Concentration-effect relationship of cisatracurium at three different dose levels in the anesthetized patient.

BACKGROUND: The linearity of cisatracurium elimination and its concentration-effect relation were determined as part of a traditional rich data study with three dose levels in patients receiving balanced anesthesia. METHODS: Forty-eight adults with American Society of Anesthesiologists status I-II were randomized to receive an intravenous bolus dose of 0.075, 0.15, or 0.30 mg/kg cisatracurium. Anesthesia was induced and maintained with nitrous oxide-oxygen, propofol, and fentanyl. The mechanical response of the adductor pollicis muscle was recorded. Arterial blood samples were collected over 8 h. Cisatracurium, laudanosine, and the monoquaternary alcohol concentrations were measured by high-performance liquid chromatography. To assess the relative contribution of the input function, a parametric (assuming elimination from both the central and peripheral compartments) and a nonparametric pharmacokinetic-pharmacodynamic model were both applied to data. RESULTS: Dose proportionality of the body disposition of cisatracurium and its two major metabolites at doses up to 0.30 mg/kg was confirmed. With the parametric approach, the effect compartment concentration at 50% block (EC50) significantly increased with the dose (136 vs. 157 vs. 209 ng/ml), whereas the effect compartment equilibration rate constant decreased (0.0675 vs. 0.0568 vs. 0.0478 min(-1)). A similar dose-dependent effect of the pharmacokinetic-pharmacodynamic relation was observed with the nonparametric approach, but the trend was 50% less pronounced. CONCLUSION: A dose-related change in pharmacokinetic-pharmacodynamic parameters was identified with both modeling approaches. A pharmacokinetic origin was ruled out, although no definite explanation of the underlying mechanism could be provided. These findings suggest that doses relevant to the anesthetic practice be used for estimation of EC50.

Crystal structure of a monocotyledon (maize ZMGlu1) beta-glucosidase and a model of its complex with p-nitrophenyl beta-D-thioglucoside.

The maize beta-glucosidase isoenzymes ZMGlu1 and ZMGlu2 hydrolyse the abundant natural substrate DIMBOAGlc (2-O-beta-D-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one), whose aglycone DIMBOA (2,4-hydroxy-7-methoxy-1,4-benzoxazin-3-one) is the major defence chemical protecting seedlings and young plant parts against herbivores and other pests. The two isoenzymes hydrolyse DIMBOAGlc with similar kinetics but differ from each other and their sorghum homologues with respect to specificity towards other substrates. To gain insights into the mechanism of substrate (i.e. aglycone) specificity between the two maize isoenzymes and their sorghum homologues, ZMGlu1 was produced in Escherichia coli, purified, crystallized and its structure solved at 2.5 Angstrom resolution by X-ray crystallography. In addition, the complex of ZMGlu1 with the non-hydrolysable inhibitor p-nitrophenyl beta-D-thioglucoside was crystallized and, based on the partial electron density, a model for the inhibitor molecule within the active site is proposed. The inhibitor is located in a slot-like active site where its aromatic aglycone is held by stacking interactions with Trp-378. Whereas some of the atoms on the non-reducing end of the glucose moiety can be modelled on the basis of the electron density, most of the inhibitor atoms are highly disordered. This is attributed to the requirement of the enzyme to accommodate two different species, namely the substrate in its ground state and in its distorted conformation, for catalysis.

Neuromuscular blockade. Inadvertent extubation of the partially paralyzed patient.

Residual neuromuscular block is common after the use of neuromuscular blocking drugs during anesthesia. Although careful reversal [table: see text] techniques usually result in adequate return of neuromuscular recovery, sometimes it is not possible to achieve full recovery of neuromuscular block. Ventilatory support and maintenance of a patent airway are required until recovery can be demonstrated. In those situations, in which some TOF fade is still obvious, the anesthesiologist should consider retaining the endotracheal tube in position; it is not a sign of failure to return a patient whose trachea is still intubated to the postanesthesia care unit. The inadvertent extubation of patients who are partially paralyzed results in increased postoperative morbidity.

Anaesthesia research is important.

The purpose of this paper is to describe the impact of anaesthesia research on clinical practice. The evolution of neuromuscular blocking drugs from the laboratory to the operating room is used as an example. Particular emphasis is given to the pioneers whose vision made this possible: H. R. Griffith and G. E. Johnson; D. Savage, J. B. Stenlake and W. C. Bowman and J. Viby-Mogensen. Our challenge is to ensure the supply of clinical scientists for the future.

The mechanism of substrate (aglycone) specificity in beta -glucosidases is revealed by crystal structures of mutant maize beta -glucosidase-DIMBOA, -DIMBOAGlc, and -dhurrin complexes.

The mechanism and the site of substrate (i.e., aglycone) recognition and specificity were investigated in maize beta-glucosidase (Glu1) by x-ray crystallography by using crystals of a catalytically inactive mutant (Glu1E191D) in complex with the natural substrate 2-O-beta-d-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOAGlc), the free aglycone DIMBOA, and competitive inhibitor para-hydroxy-S-mandelonitrile beta-glucoside (dhurrin). The structures of these complexes and of the free enzyme were solved at 2.1-, 2.1-, 2.0-, and 2.2-A resolution, respectively. The structural data from the complexes allowed us to visualize an intact substrate, free aglycone, or a competitive inhibitor in the slot-like active site of a beta-glucosidase. These data show that the aglycone moiety of the substrate is sandwiched between W378 on one side and F198, F205, and F466 on the other. Thus, specific conformations of these four hydrophobic amino acids and the shape of the aglycone-binding site they form determine aglycone recognition and substrate specificity in Glu1. In addition to these four residues, A467 interacts with the 7-methoxy group of DIMBOA. All residues but W378 are variable among beta-glucosidases that differ in substrate specificity, supporting the conclusion that these sites are the basis of aglycone recognition and binding (i.e., substrate specificity) in beta-glucosidases. The data also provide a plausible explanation for the competitive binding of dhurrin to maize beta-glucosidases with high affinity without being hydrolyzed.

The prolonged duration of rocuronium in Chinese patients.

We compared the potency and duration of action of rocuronium in Chinese and Caucasian patients during general anesthesia. Thirty-six women (18 Caucasian and 18 Chinese) and 36 children (18 Caucasian and 18 Chinese) were evaluated during the administration of propofol/fentanyl anesthesia. Patients in each age group were randomized into three subgroups to receive single doses of 0.06, 0. 12, or 0.18 mg/kg rocuronium (adults) or 0.12, 0.18, or 0.24 mg/kg rocuronium (children). Neuromuscular blockade was assessed by electromyography of the adductor pollicis after train-of-four (TOF) stimulation of the ulnar nerve. Dose response curves were constructed when maximum neuromuscular depression of the first twitch of the train (T(1)) was obtained. A second bolus dose of rocuronium was then administered to a total dose of 0.6 mg/kg. The times of spontaneous recovery to T(1) 10%, 25%, and 90% of control and to TOF 0.25, 0.50, and 0.70 were recorded. For both adults and children, recovery occurred later in Chinese than in Caucasian patients (P<0.05 for T(1) of 10%, 25%, 75%, and 90% and TOF to 0.7). The 50% effective dose was smaller in Chinese adults (125+/-63 vs. 159+/-66 microg/kg) and Chinese children (171+/-43 vs. 191+/-46 microg/kg) than in Caucasian adults and children, but the difference was not statistically significant. In adults, time to 25% T(1) recovery was 43+/-13 min in Chinese patients and 33+/-10 min in Caucasian patients (P<0.05). The corresponding values were more rapid for children: 30+/-10 and 24+/-6 min (P<0.05). We conclude that the recovery from rocuronium neuromuscular blockade was longer in Chinese compared with Caucasian patients and in adults compared with children.

The aglycone specificity-determining sites are different in 2, 4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA)-glucosidase (Maize beta -glucosidase) and dhurrinase (Sorghum beta -glucosidase).

The maize beta-glucosidase isozyme Glu1 hydrolyzes a broad spectrum of substrates in addition to its natural substrate DIMBOAGlc (2-O-beta-d-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-on e), whereas the sorghum beta-glucosidase isozyme Dhr1 hydrolyzes exclusively its natural substrate dhurrin (p-hydroxy-(S)-mandelonitrile-beta-d-glucose). To study the mechanism of substrate specificity further, eight chimeric beta-glucosidases were constructed by replacing peptide sequences within the C-terminal region of Glu1 with the homologous peptide sequences of Dhr1 or vice versa, where the two enzymes differ by 4 to 22 amino acid substitutions, depending on the length of the swapped regions. Five Glu1/Dhr1 chimeras hydrolyzed substrates that are hydrolyzed by both parental enzymes, including dhurrin, which is not hydrolyzed by Glu1. In contrast, three Dhr1/Glu1 chimeras hydrolyzed only dhurrin but with lower catalytic efficiency than Dhr1. Additional domain-swapping within the C-terminal domain of Glu1 showed that replacing the peptide (466)FAGFTERY(473) of Glu1 with the homologous peptide (462)SSGYTERF(469) of Dhr1 or replacing the peptide (481)NNNCTRYMKE(490) in Glu1 with the homologous peptide (477)ENGCERTMKR(486) of Dhr1 was sufficient to confer to Glu1 the ability to hydrolyze dhurrin. Data from various reciprocal chimeras, sequence comparisons, and homology modeling suggest that the Dhr1-specific Ser-462-Ser-463 and Phe-469 play a key role in dhurrin hydrolysis. Similar data suggest that DIMBOAGlc hydrolysis determinants are not located within the extreme 47-amino acid-long C-terminal domain of Glu1.

Molecular dynamics simulations of the d(CCAACGTTGG)(2) decamer: influence of the crystal environment.

Molecular dynamics (MD) simulations of the DNA duplex d(CCAACGTTGG)(2) were used to study the relationship between DNA sequence and structure. Two crystal simulations were carried out; one consisted of one unit cell containing two duplexes, and the other of two unit cells containing four duplexes. Two solution simulations were also carried out, one starting from canonical B-DNA and the other starting from the crystal structure. For many helicoidal parameters, the results from the crystal and solution simulations were essentially identical. However, for other parameters, in particular, alpha, gamma, delta, (epsilon - zeta), phase, and helical twist, differences between crystal and solution simulations were apparent. Notably, during crystal simulations, values of helical twist remained comparable to those in the crystal structure, to include the sequence-dependent differences among base steps, in which values ranged from 20 degrees to 50 degrees per base step. However, in the solution simulations, not only did the average values of helical twist decrease to approximately 30 degrees per base step, but every base step was approximately 30 degrees, suggesting that the sequence-dependent information may be lost. This study reveals that MD simulations of the crystal environment complement solution simulations in validating the applicability of MD to the analysis of DNA structure.

Solution 1H NMR study of the heme cavity and folding topology of the abbreviated chain 118-residue globin from the cyanobacterium Nostoc commune.

The globin from the cyanobacterium Nostoc commune, abbreviated GlbN, which appears to serve as a part of a terminal oxidase rather than as a respiratory pigment, displays relatively normal O2 binding properties, despite the highly abbreviated polypeptide chain, (118 residues) relative to more conventional globins [Thorsteinsson, M. V. , Bevan, D. R., Potts, M., Dou, Y., Eich, R. F., Hargrove, M. S., Gibson, Q. H., and Olson, J. S. (1999) Biochemistry 38, 2117-2126]. The nature of the heme cavity and the general folding topology of this cyanoglobin were investigated by solution 1H NMR to establish the extent to which, and the manner in which, this compact globin adheres to the standard globin fold. This represents by far the smallest globin subjected to structural analysis. The paramagnetic cyanomet derivative was selected because its characteristically large magnetic anisotropy imparts significant dipolar shifts which both improve resolution to greatly facilitate assignments and serve as indicators of the folding topology of the globin. Identification of the axial His 70 and highly conserved Phe 35 (CD1) determined the absolute orientation of the heme and proximal His. Sequential assignments of four helical and one loop segments, which exhibit dipolar contacts to the heme and among each other, confirm the presence of well-conserved F, G, and H helices and the FG corner. The majority of the abbreviation of the chain relative to the more conventional length globins is accommodated in the A-D helices, of which the last is completely missing. The distal residue which provides a H-bond to bound ligand is identified as Gln 43, but the expected helical position E7 could not be confirmed. His 46, placed at position E10, is found to adopt alternate orientations into, and out of, the heme cavity depending on protonation state, suggesting the presence of a Bohr effect at low pH. It is shown that the dipolar shifts exhibited by backbone protons for the assigned residues conform well to those observed for other cyanomet globins and further support a conserved Mb fold. Perturbed medium-range dipolar contacts and the pH-independent backbone proton lability of the F helix are interpreted in terms of a holoprotein which is less stable than a conventional length globin.

Monitoring and reversal of neuromuscular block.

Methods of monitoring and reversing neuromuscular blocking agents to avoid residual neuromuscular block are described. Studies have shown that if a long-acting neuromuscular blocking agent is used during anesthesia, the frequency of residual block, regardless of the method of neuromuscular monitoring, will be at least 20%. In the past 20-25 years, anesthesiologists have come to use some form of nerve stimulation to monitor the degree of residual neuromuscular block; there are various patterns of stimulation, including train-of-four (TOF) stimulation and double-burst stimulation (DBS). For both TOF stimulation and DBS, the response to the stimuli in a series fades such that the last response can be expressed as a ratio of the first. The fade to DBS is the same as that to TOF stimulation. Clinicians can clinically detect a fade in TOF response when the TOF ratio is <0.5. Fade to DBS is easier to detect than that to TOF stimulation, but, as the block recovers, the anesthesiologist's ability to detect fade decreases. Although anesthesiologists have accepted a TOF ratio of at least 0.7 as the standard, studies of vecuronium neuromuscular block have shown an impaired ventilatory response to hypoxemia and the possibility of increased risk of aspiration until the TOF ratio recovered to 0.9. The use of pancuronium and a persistent TOF ratio of 0.7 in the postanesthesia care unit was shown to be associated with a threefold greater occurrence of postoperative pulmonary complications compared with vecuronium or atracurium. Spontaneous recovery from neuromuscular block occurs through redistribution, metabolism, or buffered diffusion, but recovery can be accelerated by administration of anticholinesterase agents, such as neostigmine and edrophonium. Studies suggest that even intermediate-duration agents should be reversed. Rapacuronium is a new investigational drug with similar onset characteristics to succinylcholine and, if reversed early, similar recovery characteristics. Postoperative residual neuromuscular block is frequent, dangerous, and difficult to recognize clinically. The action of neuromuscular blocking agents should always be reversed unless there is unequivocal evidence of adequate function.

Early and late reversal of rocuronium and vecuronium with neostigmine in adults and children.

UNLABELLED: We investigated the influence of the timing of neostigmine administration on recovery from rocuronium or vecuronium neuromuscular blockade. Eighty adults and 80 children were randomized to receive 0.45 mg/kg rocuronium or 0.075 mg/kg vecuronium during propofol/fentanyl/N2O anesthesia. Neuromuscular blockade was monitored by train-of-four (TOF) stimulation and adductor pollicis electromyography. Further randomization was made to control (no neostigmine) or reversal with 0.07 mg/kg neostigmine/0.01 mg/kg glycopyrrolate given 5 min after relaxant, or first twitch (T1) recovery of 1%, 10%, or 25%. Another eight adults and eight children received 1.5 mg/kg succinylcholine. At each age, spontaneous recovery of T1 and TOF was similar after rocuronium and vecuronium administration but was more rapid in children (P < 0.05). Spontaneous recovery to TOF0.7 after rocuronium and vecuronium administration in adults was 45.7 +/- 11.5 min and 52.5 +/- 15.6 min; in children, it was 28.8 +/- 7.8 min and 34.6 +/- 9.0 min. Neostigmine accelerated recovery in all reversal groups (P < 0.05) by approximately 40%, but the times from relaxant administration to TOF0.7 were similar and independent of the timing of neostigmine administration. Recovery to T1 90% after succinylcholine was similar in adults (9.4 +/- 5.0 min) and children (8.4 +/- 1.1 min) and was shorter than recovery to TOF0.7 in any reversal group after rocuronium or vecuronium administration. Recovery from rocuronium and vecuronium blockade after neostigmine administration was more rapid in children than in adults. Return of neuromuscular function after reversal was not influenced by the timing of neostigmine administration. These results suggest that reversal of intense rocuronium or vecuronium neuromuscular blockade need not be delayed until return of appreciable neuromuscular function has been demonstrated. IMPLICATIONS: These results suggest that reversal of intense rocuronium or vecuronium neuromuscular blockade need not be delayed until return of appreciable neuromuscular function has been demonstrated. Although spontaneous and neostigmine-assisted recovery is more rapid in children than in adults, in neither is return of function as rapid as after succinylcholine administration.

Early reversal of rapacuronium with neostigmine.

BACKGROUND: Rapacuronium is a rapid-onset, short-acting neuromuscular relaxant. This multiple-center study determined neuromuscular recovery when neostigmine was given 2 or 5 min after rapacuronium. METHODS: One hundred seventeen patients were randomized to receive two different doses of rapacuronium and to receive neostigmine in two different doses and at two different times. During propofol anesthesia with nitrous oxide, oxygen, and fentanyl, 1.5 or 2.5 mg/kg rapacuronium was given 1 min before tracheal intubation. Neuromuscular block was measured by train-of-four ulnar nerve stimulation every 12 s: The adductor pollicis force of contraction was recorded mechanomyographically. Two or five minutes after rapacuronium was administered, 0.05 or 0.07 mg/kg neostigmine was administered and recovery was compared with that of control patients who received no neostigmine. RESULTS: Both doses of rapacuronium produced 100% block in all but one patient, who exhibited 97% block. Neostigmine accelerated recovery in all groups. After 1.5 mg/kg rapacuronium, the time to 25% T1 twitch recovery decreased from a mean of 16 min in control patients to mean values of 8-10 min in the treatment groups: The time to train-of-four ratio of 0.7 decreased from 38 min to 17-19 min. After 2.5 mg/kg rapacuronium, the time to 25% T1 was reduced from 23 min to 11-12 min, and the time to train-of-four ratio of 0.7 decreased from 54 min to 26-32 min. Recovery was not different among the the groups that received different doses and timing of neostigmine. CONCLUSIONS: Recovery of intense rapacuronium block was accelerated by early neostigmine administration. When given 2 min after rapacuronium, neostigmine was as effective as after 5 min, and 0.05 mg/kg neostigmine was comparable to 0.07 mg/kg neostigmine.

A cyanobacterial hemoglobin with unusual ligand binding kinetics and stability properties.

The glbN gene of the cyanobacterium Nostoc commune UTEX 584 encodes a hemoprotein, named cyanoglobin, that has high oxygen affinity. The basis for the high oxygen affinity of cyanoglobin was investigated through kinetic studies that utilized stopped-flow spectrophotometry and flash photolysis. Association and dissociation rate constants were measured at 20 degrees C for oxygen, carbon monoxide, nitric oxide, and methyl and ethyl isocyanides. The association rate constants for the binding of these five ligands to cyanoglobin are the highest reported for any naturally occurring hemoglobin, suggesting an unhindered and apolar ligand binding pocket. Cyanoglobin also shows high rates of autoxidation and hemin loss, indicating that the prosthetic group is readily accessible to solvent. The ligand binding behavior of cyanoglobin was more similar to that of leghemoglobin a than to that of sperm whale myoglobin. Collectively, the data support the model of cyanoglobin function described by Hill et al. [(1996) J. Bacteriol. 178, 6587-6598], in which cyanoglobin sequesters oxygen, and presents it to, or is a part of, a terminal cytochrome oxidase complex in Nostoc commune UTEX 584 under microaerobic conditions, when nitrogen fixation, and thus ATP demand, is maximal.

Paralysis in the critically ill: intermittent bolus pancuronium compared with continuous infusion.

OBJECTIVES: To compare recovery times from neuromuscular blockade between two groups of critically ill patients in whom pancuronium was administered by continuous infusion or intermittent bolus injection. To compare the mean pancuronium requirements (milligrams per kilogram per hour) and to assess the incidence of prolonged recovery times (>12 hrs) and residual muscle weakness. DESIGN: Prospective, observational cohort. SETTING: Intensive care unit in a university-affiliated hospital. PATIENTS: A total of 30 mechanically ventilated patients who required pharmacologic paralysis. Patients were excluded if they had renal failure (creatinine clearance <30 mL/min), heart rate >130 beats/min, hepatic failure, peripheral nerve disease or myopathy, stroke, spinal cord damage, or myasthenia gravis. INTERVENTIONS: Patients were assigned to receive pancuronium either by continuous infusion (n = 14) or intermittent bolus (n = 16). Depth of paralysis was titrated to maintain one or two responses to Train-of-Four stimulation with an accelerograph and desired clinical goals. Recovery time was defined as time from discontinuation of muscle relaxant until the amplitude of the fourth twitch, measured every 15-30 min using an accelerograph, was 70% the amplitude of the first twitch (Train-of-Four > or = 0.7). MEASUREMENTS AND MAIN RESULTS: These patients included the only three patients with status asthmaticus in our study. The groups were similar with respect to age, sex, weight, Acute Physiology and Chronic Health Evaluation II score, mode of ventilation, creatinine clearance, indications for paralysis, and duration of pancuronium administration. The median time for patients to recover from paralysis was 3.5 hrs (95% confidence interval, 1.82-5.18) in the infusion group vs. 6.3 hrs (95% confidence interval, 3.40-9.19) in the intermittent bolus group (p = .10). Less drug was administered in the intermittent group (mean, 0.02+/-0.01 mg/kg/hr) than by infusion (mean, 0.04+/-0.01 mg/kg/hr; p < .001). Six patients (five in the infusion group and one in the intermittent group) developed persistent severe muscle weakness. In addition, six different patients (three from each group) had prolonged recovery >12 hrs. CONCLUSIONS: Our study suggests that recovery time after paralysis with continuous infusion is faster than that after intermittent bolus injection. Although more pancuronium was administered in the continuous-infusion group, recovery time was not prolonged as a consequence. It is uncertain whether pancuronium given by infusion increases the risk of persistent muscle weakness.

Decreased mivacurium requirements and delayed neuromuscular recovery during sevoflurane anesthesia in children and adults.

UNLABELLED: The purpose of this study was to compare the mivacurium infusion requirements and neuromuscular recovery in adults and children during propofol/opioid and sevoflurane anesthesia. Seventy-five adult and 75 pediatric patients were randomized to receive propofol/opioid 0.5 or 1.0 minimum alveolar anesthetic concentration (MAC) (age-related) sevoflurane anesthesia. Plasma cholinesterase (PChE) activity was measured. Neuromuscular blockade was monitored by train-of-four (TOF) stimulation every 10 s and adductor pollicis electromyography. A bolus of 2 x the 95% effective dose of mivacurium (0.25 mg/kg) was followed by an infusion titrated to maintain 90%-95% blockade. Mivacurium doses were recorded every 5 min. At the end of surgery, the infusion was stopped, and recovery from mivacurium was monitored until TOF > or =0.7. PChE concentrations were within the normal range (adults 4-12 KU/L, children 6-16 KU/L) and correlated with mivacurium dose. Mivacurium infusion rates were higher in children than in adults: at 30 min, the rates in children were 13.1 +/- 6.4, 8.1 +/- 4.7, and 5.2 +/- 2.9 microg x kg(-1) x min(-1) at 0, 0.5, and 1.0 MAC sevoflurane, respectively; the corresponding rates in adults were 5.9 +/- 3.1, 4.3 +/- 1.7, and 2.9 +/- 0.7 microg x kg(-1) x min(-1) (P < 0.01). Sevoflurane decreased mivacurium requirements, maximal decreases at 45 min in children and 10 min in adults, and delayed neuromuscular function recovery. Children recovered twice as quickly as adults, achieving TOF > or =0.7 at 9.8 +/- 2.5, 11.4 +/- 2.8, and 19.6 +/- 6.3 min compared with 19.9 +/- 5.4, 26.4 +/- 8.3, and 32.9 +/- 9.8 min in adults (P < 0.0001). In conclusion, mivacurium requirements were correlated with PChE, were greater in children than in adults, and were reduced by sevoflurane. Neuromuscular recovery occurred more rapidly in children and was delayed by sevoflurane. IMPLICATIONS: The mivacurium infusion requirement to maintain constant 90%-95% neuromuscular block during anesthesia is correlated with plasma cholinesterase activity. It is increased in children and reduced by the inhaled anesthetic sevoflurane. Despite the larger dose administered to children, recovery from block occurred more rapidly in children than in adults and was delayed by sevoflurane.