Overexpression and purification of the membrane-bound cytochrome P450 2B4.

Expression of the membrane-bound cytochrome P450 2B4 by the pLW01-P450 expression vector, which utilizes a T7 promoter, is markedly improved by employing Escherichia coli strain C41(DE3) [Miroux, B., and Walker, J. (1996) J. Mol. Biol 260, 289--298; Bridges, A., Gruenke, L., Chang, Y.-T., Vasker, I., Loew, G., and Waskell, L. (1998) J. Biol. Chem. 273, 17036--17049]. Using this expression system, it was possible to routinely obtain an average of 50--60 mg and as high as 100 mg of cyt P450 2B4 per liter of cell culture in volumes of 500 ml. An improved purification procedure for cyt P450 2B4 is also described which allows recovery of 30% of the expressed protein. It was possible in one step using B-PER reagent and polyoxyethylene-9-lauryl ether to both lyse the E. coli and solubilize the expressed cyt P450. Cyt P450 2B4 with a specific content of 17 nmol/mg protein and a single band on polyacrylamide gel electrophoresis was routinely isolated. The yield of cyt P450 from the improved purification procedure is twice that from the original procedure and the purity of the recovered protein typically has a specific content of 17 nmol cyt P450/mg of protein.

Do the pharmacokinetics of vecuronium change during prolonged administration in critically ill patients?

Neuromuscular blocking drugs may be administered over several days to patients in the intensive care unit (ICU), but their pharmacokinetics have been studied at only one point in time, or assumed to be constant throughout the period of administration. We sought to determine if, in individual patients, the pharmacokinetics of vecuronium changed over the course of its administration in the ICU. In six critically ill patients, we measured plasma vecuronium concentrations during two periods: first, during initial administration of vecuronium and second, after its administration continuously for 3-6 days. A pharmacokinetic model was fitted to these plasma concentration data, and its parameters permitted to vary between the periods to determine if they had altered. Individual clearance values during the study ranged from 1.4 to 4.4 ml kg-1 min-1. During prolonged administration, vecuronium clearance increased in three and decreased in two patients. This change ranged from a 61% decrease to a 58% increase, and was not linked to any clinical factor. The steady-state volume of distribution (range 368-1765 ml kg-1; median 494 ml kg-1) did not change in any patient during the study. The change in clearance of vecuronium during its prolonged administration in critically ill patients suggests that future studies of neuromuscular blocking drugs in the ICU should take account of their changing pharmacokinetics over the course of administration.

Identification of the binding site on cytochrome P450 2B4 for cytochrome b5 and cytochrome P450 reductase.

A model of cytochrome P450 2B4, which was constructed by homology modeling with the four known crystal structures of the cytochromes P450 (Chang, T.-T., Stiffelman, O. B., Vakser, I. A., Loew, G. H., Bridges, A., and Waskell, L. (1997) Protein Eng. 10, 119-129), was used to select amino acids predicted, by computer docking studies and numerous previous biochemical and site-directed mutagenesis studies, to be involved in binding the heme domain of cytochrome b5. Twenty-four amino acid residues located on both the distal and the proximal surface of the molecule were chosen for mutagenesis. These 24 mutant proteins were expressed in Escherichia coli, purified, and characterized with respect to their ability to bind cytochrome b5 and support substrate oxidation. Seven mutants, R122A, R126A, R133A, F135A, M137A, K139A, and K433A, all on the proximal surface of cytochrome P450 2B4 near the heme ligand, were identified that exhibited decreased ability to bind cytochrome b5. All of the mutants except K433A are located in either the C or C* helices or their termini. In addition, these seven mutants and two additional mutants on the proximal surface of cytochrome P450, R422A and R443A, were shown to exhibit decreased binding to cytochrome P450 reductase. These studies indicate that the binding sites for cytochrome b5 and cytochrome P450 reductase are, as predicted, located on the proximal surface of cytochrome P450 2B4 and are partially overlapping but not identical.

Resonance Raman spectral properties and stability of manganese protoporphyrin IX cytochrome b5.

The structure and stability of cytochrome b5 reconstituted with manganese protoporphyrin IX instead of iron protoporphyrin IX has been investigated by resonance Raman spectroscopy and stopped-flow visible spectroscopy. The resonance Raman spectrum of MnIII cytochrome b5 was consistent with a high-spin hexacoordinate MnIII protoporphyrin IX structure that converted to a high-spin pentacoordinate structure at higher laser power. The resonance Raman spectrum of MnII cytochrome b5 indicated a high-spin pentacoordinate structure which was independent of laser power. Studies of the binding of MnIII protoporphyrin IX to apocytochrome b5 indicated that the MnIII-containing porphyrin bound much less tightly to the protein than did heme. Although the second-order rate constant at 20 degrees C for the association of heme with apocytochrome b5 (4.5 x 10(7) M(-1) s(-1)) was estimated to be only 1 order of magnitude higher than that with Mn protoporphyrin IX (3.3 x 10(6) M(-1) s(-1)), the dissociation of manganese substituted cytochrome b5 into the apoprotein and free Mn protoporphyrin IX occurs with a first-order rate constant of 1.2 x 10(-2) s(-1) at 20 degrees C while the dissociation of heme from cytochrome b5 at room temperature occurs 3 orders of magnitude more slowly with a first-order rate constant of 1.67 x 10(-5) s(-1) [Vergeres, G., Chen, D. Y., Wu, F.F., & Waskell, L. (1993) Arch. Biochem. Biophys. 305, 231-241]. The equilibrium dissociation constant for manganese-substituted cytochrome b5 increased with temperature from 4 nM at 20 degrees C to 14 nM at 37 degrees C. These results suggest that, in the reconstituted cytochrome P450 metabolizing system, especially in studies done with low protein concentrations (0.1 microM), and at elevated temperatures (37 degrees C), as much as 30% of the manganese-substituted cytochrome b5 may dissociate to free Mn-protoporphyrin IX and apocytochrome b5.

The stoichiometry of the cytochrome P-450-catalyzed metabolism of methoxyflurane and benzphetamine in the presence and absence of cytochrome b5.

The complete stoichiometry of the metabolism of the cytochrome b5 (cyt b5)-requiring substrate, methoxyflurane, by purified cytochrome P-450 2B4 was compared to that of another substrate, benzphetamine, which does not require cyt b5 for its metabolism. Cyt b5 invariably improved the efficiency of product formation. That is, in the presence of cyt b5 a greater percentage of the reducing equivalents from NADPH were utilized to generate substrate metabolites, primarily at the expense of the side product, superoxide. With methoxyflurane, cyt b5 addition always resulted in an increased rate of product formation, while with benzphetamine the rate of product formation remained unchanged, increased or decreased. The apparently contradictory observations of increased reaction efficiency but decrease in total product formation for benzphetamine can be explained by a second effect of cyt b5. Under some experimental conditions cyt b5 inhibits total NADPH consumption. Whether stimulation, inhibition, or no change in product formation is observed in the presence of cyt b5 depends on the net effect of the stimulatory and inhibitory effects of cyt b5. When total NADPH consumption is inhibited by cyt b5, the rapidly metabolized, highly coupled (approximately equal to 50%) substrate, benzphetamine, undergoes a net decrease in metabolism not counterbalanced by the increase in the efficiency (2-20%) of the reaction. In contrast, in the presence of the slowly metabolized, poorly coupled (approximately equal to 0.5-3%) substrate, methoxyflurane, inhibition of total NADPH consumption by cyt b5 was never sufficient to overcome the stimulation of product formation due to an increase in efficiency of the reaction.

Edrophonium increases mivacurium concentrations during constant mivacurium infusion, and large doses minimally antagonize paralysis.

BACKGROUND: Mivacurium, a nondepolarizing muscle relaxant, is metabolized by plasma cholinesterase. Although edrophonium does not alter plasma cholinesterase activity, we have observed that doses of edrophonium that antagonize paralysis from other nondepolarizing muscle relaxants are less effective with mivacurium. We speculated that edrophonium might after metabolism of mivacurium, thereby hindering antagonism of paralysis. Accordingly, we determined the effect of edrophonium on neuromuscular function and plasma mivacurium concentrations during constant mivacurium infusion. METHODS: We infused mivacurium to maintain 90% depression of adductor pollicis twitch tension and then gave edrophonium in doses ranging from 125-2,000 micrograms/kg without altering the mivacurium infusion. Peak twitch tension after edrophonium was determined to estimate the dose of edrophonium antagonizing 50% of twitch depression for antagonism of mivacurium; plasma cholinesterase activity and mivacurium concentrations before and after edrophonium were measured. Additional subjects were given 500 micrograms/kg edrophonium to antagonize continuous infusions of d-tubocurarine and vecuronium. RESULTS: With mivacurium, edrophonium increased twitch tension in a dose-dependent manner: the dose of edrophonium antagonizing 50% of twitch depression was 2,810 micrograms/kg. The largest dose of edrophonium (2,000 micrograms/kg) produced only 45 +/- 7% antagonism. Edrophonium, 500 micrograms/kg, antagonized mivacurium markedly less than it antagonized d-tubocurarine and vecuronium. Edrophonium increased plasma concentrations of the two potent stereoisomers of mivacurium 48% and 79%, these peaking at 1-2 min; plasma cholinesterase activity was unchanged. CONCLUSIONS: Edrophonium doses that antagonize d-tubocurarine and vecuronium are less effective in antagonizing the neuromuscular effects of mivacurium during constant infusion. Edrophonium increases plasma mivacurium concentrations, partly or completely explaining its limited efficacy; the mechanism by which edrophonium increases mivacurium concentrations remains unexplained. Our results demonstrate that antagonism of mivacurium by edrophonium is impaired, and therefore we question whether edrophonium should be used to antagonize mivacurium.

The pharmacokinetics and neuromuscular effects of rocuronium bromide in patients with liver disease.

To determine the effect of liver disease on the pharmacokinetics of rocuronium, the authors administered 0.6 mg/kg (twice the ED95) to 10 patients with liver disease and compared these results to values in 10 healthy surgical patients. Anesthesia was induced with thiopental and maintained with isoflurane (0.9%-1.1% end-tidal concentration) and nitrous oxide (60%). Venous blood samples were obtained for 6 h after rocuronium injection and plasma concentrations were measured using gas chromatography. Pharmacokinetic differences between groups were determined using a population-based pharmacokinetic analysis (NONMEM). Hepatic impairment did not alter the plasma clearance of rocuronium (217 +/- 21.8 mL/min, mean +/- SE, for both groups), but did increase the volume of the central compartment (5.96 +/- 1.01 L for controls, 7.87 +/- 1.33 L for patients with liver disease) and volume of distribution at steady state (16.4 L for controls, 23.4 L for patients with liver disease). In turn, elimination half-life was longer in patients with liver disease (111 min) compared to controls (75.4 min). The authors conclude that liver disease alters the pharmacokinetics of rocuronium by increasing its volume of distribution. The longer elimination half-life might result in a longer duration of action of rocuronium in patients with liver disease, particularly after prolonged administration.

The magnitude and time course of vecuronium potentiation by desflurane versus isoflurane.

BACKGROUND: Preliminary studies suggest that desflurane and isoflurane potentiate the action of muscle relaxants equally. However, variability between subjects may confound these comparisons. A crossover study was performed in volunteers on the ability of desflurane and isoflurane to potentiate the neuromuscular effect of vecuronium, to influence its duration of action, and on the magnitude and time course of reversal of potentiation when anesthesia was withdrawn. METHODS: Adductor pollicis twitch tension was monitored in 16 volunteers given 1.25 MAC desflurane on one occasion, and 1.25 MAC isoflurane on another. In eight subjects, vecuronium bolus dose potency was determined using a two-dose dose-response technique; the vecuronium infusion dose requirement to achieve 85% twitch depression also was determined. Also in these subjects, the magnitude and time course of spontaneous neuromuscular recovery were determined when the anesthetic was withdrawn while maintaining a constant vecuronium infusion. In the other eight subjects, the time course of action of 100 micrograms/kg vecuronium was determined. RESULTS: Vecuronium's ED50 and infusion requirement to maintain 85% twitch depression were 20% less during desflurane, compared to isoflurane, anesthesia; vecuronium plasma clearance was similar during the two anesthetics. After 100 micrograms/kg vecuronium, onset was faster and recovery was longer during desflurane anesthesia. When the end-tidal anesthetic concentration was abruptly reduced from 1.25 to 0.75 MAC, twitch tension increased similarly (approximately 15% of control), and time for the twitch tension to reach 90% of the final change was similar (approximately 30 min) with both anesthetics. Decreasing anesthetic concentration from 0.75 to 0.25 MAC increased twitch tension by 46 +/- 10% and 25 +/- 7% of control (mean +/- SD, P < 0.001) with desflurane and isoflurane, respectively; 90% response times for these changes were 31 +/- 10 min and 18 +/- 7 min (P < 0.05), respectively. CONCLUSIONS: Desflurane potentiates the effect of vecuronium approximately 20% more than does an equipotent dose of isoflurane.

The pharmacodynamics and pharmacokinetics of the metabolite 3-desacetylvecuronium (ORG 7268) and its parent compound, vecuronium, in human volunteers.

The pharmacology of 3-desacetylvecuronium, the principal metabolite of vecuronium, was investigated. We studied 12 healthy volunteers, each on two occasions. First they received 3-desacetylvecuronium alone and then, on a later occasion, vecuronium. Six subjects received a large dose of each drug (pharmacokinetic study), the remaining six received a small dose (pharmacodynamic study). Drug concentrations in plasma and urine were measured using capillary gas chromatography. Neuromuscular block was assessed by measuring force of contraction of the adductor pollicis. Drug plasma concentration vs. time and neuromuscular effect data were analyzed by nonlinear mixed-effects modeling. 3-Desacetylvecuronium, compared with vecuronium (median, range in parentheses), had a smaller plasma clearance, 3.51 (2.11-6.57) vs. 5.39 (5.04-7.19) ml.kg-1.min-1; a larger steady-state distribution volume, 254 (215-410) vs. 152 (111-170) ml.kg-1; a longer terminal elimination half-life 116 (44-672) vs. 34 (25-61) min and a longer mean residence time, 67 (42-145) vs. 26 (18-32) min (P < .05). Renal clearances of 3-desacetylvecuronium and vecuronium were 0.85 (0.15-1.24) and 0.58 (0.16-0.66) ml.kg-1.min-1, respectively (P < .05). Conversion to 3-desacetylvecuronium accounted for 12% of vecuronium's clearance. Concentrations of 3-desacetylvecuronium and vecuronium that produced 50% neuromuscular block were 123 (109-154) and 102 (71-123) ng.ml-1, respectively (P < .05). 3-Desacetylvecuronium is a potent neuromuscular blocking drug and may be responsible for episodes of prolonged paralysis after long-term administration of vecuronium to patients in intensive care units.

Cumulative characteristics of atracurium and vecuronium. A simultaneous clinical and pharmacokinetic study.

BACKGROUND: Cumulative effects (increased 25-75% recovery time with increasing dose) are evident with vecuronium but not with atracurium. Pharmacokinetic simulations suggest that vecuronium's cumulation occurs as recovery shifts from distribution to elimination whereas atracurium's recovery always occurs during elimination. The purpose of this study was to examine this pharmacokinetic explanation. METHODS: We assigned 12 volunteers to receive atracurium of vecuronium on three occasions during nitrous oxide-isoflurane anesthesia. Evoked adductor pollicis twitch tension was monitored. On occasion 1, the dose expected to produce 95% block (ED95) was estimated for each subject. On occasions 2 and 3, 1.2 or 3.0 multiples of ED95 were given as a bolus. Plasma was sampled for 128 min to determine muscle relaxant concentrations; pharmacodynamic modeling was used to determine effect-compartment drug concentrations (Ce). For each drug, recovery time, recovery phase half-life (rate of decrease in Ce during recovery), and Ce at 25% and 75% recovery were compared between doses. RESULTS: Atracurium's recovery time increased 2.4 +/- 2.2 min (mean +/- SD) with the larger dose, less than the increase with vecuronium (8.2 +/- 3.8 min). Atracurium's recovery phase half-life was 14.6 +/- 1.7 and 20.1 +/- 2.3 min with the small and large doses (P < 0.05); vecuronium's recovery phase half-life increased similarly from 13.5 +/- 2.3 to 18.5 +/- 5.0 min (P < 0.05). At 75% recovery, vecuronium's Ce decreased from 65 +/- 18 ng/ml with the small dose to 55 +/- 15 ng/ml with the large dose (P < 0.05). Assuming that neuromuscular junction sensitivity was constant, this difference could be explained by considering neuromuscular effects of vecuronium's metabolite, 3-desacetylvecuronium. CONCLUSIONS: Although vecuronium was cumulative (as predicted), atracurium was also slightly cumulative. Inconsistent with our hypothesis, recovery phase half-lives for both drugs increased similarly between doses; therefore, differences in cumulation were not solely explained by pharmacokinetics of the muscle relaxant. It appears that 3-desacetylvecuronium contributes to vecuronium's cumulative effect, even after usual clinical doses.

Mild intraoperative hypothermia does not change the pharmacodynamics (concentration-effect relationship) of vecuronium in humans.

To investigate the effect of mild hypothermia on the neuromuscular junction sensitivity to vecuronium, we determined the pharmacodynamics (concentration-effect relationship) of vecuronium in 10 patients (ASA physical class I or II; age range, 21-46 yr; weight range, 54-104 kg), during isoflurane-nitrous oxide-fentanyl anesthesia. Five were cooled to a mean temperature of 34.4 degrees C and five were maintained normothermic at a mean temperature of 36.8 degrees C. Neuromuscular function was monitored by measuring the evoked mechanical response of the adductor pollicis muscle after supramaximal train-of-four stimulation of the ulnar nerve at the wrist. Vecuronium, 3 micrograms.kg-1.min-1, was infused for 10 min, venous blood sampled for 60 min, and twitch tension and plasma concentration data were used to determine pharmacodynamic variables in each patient. Results for the hypothermic and normothermic groups were compared by Mann-Whitney U-test. There were no differences in any pharmacodynamic variable between the hypothermic and normothermic patients. For the hypothermic and normothermic patients, respectively, steady-state plasma concentrations of vecuronium producing 50% neuromuscular block (Css50) were 73 +/- 13 ng/mL (mean +/- SD) and 79 +/- 31 ng/mL; the rate constants for equilibration of vecuronium between the plasma and the neuromuscular junction (Keo) were 0.27 +/- 0.14 per min-1 and 0.26 +/- 0.11 per min, and the power functions representing the slope of the concentration-effect relationship (gamma) were 5.7 +/- 1.9 and 4.4 +/- 1.8.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacokinetics of rocuronium bromide (ORG 9426) in patients with normal renal function or patients undergoing cadaver renal transplantation.

To determine the effect of end-stage renal disease on the pharmacokinetics of reocuronium bromide (ORG 9426), a new nondepolarizing monoquaternary steroidal neuromuscular blocking drug, the authors administered 600 micrograms/kg rocuronium (2 x ED95) intravenously to ten patients undergoing cadaver renal transplantation and ten healthy patients undergoing elective minor surgery (controls). All patients were anesthetized with nitrous oxide (50-70% in oxygen) and isoflurane (end-tidal concentrations of 1.2 +/- 0.5% and 0.8 +/- 0.2%, mean +/- SD, for control and transplant groups, respectively). Plasma concentrations of rocuronium were determined by capillary gas chromatography. A population-based pharmacokinetic analysis (NONMEM) was used to determine typical values, standard errors, and interindividual variability for the pharmacokinetic parameters and to determine whether these values differed between control and renal transplant patients. Total plasma clearance (2.89 +/- 0.25 ml.kg-1.min-1, mean +/- SE) and volume of the central compartment (76.9 +/- 10.6 ml/kg) did not differ between control and renal transplant patients, whereas volume of distribution at steady state was greater in renal transplant patients (264 +/- 19 ml/kg) than in control patients (207 +/- 14 ml/kg). This resulted in a longer elimination half life in renal transplant patients (97.2 +/- 17.3 min) compared to controls (70.9 +/- 4.7 min). The authors conclude that renal failure and renal transplantation alter the distribution but not the clearance of rocuronium.

Persistent paralysis in critically ill patients after long-term administration of vecuronium.

BACKGROUND: The muscle relaxant vecuronium is sometimes administered to facilitate mechanical ventilation. Neuromuscular paralysis lasting up to seven days may occur after the termination of long-term administration (i.e., more than two days) of vecuronium in critically ill patients. We investigated the role of clinical factors and plasma concentrations of vecuronium and its metabolite in causing this prolonged neuromuscular blockade. METHODS: We studied 16 critically ill adult patients (8 women and 8 men) who had received vecuronium to facilitate mechanical ventilation for at least two consecutive days. Clinical factors and plasma concentrations of vecuronium and 3-desacetylvecuronium, the active metabolite of vecuronium, were compared in patients with and without prolonged neuromuscular blockade. In addition, we performed detailed pharmacokinetic studies in the patients without prolonged neuromuscular blockade. RESULTS: Seven of the 16 patients had prolonged neuromuscular blockade, lasting from six hours to more than seven days, after the termination of vecuronium therapy. These seven patients, six of whom were women, had higher plasma magnesium concentrations and lower arterial blood pH values than the nine patients without prolonged neuromuscular blockade. They also had higher plasma concentrations of 3-desacetylvecuronium and a higher frequency of renal failure (seven of seven patients vs. four of nine patients, P less than 0.03). In the patients without prolonged neuromuscular blockade, the mean (+/- SD) plasma clearance, elimination half-life, and volume of distribution of vecuronium were 2.5 +/- 1.0 ml per kilogram of body weight per minute, 299 +/- 154 minutes, and 1.1 +/- 0.6 liters per kilogram, respectively. CONCLUSIONS: Prolonged neuromuscular blockade after the termination of long-term treatment with vecuronium is associated with metabolic acidosis, elevated plasma magnesium concentrations, female sex, and probably more important, the presence of renal failure and high plasma concentrations of 3-desacetylvecuronium.

Fluoride metabolites after prolonged exposure of volunteers and patients to desflurane.

We examined the metabolism of desflurane in 13 healthy volunteers given 7.35 +/- 0.81 MAC-hours (mean +/- SD) of desflurane and 26 surgical patients given 3.08 +/- 1.84 MAC-hours (mean +/- SD). Markers of desflurane metabolism included fluoride ion measured via an ion-specific electrode, nonvolatile organic fluoride measured after sodium fusion of urine samples, and trifluoroacetic acid determined by a gas chromatographic-mass spectrometric method. In both volunteer and patient groups, postanesthesia serum fluoride ion concentrations did not differ from background fluoride ion concentrations. Similarly, postanesthesia urinary excretion of fluoride ion and organic fluoride in volunteers was comparable to preanesthesia excretion rates. However, small but significant levels of trifluoroacetic acid were found in both serum and urine from volunteers after exposure to desflurane. A peak serum concentration of 0.38 +/- 0.17 mumol/L of trifluoroacetic acid and a peak urinary excretion rate of 0.169 +/- 0.107 mumol/h were detected in volunteers at 24 h after desflurane exposure. Although these increases in trifluoroacetic acid after exposure to desflurane were statistically significant, they are approximately 10-fold less than levels seen after exposure to isoflurane. Thus, desflurane strongly resists biodegradation, but a small amount is metabolized in humans.

Pharmacokinetics, neuromuscular effects, and biodisposition of 3-desacetylvecuronium (Org 7268) in cats.

The pharmacokinetics, biodisposition, and neuromuscular blocking properties of 3-desacetylvecuronium were studied in 17 adult cats. Animals were divided into three groups: five cats with kidney failure induced by bilateral ligation of the renal pedicles, six cats with galactosamine-induced fulminant hepatitis, and six control cats. An intravenous bolus of 300 micrograms.kg-1 of 3-desacetylvecuronium was rapidly injected into the jugular vein. Arterial blood, urine, and bile samples were collected at regular intervals for 6 h in control cats and for 8 h in cats with kidney or liver failure. The liver was excised for analysis at the end of the experiment. In cats with renal failure, 3-desacetylvecuronium pharmacokinetic and pharmacodynamic variables did not differ from those in control cats. In cats with liver failure, plasma clearance was significantly less and mean residence time greater than in control cats (2.8 +/- 0.6 vs. 14.1 +/- 6.5 ml.kg-1.min-1 and 334 +/- 225 vs. 49 +/- 29 min, mean +/- SD, respectively). Volume of distribution at steady state in cats with liver failure and in control cats was not different. Also, in cats with liver failure, the duration of action and recovery index of 3-desacetylvecuronium was significantly greater than in control cats (168 +/- 62 vs. 82 +/- 32 min, and 39 +/- 19 vs. 10 +/- 4 min, respectively). Onset time of neuromuscular blockade was similar in all three groups. Total recovery of 3-desacetylvecuronium, for all three groups, in urine, bile, and liver was 90 +/- 11% (mean +/- SD). In control cats, 70 +/- 18% of 3-desacetylvecuronium was recovered in bile and liver and 19 +/- 14% in urine. No 3,17-bidesacetylvecuronium (a putative 3-desacetylvecuronium metabolite) was detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Factors associated with obtaining nipple aspirate fluid: analysis of 1428 women and literature review.

Studies of cytologic and biochemical constituents of nipple aspirates of breast fluid have contributed to understanding the natural history of benign and malignant breast disease. We conducted multivariate analyses using 1428 women from a recent case-control study of breast disease to determine which factors were independently associated with the ability to obtain breast fluid from nonlactating women. We then compared results from these analyses to the results from five previous studies that also used the aspiration technique of Sartorius. Four factors were consistently associated across studies with increased ability to obtain breast fluid: 1) age up to 35 to 50 years; 2) earlier age at menarche; 3) non-Asian compared to Asian ethnicity; and 4) history of lactation. Exogenous estrogen use, endogenous estrogen concentrations, phase of menstrual cycle, family history of breast cancer, type of menopause, and less than full-term pregnancy consistently did not influence ability to obtain fluid. New findings from this study shed light on some apparently contradictory findings from the previous studies. In particular, this study showed that the effects of age on ability to obtain fluid appeared to be independent of the effects of menopause. Furthermore, discrepancies in previous findings on the effects of parity on ability to obtain fluid may be explained by our finding that the increased ability to obtain fluid from parous compared to nulliparous women applied only to parous women who had breastfed.