Characterization of a flavoprotein oxidase from opium poppy catalyzing the final steps in sanguinarine and papaverine biosynthesis.

Benzylisoquinoline alkaloids are a diverse class of plant specialized metabolites that includes the analgesic morphine, the antimicrobials sanguinarine and berberine, and the vasodilator papaverine. The two-electron oxidation of dihydrosanguinarine catalyzed by dihydrobenzophenanthridine oxidase (DBOX) is the final step in sanguinarine biosynthesis. The formation of the fully conjugated ring system in sanguinarine is similar to the four-electron oxidations of (S)-canadine to berberine and (S)-tetrahydropapaverine to papaverine. We report the isolation and functional characterization of an opium poppy (Papaver somniferum) cDNA encoding DBOX, a flavoprotein oxidase with homology to (S)-tetrahydroprotoberberine oxidase and the berberine bridge enzyme. A query of translated opium poppy stem transcriptome databases using berberine bridge enzyme yielded several candidate genes, including an (S)-tetrahydroprotoberberine oxidase-like sequence selected for heterologous expression in Pichia pastoris. The recombinant enzyme preferentially catalyzed the oxidation of dihydrosanguinarine to sanguinarine but also converted (RS)-tetrahydropapaverine to papaverine and several protoberberine alkaloids to oxidized forms, including (RS)-canadine to berberine. The K(m) values of 201 and 146 µm for dihydrosanguinarine and the protoberberine alkaloid (S)-scoulerine, respectively, suggested high concentrations of these substrates in the plant. Virus-induced gene silencing to reduce DBOX transcript levels resulted in a corresponding reduction in sanguinarine, dihydrosanguinarine, and papaverine accumulation in opium poppy roots in support of DBOX as a multifunctional oxidative enzyme in BIA metabolism.

Isolation and characterization of a cDNA encoding (S)-cis-N-methylstylopine 14-hydroxylase from opium poppy, a key enzyme in sanguinarine biosynthesis.

Sanguinarine is a benzo[c]phenenthridine alkaloid with potent antimicrobial properties found commonly in plants of the Papaveraceae, including the roots of opium poppy (Papaver somniferum). Sanguinarine is formed from the central 1-benzylisoquinoline intermediate (S)-reticuline via the protoberberine alkaloid (S)-scoulerine, which undergoes five enzymatic oxidations and an N-methylation. The first four oxidations from (S)-scoulerine are catalyzed by cytochromes P450, whereas the final conversion involves a flavoprotein oxidase. All but one gene in the biosynthetic pathway from (S)-reticuline to sanguinarine has been identified. In this communication, we report the isolation and characterization of (S)-cis-N-methylstylopine 14-hydroxylase (MSH) from opium poppy based on the transcriptional induction in elicitor-treated cell suspension cultures and root-specific expression of the corresponding gene. Along with protopine 6-hydroxylase, which catalyzes the subsequent and penultimate step in sanguinarine biosynthesis, MSH is a member of the CYP82N subfamily of cytochromes P450. The full-length MSH cDNA was expressed in Saccharomyces cerevisiae and the recombinant microsomal protein was tested for enzymatic activity using 25 benzylisoquinoline alkaloids representing a wide range of structural subgroups. The only enzymatic substrates were the N-methylated protoberberine alkaloids N-methylstylopine and N-methylcanadine, which were converted to protopine and allocryptopine, respectively.

The Toxicity and Attenuation Methods of Toxic Chinese Materia Medica for its Reasonable Application: A Review.

Over a millennia, traditional Chinese medicine (TCM) has been used to treat various diseases in China. In recent years, more and more Chinese materia medica (CMM) have been studied in scientific research projects, applied in clinical practice, and their extracts have even appeared in some health products. However, the toxicity of some CMM is often overlooked, including hepatotoxicity, nephrotoxicity, neurotoxicity, cardiotoxicity, etc. In this review, the toxic components and their toxicological mechanisms of some toxic CMM were listed according to the chemical structure classification of toxic components. Afterwards, the traditional methods (processing and compatibility) and modern methods (structural modification, biotransformation, etc.) of attenuation of CMM were discussed. Since ancient times, it has been said that "fight fire with fire, fight poison with poison," and toxic CMM are of great significance in the treatment of difficult and severe diseases. The rational application of toxic CMM and their components in clinical practice was also exemplified in this review. While the pharmacological effects of TCMs have been emphasized, the scientific attenuation and rational application of toxic components should be concerned. We hope this review can provide a reference for future related research.

Integration of deep transcriptome and proteome analyses reveals the components of alkaloid metabolism in opium poppy cell cultures.

BACKGROUND: Papaver somniferum (opium poppy) is the source for several pharmaceutical benzylisoquinoline alkaloids including morphine, the codeine and sanguinarine. In response to treatment with a fungal elicitor, the biosynthesis and accumulation of sanguinarine is induced along with other plant defense responses in opium poppy cell cultures. The transcriptional induction of alkaloid metabolism in cultured cells provides an opportunity to identify components of this process via the integration of deep transcriptome and proteome databases generated using next-generation technologies. RESULTS: A cDNA library was prepared for opium poppy cell cultures treated with a fungal elicitor for 10 h. Using 454 GS-FLX Titanium pyrosequencing, 427,369 expressed sequence tags (ESTs) with an average length of 462 bp were generated. Assembly of these sequences yielded 93,723 unigenes, of which 23,753 were assigned Gene Ontology annotations. Transcripts encoding all known sanguinarine biosynthetic enzymes were identified in the EST database, 5 of which were represented among the 50 most abundant transcripts. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of total protein extracts from cell cultures treated with a fungal elicitor for 50 h facilitated the identification of 1,004 proteins. Proteins were fractionated by one-dimensional SDS-PAGE and digested with trypsin prior to LC-MS/MS analysis. Query of an opium poppy-specific EST database substantially enhanced peptide identification. Eight out of 10 known sanguinarine biosynthetic enzymes and many relevant primary metabolic enzymes were represented in the peptide database. CONCLUSIONS: The integration of deep transcriptome and proteome analyses provides an effective platform to catalogue the components of secondary metabolism, and to identify genes encoding uncharacterized enzymes. The establishment of corresponding transcript and protein databases generated by next-generation technologies in a system with a well-defined metabolite profile facilitates an improved linkage between genes, enzymes, and pathway components. The proteome database represents the most relevant alkaloid-producing enzymes, compared with the much deeper and more complete transcriptome library. The transcript database contained full-length mRNAs encoding most alkaloid biosynthetic enzymes, which is a key requirement for the functional characterization of novel gene candidates.

Investigation of the relaxant effects of pancuronium, rocuronium, vecuronium and mivacurium on rat thoracic aorta.

BACKGROUND AND OBJECTIVE: Pancuronium, vecuronium, mivacurium and rocuronium are nondepolarizing neuromuscular blocking agents, which are competitive antagonists against acetylcholine at nicotinic receptors, and considered to have no direct actions on vascular smooth muscle. We aimed to investigate the relaxant effects and possible underlying mechanisms of these agents on isolated rat thoracic aorta. METHODS: The preparations were precontracted with prostaglandin F2alpha (10(-7) mol l(-1)) and pancuronium (10(-7)-10(-4) mol l(-1)), rocuronium (10(-7)-10(-4) mol l(-1)), vecuronium (10(-7)-10(-4) mol l(-1)) and mivacurium (10(-7)-10(-4) mol l(-1)) added at cumulative concentrations in the presence or absence of a prostaglandin synthesis inhibitor, indomethacin (10(-6) M), and a nitric oxide synthesis inhibitor, N(omega)-nitro-L-arginine methylester (3 x 10(-5)). The same protocol was applied to both endothelia (+) and endothelia (-) aortic rings. The preparations precontracted with prostaglandin F2alpha (10(-7) mol l(-1)) were stimulated with electrical field stimulation at a frequency of 10 Hz as square-wave pulses of 50 V (0.2 ms) in the presence of a noradrenaline reuptake inhibitor desipramine (10(-7) mol l(-1)) and a nonselective beta-blocker propranolol (10(-6) mol l(-1)). Drugs were added at ineffective concentration of 10(-7) mol l(-1). Tetrodotoxin (10(-7) mol l(-1)) was added to test whether the changes were dependent on the neuronal response. RESULTS: Pancuronium and rocuronium relaxed aortic rings precontracted by prostaglandin F2alpha in a dose-dependent manner, but vecuronium and mivacurium did not. The relaxation effect of pancuronium and rocuronium was endothelium independent because there was not a significant response difference from the endothelium-denuded group. CONCLUSION: In conclusion, their relaxation effect may be due to an increase in prostaglandin synthesis. The increased relaxation effect of these agents at electrical field stimulation may be by the decreasing effect of noradrenaline reuptake from nerve endings because a noradrenaline reuptake inhibitor desipramine did not change this effect. Also, these neuromuscular agents may affect beta-receptors, because a nonselective beta-blocker agent, propranolol, decreased their electrical field stimulation-induced relaxations.

LC/MS/MS analysis of quaternary ammonium drugs and herbicides in whole blood.

Quaternary ammonium drugs (atracurium, bretylium, edrophonium, ipratropium, mivacurium, neostigmine, pancuronium and rocuronium) and herbicides (difenzoquat, diquat and paraquat) in human whole blood were analysed by LC/MS/MS with positive electrospray ionisation (ESI), following extraction with Bond Elut LRC-CBA cartridges. Internal standards were benzyldimethylphenylammonium chloride monohydrate and ethyl viologen for drug and herbicide analysis, respectively. Ion-pair chromatography used heptafluorobutyric acid (15 mM)-ammonium formate (20 mM) buffer adjusted to pH 3.30 with formic acid and a linear gradient from 5 to 90% methanol run over 18 min. Recoveries ranged from 79.7 to 105.1%, detection limits were between 3.6 and 20.4 ng/ml and the intra- and inter-day precisions were less than 18.6% at a concentration of 10 ng/ml. The method was applied to a case of accidental paraquat poisoning in which the concentration of paraquat in blood was 0.64 mg/l, which is within the range associated with fatal paraquat poisoning.

Anesthesiologist suicide with atracurium.

Atracurium is a nondepolarizing skeletal muscle relaxant used to facilitate endotracheal intubation and to induce skeletal muscle relaxation during surgery or mechanical ventilation. The drug undergoes a spontaneous non-enzymatic biotransformation, yielding laudanosine and an acrylate moiety. This report documents the case of a 45-year-old anesthesiologist who was found dead at the hospital where he worked. The victim was known to be depressed and undergoing treatment with venlafaxine. An empty syringe was found near the body. Toxicological analysis revealed the presence of laudanosine in the syringe, 0.6 mg/L of laudanosine in heart blood, 0.3 mg/L in urine, and 0.02 mg/L in vitreous humor. Meanwhile, concentrations of venlafaxine and O-desmethyl-venlafaxine, its active metabolite, were 0.7 and 1.1 mg/L in heart blood, 1.7 and 5.2 mg/L in urine, 0.5 and 0.7 mg/L in vitreous humor, and 400 and 20 mg in gastric content, respectively. All drugs and metabolites involved in the case were detected using gas chromatography with nitrogen-phosphorus detection (GC-NPD) and confirmed using GC-mass spectrometry in full scan mode after solid-phase extraction using Bond-Elut Certify columns. Additional high-performance liquid chromatography coupled to diode-array detection screening also obtained the same results. Quantitation of laudanosine and venlafaxine together with its metabolite was carried out using GC-NPD. No other drugs, including ethanol, were detected. Recoveries for laudanosine and venlafaxine were 89% and 86%, respectively, at 0.5 mg/L; intraday and interday precisions were 2% and 6%, and 3% and 7%, respectively; and limits of detection and quantitation were 6 and 20 ng/mL and 18 and 59 ng/mL, respectively. The linearity of the blood calibration curves was excellent for both drugs with r(2) values of > 0.999 (range 0.1-2.0 mg/L). Based on the autopsy findings, case history, and toxicology results, the forensic pathologists ruled that the cause of death was an overdose of atracurium, and the manner of death was suicide.

Neuromuscular blocking agents inhibit receptor-mediated increases in the potassium permeability of intestinal smooth muscle.

The neuromuscular blocking agents tubocurarine, atracurium and pancuronium have been tested for their ability to inhibit receptor-mediated increases in the K+ permeability of intestinal smooth muscle. All three agents, as well as the bee venom peptide apamin, reduced both the resting efflux of 86Rb and the increase in efflux caused by the application of either bradykinin (1 microM) or an alpha 1-adrenoceptor agonist, amidephrine (20 microM), to depolarized strips of guinea-pig taenia caeci. This suggested that like apamin, the neuromuscular blocking agents inhibit the Ca2+-dependent K+ permeability (PK(Ca] mechanism which in this tissue is activated by a variety of membrane receptors. The concentrations (IC50S) of atracurium, pancuronium and (+)-tubocurarine which reduced the effect of amidephrine on 86Rb efflux by 50% were 12, 37 and 67 microM respectively. Also in keeping with an ability to block PK(Ca), the neuromuscular blockers and apamin reduced the inhibition by amidephrine and bradykinin of physalaemin-mediated contractions of the taenia caeci. The IC50 values were 15, 31 and 120 microM for atracurium, tubocurarine and pancuronium respectively, and 2.3 nM for apamin. Each of the neuromuscular blockers, and apamin, increased the spontaneous contractions of the rabbit duodenum and blocked the inhibitory effect of amidephrine thereon. It is concluded that the PK(Ca) mechanism in the longitudinal smooth muscle of the intestine It is concluded that the PK(Ca) mechanism in the longitudinal smooth muscle of the intestine resembles that of hepatocytes and sympathetic ganglion cells in its susceptibility to inhibition by neuromuscular blocking agents, as well as by apamin.

Isobolographic analysis of non-depolarising muscle relaxant interactions at their receptor site.

Administration of certain combinations of non-depolarising muscle relaxants produces greater than expected neuromuscular blockade. Synergistic effects may be explained by drug interactions with the postsynaptic muscle nicotinic acetylcholine receptor. To investigate this hypothesis, the adult mouse muscle nicotinic acetylcholine receptor (alpha(2)beta delta epsilon) was heterologously expressed in Xenopus laevis oocytes and activated by the application of acetylcholine (10 microM). The effects of five individually applied muscle relaxants and six combinations of structurally similar and dissimilar compounds were studied. Drug combinations containing equipotent concentrations of two agents were tested and dose-response curves were determined. All compounds tested alone and in combination produced rapid and readily reversible, concentration-dependent inhibition. Isobolographic and fractional analyses indicated additive interactions for all six tested combinations. These findings suggest that synergistic neuromuscular blocking effects, observed for the administration of certain combinations of muscle relaxants, do not result from purely postsynaptic binding events at the muscle nicotinic acetylcholine receptor, but rather from differential actions on pre- and postsynaptic sites.

Atracurium and intraocular pressure.

The effect of atracurium on intraocular pressure was studied by comparing it with pancuronium in a randomised controlled trial. The intraocular pressure was measured in patients undergoing cataract surgery before administration of the muscle relaxant, at 1, 3, and 5 minutes after its administration, and at 1 minute after tracheal intubation. Atracurium was found to decrease intraocular pressure to a significantly greater degree than pancuronium. The intraocular pressure after tracheal intubation was found to be significantly higher than that measured immediately after induction of anaesthesia. The authors conclude that atracurium provides an acceptable alternative to pancuronium for ophthalmic surgery but does not overcome the ocular hypertensive effect of tracheal intubation.

Identification of laudanosine, an atracurium metabolite, following a fatal drug-related shooting.

In a case involving a fatal shooting, toxicology tests on blood and urine demonstrated the presence of cocaine metabolites and a large amount of an unidentified compound. This compound was subsequently identified by mass spectral, gas chromatographic, and thin layer chromatographic tests as laudanosine, a metabolite of the skeletal muscle relaxant atracurium which was administered during emergency surgery. Identification was confirmed by comparison with commercially available standards. Because of the difficulty associated with isolating and chromatographing highly water-soluble compounds, recognition of this artifact is a useful tool in identifying these cases.

Neuromuscular blocking agents in the emergency department.

Neuromuscular blocking agents (NMBAs) are utilized frequently in the emergency department (ED). We begin with a brief history of neuromuscular blockade, then review the indications and guidelines for its use in the emergency department setting. The relevant agents will be discussed focusing on dosage, side effects, and adverse reactions. Special attention will be paid to succinylcholine, the drug most commonly employed in the ED setting, followed by a summary of the nondepolarizing agents currently available, in particular the four shorter-acting agents that are most appropriate for administration in the ED.

Economic considerations in the use of neuromuscular blocking drugs.

The acceptance of new and increasingly expensive technologies is a major component of the rising costs of health care. While the practice of anesthesia has been relatively immune from the effects of cost containment, it is inevitable that practitioners will have to justify costly practices. Available pharmacoeconomic methods can be applied to the use of all anesthetic drugs, particularly neuromuscular blocking drugs. Cost-effectiveness analysis allows the practicing anesthesiologist to prioritize the use of neuromuscular blocking drugs to maximize their benefit while reducing unnecessary costs.

The impact of price labeling of muscle relaxants on cost consciousness among anesthesiologists.

STUDY OBJECTIVE: To determine whether placing price labels on the vial caps of muscle relaxants increases cost consciousness among anesthesiologists. DESIGN: Retrospective study. SETTING: University hospital departments of anesthesia and pharmacy. MEASUREMENTS AND MAIN RESULTS: We placed price labels on the vial caps of all muscle relaxants for a study period of 1 year. At the beginning of the investigation, we informed the anesthesiologists of the study, discussed the prices for different muscle relaxants, and encouraged utilizing less expensive muscle relaxants whenever possible without compromising patient care. The price labels on the vial caps served as visual reminders of the various costs of muscle relaxants during daily practice. We compared the total amount spent on each muscle relaxant during the period from October 1993 to September 1994 with the period from October 1994 to September 1995. The total number of surgical cases from October 1993 to September 1994 and from October 1994 to September 1995 was unchanged and equaled 20,389 and 20,358 cases, respectively. Expenditures for pancuronium increased 104.1%. Total expenditure decreased by 12.5%, with a net savings of $47,111. CONCLUSION: Expenditures for the less costly pancuronium increased while expenditures for vecuronium and atracurium decreased. Price labeling of muscle relaxants in conjunction with education reduces total pharmacy expenditure on muscle relaxants.

[Relaxation and the electromyographic identification of the recurrent laryngeal nerve]. / Relaxierung und elektromyographische Identifikation des N. laryngeus recurrens.

INTRODUCTION: Electromyography for the identification of the recurrent laryngeal nerve is gaining ever more acceptance in thyroid surgery. Relaxation of the patient, e.g., to improve intubation conditions for anesthesia, carries a potential risk for error. METHOD: After definite identification of the recurrent laryngeal nerve and the vagus nerve (Neurosign 100, Magstim Co., Wales), ten consecutive patients were relaxed with mivacurium and atracurium besylate at a weight-dependent ED95 dosage. After peripheral relaxation was achieved (TOF-Watch, Organon Teknika Corp., at the adductor muscle of the thumb), the signal derived via the vocal muscle was assessed acoustically in 3-min intervals by the surgeon and graphically recorded by computer (EWACS, Inomed Co.). RESULTS: Complete peripheral relaxation was attained with mivacurium after 3-7 min and with atracurium after 7-11 min. A decrease in amplitude of the vocal muscle signal of >60% was recognized by the surgeon as a weakened signal and could be confirmed during an average duration of 13.3 min with mivacurium (maximum: 37 min) and 17.7 min with atracurium besylate (maximum: 23 min), respectively. Complete obliteration of the acoustic signal (<20% of the initial signal) occurred in three of six patients treated with mivacurium and in four of four patients treated with atracurium. CONCLUSIONS: The accuracy of electromyography of the recurrent laryngeal nerve can be substantially impaired by the administration of relaxants. If this medication cannot be dispensed with, the surgeon must be aware of the situation. In these cases, a peripheral relaxometer should also be employed to monitor relaxation as it subsides.

Muscle relaxants for out-patient procedures.

The requirements for muscle relaxation in out-patient anesthesia are: a short total relaxation enabling the induction of an endotracheal tube followed by a period of 15-20 minutes of clinical relaxation. Reversal should be spontaneous. The new short acting muscle relaxants vecuronium and atracurium enable such a procedure, provided small doses are used (0.045 mg/kg for vecuronium or 0.2 mg/kg for atracurium. These small doses require a longer waiting time before intubation is easily performed (5 min for vecuronium and 6 min for atracurium). Spontaneous reversal of these small doses is achieved within 20-30 minutes. Potentiation of the relaxation by inhalational agents is not recommended.

[Determination of the dose-response curve for atracurium dibesylate in the anesthetized adult]. / Détermination de la courbe dose-effet du dibésylate d'atracurium chez l'homme adulte anesthésié.

The mechanical response of the adductor pollicis to a 0.15 Hz stimulation of the ulnar nerve was studied in 35 unpremedicated adult patients (mean age 38 yr) under general anaesthesia using thiopentone, fentanyl and a N2O/O2 mixture under mechanical ventilation. PaCO2, pH, K, Ca, Mg plasma levels and temperature were in the normal range. Each patient received a single bolus of atracurium dibesylate: 0.10 mg . kg-1 (n = 11), 0.15 mg . kg-1 (n = 10), 0.20 mg . kg-1 (n = 11) or 0.30 mg . kg-1 (n = 4). The dose-response curve was constructed using the log-probit method for 0.10, 0.15, 0.20 mg . kg-1 doses, giving neuromuscular blocks greater than 0% and less than 0.20 mg . kg-1. The 0.20 mg . kg-1 dose had an onset time of 6.1 +/- 0.6 min, duration 0-90% of 34.3 +/- 3.2 min and a recovery index 25-75% of 10.9 +/- 1.0 min. The 0.3 mg . kg-1 dose resulted in onset time of 4.7 +/- 1.3 min, duration of 39.9 +/- 3.7 min and a recovery index of 10.7 +/- 1.8 min. Thus atracurium dibesylate seemed to be an agent of intermediate potency. Onset time was approximately the same as that for other non-depolarizing neuromuscular blocking drugs, but duration of action and recovery index were quite shorter, except for vecuronium bromide.

[Clinical use in adults of 2 new muscle relaxants: atracurium and vecuronium]. / Utilisation clinique chez l'adulte de deux nouveaux myorelaxants: l'atracurium et le vécuronium.

115 general and urologic surgery adult patients, ASA class I-II, were divided in four groups according to initial bolus and relaxant used: group A atracurium 0.6 mg X kg-1, group B 0.5 mg X kg-1, group C vecuronium 0.1 mg X kg-1 and group D pancuronium 0.1 mg X kg-1. When the single twitch recovered to 25% of control height (T25), subgroups were individualized depending on whether repeat doses of 1/3 of initial bolus were given or not, and whether reversal was spontaneous or obtained by a standard dose of neostigmine 2.5 mg and atropine 1.25 mg. By ulnar nerve stimulation at the wrist, the force of thumb adduction was recorded on a polygraph; single twitch (tw), train of four (tof) and ratio tof 4/1 (Rtof) were measured. Anaesthesia was induced with thiopentone and fentanyl without premedication and maintained with fentanyl and N2O in oxygen; the trachea was intubated once the block was at its maximum. The onset time of maximal block was 5 min for groups A, B and C, and 7.9 min for group D. T25 was 39.9 +/- 8.5 min for group A, 34.4 +/- 9.7 min for group B, 28.9 +/- 9.9 min for group C and 70.7 +/- 25.9 min for group D. A Rtof equal to 75% was achieved in less than 65 min with atracurium and vecuronium, but much later with pancuronium. Reversal at T25 was efficient, but not really required, for atracurium and vecuronium, but necessary and useful for pancuronium.(ABSTRACT TRUNCATED AT 250 WORDS)

Histaminoid responses to atracurium, vecuronium and tubocurarine.

Sixty patients scheduled for elective surgery underwent intradermal testing with 0.1 ml of the following solutions diluted in 0.9% saline: vecuronium and tubocurarine (1 in 1,000), atracurium (1 in 1,000 and 1 in 10,000), thiopentone (1 in 100) and also a 0.9% saline control. Thirty minutes later, an area of erythema of greater than 1.5 cm, or a wheal exceeding 1.0 cm in diameter, was recorded as a positive reaction. The patients then randomly received equipotent doses of atracurium, vecuronium or tubocurarine during a standardized anaesthetic induction. Any cutaneous reaction and the percentage fall in systolic pressure three minutes after administration of the relaxant were recorded. In 51 patients plasma IgE levels were measured. The incidence of positive cutaneous reactions to intradermal and intravenous relaxants was significantly different with each agent (p less than 0.01). The percentage fall in systolic pressure after tubocurarine was significantly different relative to the other two agents (p less than 0.01). This was regarded as reflecting potency in releasing histamine and placed the relaxants in the same order: tubocurarine, atracurium and vecuronium. The response to intradermal administration was no guide to the subsequent response after intravenous administration of the three relaxants. IgE levels below 15 IU X ml-1 occurred significantly more often in females and were associated with a significantly higher incidence of cutaneous reactions after intradermal atracurium (1 in 1,000 and 1 in 10,000) (p less than 0.05 and 0.001 respectively) and tubocurarine (1 in 1,000). With these two agents, generalized flushing after intravenous administration was also more common in this group, relative to the normal/high IgE group.