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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.

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 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.

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.

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)

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.

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.

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.

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)

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.

A method for the simultaneous quantitation of imipramine and its N-demethylated metabolite desipramine at the nanogram level in a single gas chromatograph peak is presented, utilizing gas chromatography-mass spectrometry with selected ion recording. The assay is specific and quantitation is achieved using [2H4]imipramine as the internal standard. The method involves the in situ methylation of desipramine with [2H2]formaldehyde and sodium borohydride to give [2H2]imipramine. Quantitation is then achieved by selected ion recording at m/z 280, 282 and 284, whence the ratio of the ion currents at 280/284 and at 282/284 gives the quantities of imipramine and desipramine respectively.

A selected ion monitoring gas chromatographic/mass spectrometric method for the quantitative determination of benzene in air, breath, and blood was developed utilizing a headspace assay with benzene-d3 as an internal standard. Limits of detection for 2 ng/mL in blood and 0.1 ppb in a 5-L sample of air or breath were attained. The influence of contamination by background benzene on the analytical process was studied carefully. For cases where background contamination could not be adequately controlled, the assay was modified for the quantitative determination of labelled benzenes six mass units heavier than natural benzene (benzene-d6 or benzene-13C6). Use of the method for the analysis of natural benzene was illustrated for the measurement of background levels in urban smokers and nonsmokers.

Studies on the metabolism of nicotine by rabbit liver microsomal fractions in the presence of 0.01 M sodium cyanide have led to the characterization of two isomeric cyanonicotine compounds. The locations of the cyano groups were established by GC--EIMS analyses of the deuterium-labeled products obtained from the specifically deuterium-labeled substrates (S)-nicotine-5',5'-d2, (R,S)-nicotine-2',5',5'-d3, and (R,S)-nicotine-N-methyl-d3. One cyano adduct was shown to be 5'-cyanonicotine, a product previously isolated from similar microsomal preparations. The second cyano adduct was shown to be N-cyanomethyl)nornicotine; this structure assignment was confirmed by synthesis. Formation of N-cyanomethyl)nornicotine appears to occur, at least in part, without prior nitrogen--carbon bond cleavage, implicating the in situ generation of the N-methyleniminium species during the course of metabolic oxidative N-demethylation of nicotine.

Glucose is an important substrate for myocardial metabolism. This study was designed to determine the effect of circulating metabolic substrates on myocardial glucose extraction and to determine the metabolic fate of glucose in normal human myocardium. Coronary sinus and arterial catheters were placed in 23 healthy male volunteers. [6-14C]Glucose was infused as a tracer in 10 subjects. [6-14C]Glucose and [U-13C]lactate were simultaneously infused in the other 13 subjects. Simultaneous blood samples were obtained for chemical analyses of glucose, lactate, and free fatty acids and for the the isotopic analyses of glucose and lactate. Glucose oxidation was assessed by measuring myocardial 14CO2 production. The amount of glucose extracted and oxidized by the myocardium was inversely correlated with the arterial level of free fatty acids (r = -0.71; P less than 0.0001). 20% (range, 0-63%) of the glucose extraction underwent immediate oxidation. Chemical lactate analysis showed a net extraction of 26.0 +/- 16.4%. However, isotopic analysis demonstrated that lactate was being released by the myocardium. In the 13 subjects receiving the dual-carbon-labeled isotopes, the lactate released was 0.09 +/- 0.04 mumol/ml and 49.5 +/- 29.5% of this lactate was from exogenous glucose. This study demonstrates that the circulating level of free fatty acids plays a major role in determining the amount of glucose extracted and oxidized by the normal human myocardium. Only 20.1 +/- 19.4% of the glucose extracted underwent oxidation, and 13.0 +/- 9.0% of the glucose extracted was metabolized to lactate and released by the myocardium. Thus, 60-70% of the glucose extracted by the normal myocardium is probably stored as glycogen in the fasting, resting state.

A selected ion monitoring gas chromatography/mass spectrometric method for the quantitative determination of 3,4,4'-trichlorocarbanilide (TCC) and its major metabolites (the 2'-hydroxy sulfate and the N- and N'-glucuronides) in human plasma and urine was developed using the deuterium-labelled compounds as internal standards. Limits of detection of 3 ng/mL in urine for the N-glucuronides and of 1.5 ng/mL in plasma for the 2'-hydroxy sulfate were attained. Use of the method was illustrated in a study in human subjects employing TCC-containing bar soaps.

Because cholesterol 5,6-epoxides have been reported to be mutagenic, carcinogenic, and cytotoxic, we investigated the relationship of these substances in breast fluid to histopathologically defined breast disease. We measured cholesterol and its oxidation product, 5 beta,6 beta-epoxide, in breast fluids from 68 women with biopsied benign breast disease (BBD) and 135 women with no history of breast disease (controls). Each biopsy was classified according to the most severe epithelial change: (a) nonproliferative epithelia; (b) hyperplasia without atypia; or (c) hyperplasia with atypia. Similar to our previous findings in control women, breast fluid cholesterol and beta-epoxide concentrations in women with BBD were associated with factors of interest in relation to breast cancer: concentrations increased with age and were higher in white than nonwhite women and in women who were past or current smokers; concentrations were lower in women who had given birth or breastfed within 2 yr. Increased breast fluid cholesterol and beta-epoxide concentrations were significantly associated with proliferative BBD (hyperplasia with or without atypia) compared to controls. After adjustment for covariates, the odds ratio for proliferative BBD associated with detectable versus nondetectable beta-epoxide concentrations was 8.5 (95% confidence intervals, 1.1, 68.8). Our findings suggest that the histological progression from normal epithelium to hyperplasia without atypia to atypical hyperplasia is associated with progressively increasing concentrations of both cholesterol and cholesterol beta-epoxide.

A selected ion monitoring gas chromatographic mass spectrometric assay for trifluoroacetic acid was developed for the study of the metabolism of the volatile anesthetic agent halothane by in vitro preparations. The assay uses a headspace sampling technique after formation of methyl esters with dimethyl sulfate and sulfuric acid. Pentafluoropropionic acid proved to be a suitable internal standard, although care is required in the preparation of the calibration standards so that they reflect the composition and treatment of the samples. Contamination of the samples, possibly with trifluoroacetic acid itself, was found to be the primary factor in limiting the sensitivity of the assay for the metabolism of halothane. Generally, trifluoroacetic acid could be determined at levels as low as 1 microM in 50-100 microliters of incubate.

An improved analog four-channel selected ion recording system is described, in which major modifications permit a decrease in the dwell time to 33 ms per channel, thus minimizing the mass cycling error. Synchronization with a sinusoidal sweep voltage superimposed on the normal accelerating voltage (8 kV) enables two channels to be monitored simultaneously in real time and each mass to be recorded continuously. These improvements allow measurement of ion current ratios with a precision of 0.2% over a wide dynamic range, permitting accurate determination of isotopic enrichment in biomedical assays even when this enrichment is derived from a single label. Use of the system is illustrated by the analysis of palmitate turnover in dogs (using [1--13C] palmitic acid) with an average standard deviation corresponding to the detection of 0.04% excess of [1--13C] palmitate.

Metabolic N-hydroxylation of the potent psychotomimetic amine 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (5) by rabbit liver microsomal preparations has been investigated. Synthetic hydroxylamine 8 was obtained by sequential reduction of the corresponding nitropropene 10 with sodium borohydride followed by zinc reduction of the resulting nitropropane 11. Compound 8 in water (pH 7.4) was rapidly air oxidized to oxime 12; this oxidation was completely blocked by rabbit liver microsomes. Microsomal incubations of amine 5 or its bis(methoxy-d3)hexadeuterio analog 5-d6 resulted in the formation of 8 and 8-d6, respectively, identified as their bis(trifluoroacetyl) derivatives by GLC-MS. Quantitative estimations of metabolite formation employing selected ion monitoring with the aid of an accelerating voltage alternator were accomplished by stable isotope dilution analyses with 5-d6 as substrate and 8-d0 as internal standard. Similar analyses starting with "pseudoracemates" (R)-5-d0:(S)-5-d6 or (R)-5-d6:(S)-5-d0 as substrates established metabolite 8 to be enriched with its R enantiomer.

In nipple aspirates of breast fluid from nonpregnant healthy women, cholesterol and cholesterol epoxide levels were determined with gas-liquid chromatography and mass spectrophotometric techniques. Cholesterol levels were found to be elevated above plasma levels averaging 2200 +/- 1995 (S.D.) mg/dl and showing progressive increases in mean breast fluid cholesterol levels with advancing age, averaging 187, 1957, and 3554 mg/dl in women of age groups 20 to 29, 30 to 39, an 40 to 49 years, respectively. Cholesterol epoxide was detected in a significant number of women who yielded high levels of breast fluid cholesterol. Cholesterol epoxide has been reported by other workers to have transforming activity for embryo hamster cells and to be carcinogenic in animals. The findings lend support to our hypothesis and observation that the human breast secretes mutagenic and cancer-promoting substances which may have relevance in studies of the etiology of benign breast disease and cancer.

alpha-1-Antitrypsin is a serum protein that inhibits many proteolytic enzymes. Recently, it was suggested that the alpha-1-antitrypsin-trypsin complex is an acyl ester analogous to the acyl intermediate that forms between trypsin and its substrates. In previous work we showed that the alpha-1-antitrypsin-trypsin complex can be split at high pH, releasing a component of alpha-1-antitrypsin. This component had a new carboxyl-terminal lysine, and it had lost a peptide of about 4000 daltons. In order to determine whether the alpha-1-antitrypsin is bound to trypsin through the new carboxy-terminal lysine, as would be expected if the above hypothesis is correct, we split the complex in the presence of 18OH-. When the new carboxy-terminal lysine was cleaved with carboxypeptidase B, singly labeled, doubly labeled, and unlabeled lysine were recovered. These data support the hypothesis that the alpha-1-antitrypsin-trypsin complex is an acyl ester or a tetrahedral precursor that is transformed into the acyl ester form at high pH. If other enzymes are bound by a similar mechanism, the methods used may be useful in determining which amino acids on alpha-1-antitrypsin bind covalently to each enzyme.

We measured levels of cholesterol and its oxidation products, 5,6 alpha- and beta-epoxides and their common hydrolysis product cholestane triol, in breast fluids of women without breast disease, compared these levels to serum cholesterol levels, and explored associations of these breast fluid measurements with known breast cancer risk factors and other characteristics. Subjects were 105 women with no history of breast disease from whom breast fluid could be obtained by nipple aspiration. The four breast fluid measurements were significantly correlated with each other (P less than 0.0001) but none was correlated with serum cholesterol. In subsequent analyses restricted to breast fluid cholesterol and cholesterol beta-epoxide, cholesterol levels (but not beta-epoxide levels) increased with age and were higher in white than nonwhite women. Both measurements were low in women who were lactating, who were parous, or who had breast-fed. The lower levels among parous women persisted for at least 2 years postpartum or postlactation. Because breast fluid levels of cholesterol beta-epoxide are reduced for some time following a birth or cessation of lactation, the alveolar-ductal systems of parous women presumably have less cumulative exposure to this potentially carcinogenic substance. This biochemical mechanism may, in part, explain the observed reduction of breast cancer risk associated with parity.

A method for the quantitative determination of chlorpromazine and five of its major metabolites in a single sample of biological fluid in the ng/ml range has been developed utilizing gas chromatography/mass spectrometry with selected ion recording. The assay is highly specific and quantification is accomplished by an inverse stable isotope dilution technique, using deuterium-labeled variants of the compounds as internal standards. In this way the concentrations of chlorpromazine and five of its major metabolites (the sulfoxide, the N-oxide, the monodemethylated, the didemethylated, and the 7-hydroxylated compounds) can be determined in biological fluids. Levels in humans have been measured both in plasma and in red blood cells and are compared to those found in related in vitro studies.

A method has been developed for the quantitative determination of cholesterol and three of its major oxidative metabolites (the 5 alpha,6 alpha-epoxide, the 3 beta,5 alpha,6 beta-triol, and the 5 beta,6 beta-epoxide) in a single sample of human breast fluid (2-50 microliters), using gas chromatography/mass spectrometry with selected ion monitoring. High specificity and reliable quantification is achieved by the use of the inverse stable isotope dilution method, employing deuterium-labeled variants of the compounds as internal standards.

A sensitive assay for trifluoroacetic acid, the major product of the oxidative metabolism of halothane, has been developed to study the biotransformation of halothane. A selected ion monitoring gas chromatographic mass spectrometric assay measured trifluoroacetic acid levels as low as 1 microM in 100 microliter of reaction mixture. This assay was used to quantitate halothane metabolism in human and rabbit microsomal systems and with purified proteins. Trifluoroacetic acid production was examined as a function of the concentration of substrate present, the amount of microsomal protein used and the length of reaction time. Halothane metabolism in microsomes was linear for at least 30 min, and up to a microsomal protein concentration of 1 mg/ml. In rabbits, phenobarbital and imidazole induced the microsomal metabolism of halothane 7.36- and 18.2-fold, respectively. Imidazole was used because it is a potent inducer of cytochrome P-450 isozyme 3a which is also induced by ethanol. The cytochrome P-450 in microsomes from a single human subject metabolized halothane at a rate comparable to that found in microsomes from phenobarbital- and imidazole-pretreated rabbits. The purified phenobarbital and imidazole inducible cytochromes P-450, isozymes 2 and 3a, catalyzed the oxidation of halothane to trifluoroacetic acid. Cytochrome b5 stimulated the isozyme 3a-catalyzed oxidation of halothane by 19-fold, whereas isozyme 2 catalyzed oxidation was increased 4.3-fold. Antibodies to cytochrome P-450 3a inhibited halothane metabolism by 90% in microsomes from imidazole-pretreated rabbits, suggesting that isozyme 3a catalyzes halothane metabolism in imidazole-pretreated rabbits. In conclusion, the oxidation of halothane to trifluoroacetic acid by cytochrome P-450 isozymes 3a and 2 is enhanced markedly by cytochrome b5.

The major unknown component present in the plasma of all subjects given thioridazine (la) or mesoridazine (lb) has been identified by TLC, GC, and mass spectrometry as thioridazine side-chain sulfone (sulforidazine) (1c).

An acetylenic C-glucuronide of the sedative-hypnotic drug ethchlorvynol was isolated from rabbit urine as the major metabolite. The C-glucuronide represents a novel metabolic pathway for acetylenes and is a rare example of the formation of a carbon-glucuronide bond in mammalian systems.

A stable isotope tracer method has been developed for studying lactate metabolism in humans. The method uses lactic acid triple labeled with 13C as the tracer. The stable isotope is infused to attain a level of approximately 1.5% of that of the circulating unlabeled lactate. Following the isolation of lactic acid from the blood, the percentage of triple labeled (13C)lactate is measured using gas chromatography mas spectrometry. We compared this method with tracer methodology using [14C]lactate and found comparable results.

"New" glucose production has been measured in 54 infants and children for the first time by continuous three-to-four-hour influsion of the safe, nonradioactive tracer 6,6-dideuteroglucose. The use of combined gas chromatography--mass spectrometry with monitoring of selected ions allowed deuterium enrichment in blood glucose to be measured on microliter samples with an error of less than 2 per cent. In the young child, glucose production increased in a slightly curvilinear manner from 1 kg. to 25 kg. body weight, when it reached 140 mg. per minute, almost the adult value of 173 mg. per minute (2.28 +/- 0.23 mg./kg.-min., mean +/- S.E.). Normalized for weight, glucose production in premature infants was 5.46 +/- 0.31 mg./kg.-min., in term neonates averaged 6.07 +/- 0.27 mg./kg.-min., in children below the age of six years was 7.1 +/- 0.27 mg./kg.-min., and in late childhood averaged 5.4 +/- 0.28 mg./kg.-min. Relative to estimated brain weight, however, glucose production was essentially linear from the 1-kg. premature infant to the 80-kg. adult. These data, the first measurements of "new" glucose production in childhood, suggest that brain size may be a principal determinant of those factors that regulate hepatic glucose output throughout life.

Benzene levels in human breath and in ambient air were compared in the urban area of San Francisco (SF) and in a more remote coastal pristine setting of Stinson Beach, Calif. (SB). Benzene analysis was done by gas chromatography-mass spectroscopy (GC-MS). Ambient benzene levels were sevenfold higher in SF (2.6 +/- 1.3 ppb, n = 25) than SB (0.38 +/- 0.39 ppb, n = 21). In SF, benzene in smokers' breath (6.8 +/- 3.0 ppb) was greater than in nonsmokers' breath (2.5 +/- 0.8 ppb) and smokers' ambient air (3.3 +/- 0.8 ppb). In SB the same pattern was observed: benzene in smokers' breath was higher than in nonsmokers' breath and ambient air. Benzene in SF nonsmokers' breath was greater than in SB nonsmokers' breath. Marijuana-only smokers had benzene breath levels between those of smokers and nonsmokers. There was little correlation between benzene in breath and number of cigarettes smoked, or with other benzene exposures such as diet. Of special interest was the finding that benzene in breath of SF nonsmokers (2.5 +/- 0.8 ppb) was greater than that in nonsmokers ambient air (1.4 +/- 0.1 ppb). The same was true in SB, where benzene in nonsmokers breath was greater than ambient air (1.8 +/- 0.2 ppb versus 1.0 +/- 0.1 ppb on d 1 and 1.3 +/- 0.3 ppb versus 0.23 +/- 0.18 ppb on d 2). This suggests an additional source of benzene other than outdoor ambient air.

A new method based on selected ion monitoring chemical ionization mass spectrometry was developed to measure the quaternary ammonium neuromuscular blocking agents, pancuronium and vecuronium, in serum. An ion-pair extraction procedure is utilized to separate the compounds of interest from biological fluids. The intensities of the ion currents produced by the bisamines formed from the drugs and the corresponding deuterated internal standards through thermolytic dequaternization are monitored. The assay shows good linearity over the range of 1-500 ng/ml. This assay has been utilized in a variety of clinical pharmacokinetic studies involving surgical pediatric, geriatric and obstetric patients requiring anesthesia.

Using a combination of gas chromatography, mass spectrometry, and selected ion recording techniques, we have identified nicotine and its major metabolite, continine, in the breast fluid of nonlactating women smokers. As little as 25 picograms could be measured by using the deuterated variants, [5',5'-2H]nicotine and [3,3-2H]cotinine, both as internal standards and as carriers in an inverse isotope dilution method.

Simultaneous lactate production and extraction have been previously demonstrated in the myocardium in patients with coronary artery disease. To quantitate this lactate production and determine its source, dual carbon-labeled isotope experiments were performed. L-[1,2,3-13C3] lactate and D-[6-14C] glucose were infused in 10 patients with significant coronary artery disease. Metabolic samples were obtained at rest and during atrial pacing. Despite net chemical myocardial lactate extraction in the 10 patients at rest and no evidence of clinical ischemia, the L-[1,2,3-13C3] lactate analysis demonstrated that lactate was being released by the myocardium. During atrial pacing, seven patients did not develop clinical symptoms of ischemia, and the chemical lactate analysis showed net lactate extraction. However, tracer analysis demonstrated that there was a significant increase in the lactate released during atrial pacing (from 6.9 +/- 2.3 to 16.2 +/- 10.1 mumol/min) (p less than 0.05). In these seven patients, circulating glucose was the source of 23 +/- 15% of the lactate released at rest, and there was no significant change during pacing. The remaining three patients had mild chest pain and net chemical lactate production during pacing. Lactate release detected by the tracer increased from 5.7 +/- 3.0 mumol/min at rest to 50.9 +/- 16.8 mumol/min during pacing (p less than 0.01). In these patients, the contribution of glucose to lactate production increased significantly during pacing-induced clinical ischemia from 25 +/- 22 to 67 +/- 14% (p less than 0.005). Thus, dual carbon-labeled isotopic experiments are powerful tools for investigating myocardial metabolic pathways.(ABSTRACT TRUNCATED AT 250 WORDS)

The metabolism of chlorpromazine by microsomes in vitro has been examined with extracts from normal liver and small intestinal mucosa of man and guinea pigs. A GC-MS approach has been utilized to measure primary metabolites generated by these extracts, including the S-oxide, N-oxide, 7-hydroxyl, desmethyl, and didesmethyl species. In short term incubations (less than 30 min), the measured metabolites accounted for at least 90% of the substrate utilized. Chlorpromazine metabolism differed strikingly both between species and between hepatic and intestinal tissues of the same species. Guinea pig hepatic microsomes were the most active of the preparations studied, producing relatively large amounts of N-oxide. By contrast, human hepatic microsomes produced the 7-hydroxyl metabolite predominantly, with minimal formation of N-oxide. Extracts of guinea pig intestinal mucosa formed the desmethyl and S-oxide products; an extract of duodenal mucosa from a healthy accident victim exhibited minimal metabolism of chlorpromazine. The kinetics of metabolite formation and studies with inhibitors of cytochrome P-450 suggested the involvement of multiple microsomal enzymes in chlorpromazine metabolism.