaesthesia following extubation may make aspiration of pharyngeal contents more likely. There is little evidence in the literature to suggest that this is a significant problem, but it seems intuitive that the child with diminished protective reflexes may be at higher risk.

In summary, this study supports the plausibility of this technique for topical anaesthesia of the larynx of anaesthetised children. This supports the continued use of this technique and we plan to carry out further research based upon these findings to determine whether this local anaesthesia of the

  CO-AUTHOR DETAILS Naomi Skeahan: Paediatric anaesthesia fellow, Royal Children's Hospital, Melbourne, Australia. Suzette Sheppard: Anaesthesia Research Project Officer, Murdoch Children's Research Institute, Melbourne, Australia Philip Ragg, Nick Martin, Ian McKenzie and Andrew Davidson: Staff Anaesthetists, Royal Children's Hospital, Melbourne, Australia.

  Oral administration of lignocaine without use of a laryngoscope frequently results in widespread coverage of key laryngeal structures and may reduce the risk of coughing.

  as occasional use of additional procedures such as lifting the patient's head, or posterior depression of the larynx (dunking) with the intention of enhancing delivery of the solution to the desired anatomical structure. The advantages of this technique include simplicity, and avoidance of the need for laryngoscopy to allow application of the local anaesthetic.

  plausibility for further study to examine the effectiveness of the technique in terms of relevant clinical outcomes.

  consent and approval from the Human Research Ethics Committee at the Royal Children's Hospital in Melbourne, we recruited sixty three healthy children (ASA 1 or 2), aged between 6 months and 16 years, who were scheduled to undergo elective surgery for which they would require a tracheal tube. Children with a contraindication to the use of either of the study drugs, or an anticipated difficult laryngoscopy were excluded.Children were stratified into five age groups (6 to 12 months; 1 to 2 years; 2 to four years; 4 to 8 years; and 8 to 16 years) and randomised to receive one of two volumes of lignocaine (0.05 or 0.15 ml/kg), and one of three additional manoeuvres following application of the local anaesthetic (no additional manoeuvre; head lift; or laryngeal dunk). Head lift was performed by lifting the child's head until the chin touched the chest for three seconds and then returning it to the neutral position. Laryngeal dunk was performed by applying standard cricoid pressure for three seconds. Methylene blue was mixed with the lignocaine to act as a tissue stain. Two percent lignocaine was mixed with 1% methylene blue in a 9:1 ratio by volume, resulting in a solution containing 1.8 percent lignocaine and 0.1 percent methylene blue. A maximum volume of four millilitres was used. The primary outcome measure was complete staining of four laryngeal structures: the vocal cords; piriform fossae; epiglottis; and vallecula. The secondary outcome measure was the frequency of airway complications after dye up until the start of surgery, including: coughing; laryngospasm; oxygen desaturation (below 90 percent); and bradycardia (less than 80 percent of baseline rate).All observations during the study were made by one of two principal investigators. The choice of anaesthetic technique was decided by the anaesthetist. Once the patient was anaesthetised, one investigator poured the predetermined volume of lignocaine/methylene blue into the back of the child's mouth over a few seconds using a syringe without needle and performed any additional manoeuvre required by randomisation. The facemask was then reapplied to the child's face and the child ventilated or allowed to breathe as normal. When the anaesthetist was satisfied that the child was adequately anaesthetised to allow intubation, the other blinded investigator perfomed standard direct laryngoscopy to assess the staining of the vocal cords, epiglottis, vallecula and piriform fossae. Staining was classified as complete, partial or unstained. The investigator or anaesthetist then inserted the tracheal tube and surgery commenced as normal. The degree of staining was recorded on a case report form along with demographic information, details of the anaesthetic technique and details of adverse events.

  in each age group; 60 children for each volume of lignocaine; and 40 children for each additional manoeuvre. However, recruitment was slower than expected and after studying 63 children the principal investigator's rotation at the hospital came to an end. It was therefore decided that the study should be stopped at this stage. The analysis plan was to report the frequency of full staining in each combination group and depending on the spread of results, perform more sophisticated post-hoc analysis examining the difference between groups if possible. Such post-hoc analyses were performed using the Chi squared test, with Yates' correction for continuity when expected frequency was less than five.

  ranged from seven months to 15 years, and weight ranged from seven to 109 kilograms. In the five different age groups (six to 12 months; 1 to 2 years; 2 to 4 years; 4 to 8 years and 8 to 16 years) there were 6; 6; 7; 21 and 23 children respectively. In the low volume and high volume groups, there were 31 and 32 children respectively. For the different techniques (no additional technique; head lift and laryngeal dunk) there were 21; 20; and 22 children respectively.No substantial differences were noted between the groups in terms of patient demographics or anaesthetic technique, including use of muscle relaxation.The time between instillation of solution and laryngoscopy ranged from 30 seconds to four minutes (median time one minute). The primary outcome measure was complete staining of four laryngeal structures (vocal cords, piriform fossae, epiglottis and vallecula). Fifty three of the 63 subjects had complete staining of all four areas. Four children had one area unstained and all others had at least partial staining of the four structures. All children had complete staining of the piriform fossae. Eight children had only partial staining of the epiglottis, one child had an unstained vallecula and three children had no staining of the vocal cords. The details of the 10 children in whom there was incomplete staining are shown in Table2.

  3); or the use of additional techniques (p=0.9). The secondary outcome measure was the frequency of adverse events during induction of anaesthesia (cough, laryngospasm and drop in oxygen saturation below 90 percent). Nine children coughed, two during intubation and seven following intubation. No children developed laryngospasm or bradycardia. Two children desaturated below 90 percent, although in both cases this was attributed to anaesthetic technique, not an airway complication. Details of the nine children who coughed are shown in Table 3. Coughing was more likely in children with incomplete staining (five of the 53 children with complete staining and in four of the 10 children with incomplete staining; risk ratio 0.24; 95% confidence intervals 0.08 to 0.7; p=0.03). All nine children who coughed had received the low volume of lignocaine (p=0.003). Coughing occurred in seven children who had received a head lift, none who had received a laryngeal dunk and two patients in which no additional technique was used (p=0.02).Follow up of children after their procedure revealed no complications apart from temporary residual oropharyngeal staining. There were no reported episodes of aspiration or difficulty with drinking or eating in the postoperative period.

  that oral administration of lignocaine without use of a laryngoscope frequently results in complete coverage of key laryngeal structures. Fifty three of 63 children had completely stained larynxes from this technique, and the other 10 children all had at least partial staining of the larynx.

  Murthy BV, Barrett PJ et al. Comparison of the effects of topical lignocaine spray applied before or after induction of anaesthesia on the pressor response to direct laryngoscopy and intubation. Eur J Anaesthesiol 1999;16(1): 7-10. 8 Hamaya Y, Dohi S. Differences in cardiovascular response to airway stimulation at different sites and blockade of the responses by lidocaine. Anesthesiol 2000; 93(1): 95-103. 9 Stoelting RK. Circulatory response to laryngoscopy and tracheal intubation with or without prior oropharyngeal viscous lidocaine. Anesth Analg 1977; 56(5): 618-21.

Table 1

 1 Randomisation details and degree of laryngeal staining

	Age		Technique		Volume	Number of patients
	category number	category number	category	number	Incompletely	Completely
		of	of	(ml/kg)	of	stained	stained
	patients	patients		patients
			None	2	0.05 0.15	1 1		1 1
	6 -12 months	6	Head lift	1	0.05 0.15	1 0		1
			Laryngeal dunk	3	0.05 0.15	1 2		1 2
			None	2	0.05 0.15	1 1	1	1
	1 -2 years	6	Head lift	2	0.05 0.15	1 1		1 1
			Laryngeal dunk	2	0.05 0.15	1 1	1	1
			None	1	0.05 0.15	0 1	1
	2 -4 years	7	Head lift	3	0.05 0.15	2 1	1	1 1
			Laryngeal dunk	3	0.05 0.15	2 1		2 1
			None	8	0.05 0.15	4 4		4 4
	4 -8 years	21	Head lift	6	0.05 0.15	3 3	1	3 2
			Laryngeal dunk	7	0.05 0.15	3 4	1	2 4
			None	8	0.05 0.15	4 4		4 4
	8 -16 years	23	Head lift	8	0.05 0.15	4 4	2	2 4
			Laryngeal dunk	7	0.05 0.15	3 4	2	1 4

Table 2

 2 Details of the 10 children with incomplete staining

	F o r
	P
	e
	e r
	R
	e
	v i e
	w

Table 3

 3 Details of the nine children who coughed How participants were allocated to interventions (e.g., "random allocation", "randomized", or "randomly assigned"). (denominator) in each group included in each analysis and whether the analysis was by "intention-totreat". State the results in absolute numbers when feasible (e.g., 10/20, not 50%).Outcomes and estimation 17For each primary and secondary outcome, a summary of results for each group, and the estimated effect size and its precision (e.g., 95% confidence interval).

	Child	Age	Volume	Staining Technique
		(years)	(mL/Kg)
	1	4 to 8	0.05	complete	None
	2	8 to 16	0.05	incomplete Head lift
	3	4 to 8	0.05	complete	Head lift
	4	8 to 16	0.05	complete	Head lift
	5	2 to 4	0.05	complete	Head lift
	6	8 to 16	0.05	complete	Head lift
	7	1 to 2	0.05	incomplete	None
	8	4 to 8	0.05	incomplete head lift
	9	8 to 16	0.05	incomplete head lift

www.consort-statement.org

ACKNOWLEDGEMENTS

We would like to thank Associate Professor Donnath at the Murdoch Children's Research Institute in Melbourne for her advice on study design and statistics.