Identification of nicotinic acetylcholine receptor amino acids photolabeled by the volatile anesthetic halothane.

To identify inhalational anesthetic binding domains in a ligand-gated ion channel, we photolabeled nicotinic acetylcholine receptor (nAChR)-rich membranes from Torpedo electric organ with [(14)C]halothane and determined by Edman degradation some of the photolabeled amino acids in nAChR subunit fragments isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance liquid chromatography. Irradiation at 254 nm for 60 s in the presence of 1 mM [(14)C]halothane resulted in incorporation of approximately 0.5 mol of (14)C/mol of subunit, with photolabeling distributed within the nAChR extracellular and transmembrane domains, primarily at tyrosines. GammaTyr-111 in ACh binding site segment E was labeled, while alphaTyr-93 in segment A was not. Within the transmembrane domain, alphaTyr-213 within alphaM1 and deltaTyr-228 within deltaM1 were photolabeled, while no labeled amino acids were identified within the deltaM2 ion channel domain. Although the efficiency of photolabeling at the subunit level was unaffected by agonist, competitive antagonist, or isoflurane, state-dependent photolabeling was seen in a delta subunit fragment beginning at deltaPhe-206. Labeling of deltaTyr-212 in the extracellular domain was inhibited >90% by d-tubocurarine, whereas addition of either carbamylcholine or isoflurane had no effect. Within M1, the level of photolabeling of deltaTyr-228 with [(14)C]halothane was increased by carbamylcholine (90%) or d-tubocurarine (50%), but it was inhibited by isoflurane (40%). Within the structure of the nAChR transmembrane domain, deltaTyr-228 projects into an extracellular, water accessible pocket formed by amino acids from the deltaM1-deltaM3 alpha-helices. Halothane photolabeling of deltaTyr-228 provides initial evidence that halothane and isoflurane bind within this pocket with occupancy or access increased in the nAChR desensitized state compared to the closed channel state. Halothane binding at this site may contribute to the functional inhibition of nAChRs.

Bioactivation and cytotoxicity of 1,1-dichloro-2,2,2-trifluoroethane (HCFC-123) in isolated rat hepatocytes.

The bioactivation and cytotoxicity of 1,1-dichloro-2,2,2-trifluoroethane (HCFC-123), a replacement for some ozone-depleting chlorofluorocarbons, were investigated using freshly isolated hepatocytes from non-induced male rats. A time- and concentration-dependent increase in the leakage of lactate dehydrogenase and a concentration-dependent loss of total cellular glutathione were observed in cells incubated with 1, 5 and 10 mM HCFC-123 under normoxic or hypoxic (about 4% O2) conditions. Lactate dehydrogenase leakage was completely prevented by pretreating the cell suspension with the free radical trapper N-t-butyl-alpha-phenylnitrone. The aspecific cytochrome P450 (P450) inhibitor, metyrapone, totally prevented the lactate dehydrogenase leakage from hepatocytes, while two isoform-specific P450 inhibitors, 4-methylpyrazole and troleandomycin (a P450 2E1 and a P450 3A inhibitor, respectively), provided a partial protection against HCFC-123 cytotoxicity. Interestingly, pretreatment of cells with glutathione depletors, such as phorone and diethylmaleate, did not enhance the HCFC-123-dependent lactate dehydrogenase leakage. Two stable metabolites of HCFC-123, 1-chloro-2,2,2-trifluoroethane and 1-chloro-2,2-difluoroethene, were detected by gas chromatography/mass spectrometry analysis of the head space of the hepatocyte incubations carried out under hypoxic and, although at a lower level, also normoxic conditions, indicating that reductive metabolism of HCFC-123 by hepatocytes had occurred. The results overall indicate that HCFC-123 is cytotoxic to rat hepatocytes under both normoxic and hypoxic conditions, due to its bioactivation to reactive metabolites, probably free radicals, and that P450 2E1 and, to a lower extent, P450 3A, are involved in the process.

Human halothane metabolism, lipid peroxidation, and cytochromes P(450)2A6 and P(450)3A4.

OBJECTIVE: Halothane undergoes both oxidative and reductive metabolism by cytochrome P450 (CYP), respectively causing rare immune-mediated hepatic necrosis and common, mild subclinical hepatic toxicity. Halothane also causes lipid peroxidation in rodents in vitro and in vivo, but in vivo effects in humans are unknown. In vitro investigations have identified a role for human CYPs 2E1 and 2A6 in oxidation and CYPs 2A6 and 3A4 in reduction. The mechanism-based CYP2E1 inhibitor disulfiram diminished human halothane oxidation in vivo. This investigation tested the hypotheses that halothane causes lipid peroxidation in humans in vivo, and that CYP2A6 or CYP3A4 inhibition can diminish halothane metabolism. METHODS: Patients (n = 9 each group) received single doses of the mechanism-based inhibitors troleandomycin (CYP3A4), methoxsalen (CYP2A6) or nothing (controls) before a standard halothane anaesthetic. Reductive halothane metabolites chlorotrifluoroethane and chlorodifluoroethylene in exhaled breath, fluoride in urine, and oxidative metabolites trifluoroacetic acid and bromide in urine were measured for 48 h postoperatively. Lipid peroxidation was assessed by plasma F2-isoprostane concentrations. RESULTS: The halothane dose was similar in all groups. Methoxsalen decreased 0- to 8-h trifluoroacetic acid (23 +/- 20 micromol vs 116 +/- 78 micromol) and bromide (17 +/- 11 micromol vs 53 +/- 49 micromol) excretion (P < 0.05), but not thereafter. Plasma F2-isoprostanes in controls were increased from 8.5 +/- 4.5 pg/ml to 12.5 +/- 5.0 pg/ml postoperatively (P < 0.05). Neither methoxsalen nor troleandomycin diminished reductive halothane metabolite or F2-isoprostane concentrations. CONCLUSIONS: These results provide the first evidence for halothane-dependent lipid peroxidation in humans. Methoxsalen effects on halothane oxidation confirm in vitro results and suggest limited CYP2A6 participation in vivo. CYP2A6-mediated, like CYP2E1-mediated human halothane oxidation, can be inhibited in vivo by mechanism-based CYP inhibitors. In contrast, clinical halothane reduction and lipid peroxidation were not amenable to suppression by CYP inhibitors.

Human reductive halothane metabolism in vitro is catalyzed by cytochrome P450 2A6 and 3A4.

The anesthetic halothane undergoes extensive oxidative and reductive biotransformation, resulting in metabolites that cause hepatotoxicity. Halothane is reduced anaerobically by cytochrome P450 (P450) to the volatile metabolites 2-chloro-1,1-difluoroethene (CDE) and 2-chloro-1,1,1-trifluoroethane (CTE). The purpose of this investigation was to identify the human P450 isoform(s) responsible for reductive halothane metabolism. CDE and CTE formation from halothane metabolism by human liver microsomes was determined by GC/MS analysis. Halothane metabolism to CDE and CTE under reductive conditions was completely inhibited by carbon monoxide, which implicates exclusively P450 in this reaction. Eadie-Hofstee plots of both CDE and CTE formation were nonlinear, suggesting multiple P450 isoform involvement. Microsomal CDE and CTE formation were each inhibited 40-50% by P450 2A6-selective inhibitors (coumarin and 8-methoxypsoralen) and 55-60% by P450 3A4-selective inhibitors (ketoconazole and troleandomycin). P450 1A-, 2B6-, 2C9/10-, and 2D6-selective inhibitors (7,8-benzoflavone, furafylline, orphenadrine, sulfaphenazole, and quinidine) had no significant effect on reductive halothane metabolism. Measurement of product formation catalyzed by a panel of cDNA-expressed P450 isoforms revealed that maximal rates of CDE formation occurred with P450 2A6, followed by P450 3A4. P450 3A4 was the most effective catalyst of CTE formation. Among a panel of 11 different human livers, there were significant linear correlations between the rate of CDE formation and both 2A6 activity (r = 0.64, p < 0.04) and 3A4 activity (r = 0.64, p < 0.03). Similarly, there were significant linear correlations between CTE formation and both 2A6 activity (r = 0.55, p < 0.08) and 3A4 activity (r = 0.77, p < 0.005). The P450 2E1 inhibitors 4-methylpyrazole and diethyldithiocarbamate inhibited CDE and CTE formation by 20-45% and 40-50%, respectively; however, cDNA-expressed P450 2E1 did not catalyze significant amounts of CDE or CTE production, and microsomal metabolite formation was not correlated with P450 2E1 activity. This investigation demonstrated that human liver microsomal reductive halothane metabolism is catalyzed predominantly by P450 2A6 and 3A4. This isoform selectivity for anaerobic halothane metabolism contrasts with that for oxidative human halothane metabolism, which is catalyzed predominantly by P450 2E1.

Isoflurane acts as an inhibitor of oxidative dehalogenation while acting as an accelerator of reductive dehalogenation of halothane in guinea pig liver microsomes.

The effects of isoflurane, 1-chloro-2,2,2-trifluoroethyl difluoromethyl ether, on the oxidative metabolism of halothane to produce trifluoroacetic acid (TFA) and on the reductive metabolism of halothane to produce chlorodifluoroethylene (CDE) and chlorotrifluoroethane (CTE) in liver microsomes of guinea pig were examined. Isoflurane enhanced the production of CDE and CTE and inhibited the production of TFA. Isoflurane enhanced cytochrome P450 reduction and formation of an intermediate complex with cytochrome P450 without enhancement of NADPH-cytochrome P450 reductase (EC 1.6.2.4) activity. We conclude that isoflurane interacts with cytochrome P450 to prevent the formation of the halothane-cytochrome P450 complex, causing inhibition of the oxidative dehalogenation. This interaction of isoflurane enhances the reduction of cytochrome P450 and the formation of a reductive intermediate-cytochrome P450 complex under anaerobic conditions causing reductive dehalogenation of halothane.

Metabolism of 1-fluoro-1,1,2-trichloroethane, 1,2-dichloro-1,1-difluoroethane, and 1,1,1-trifluoro-2-chloroethane.

1-Fluoro-1,1,2-trichloroethane (HCFC-131a), 1,2-dichloro-1,1-difluoroethane (HCFC-132b), and 1,1,1-trifluoro-2-chloroethane (HCFC-133a) were chosen as models for comparative metabolism studies on 1,1,1,2-tetrahaloethanes, which are under consideration as replacements for ozone-depleting chlorofluorocarbons (CFCs). Male Fischer 344 rats were given 10 mmol/kg ip HCFC-131a or HCFC-132b or exposed by inhalation to 1% HCFC-133a for 2 h. Urine collected in the first 24 h after exposure was analyzed by 19F NMR and GC/MS and with a fluoride-selective ion electrode for the formation of fluorine-containing metabolites. Metabolites of HCFC-131a included 2,2-dichloro-2-fluoroethyl glucuronide, 2,2-dichloro-2-fluoroethyl sulfate, dichlorofluoroacetic acid, and inorganic fluoride. Metabolites of HCFC-132b were characterized as 2-chloro-2,2-difluoroethyl glucuronide, 2-chloro-2,2-difluoroethyl sulfate, chlorodifluoroacetic acid, chlorodifluoroacetaldehyde hydrate, chlorodifluoroacetaldehyde-urea adduct, and inorganic fluoride. HCFC-133a was metabolized to 2,2,2-trifluoroethyl glucuronide, trifluoroacetic acid, trifluoroacetaldehyde hydrate, trifluoroacetaldehyde-urea adduct, inorganic fluoride, and a minor, unidentified metabolite. With HCFC-131a and HCFC-132b, glucuronide conjugates of 2,2,2-trihaloethanols were the major urinary metabolites, whereas with HCFC-133a, a trifluoroacetaldehyde-urea adduct was the major urinary metabolite. Analysis of metabolite distribution in vivo indicated that aldehydic metabolites increased as fluorine substitution increased in the order HCFC-131a < HCFC-132b < HCFC-133a. With NADPH-fortified rat liver microsomes, HCFC-133a and HCFC-132b were biotransformed to trifluoroacetaldehyde and chlorodifluoroacetaldehyde, respectively, whereas HCFC-131a was converted to dichlorofluoroacetic acid. No covalently bound metabolites were detected by 19F NMR spectroscopy.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification and quantification of fluorine-containing metabolites of 1-chloro-2,2,2-trifluoroethane (HCFC133A) in the rat by 19F-NMR spectroscopy.

1-Chloro-2,2,2-trifluorethane (HCFC133a) causes a reduction in testis weight and germinal epithelial cell atrophy in the rat following exposure by inhalation at concentrations of 10,000 ppm and above. Following administration by gavage, an increased incidence of Leydig cell tumors of the testis was seen. The metabolism of HCFC133a has been investigated in respect to the known toxicity of this compound. Male rats were exposed by inhalation to an atmosphere of 50,000 ppm HCFC133a for a period of 6 hr. Analysis of urine, collected during the exposure period and up to 48 hr following exposure, by 19F-NMR spectroscopy identified 2,2,2-trifluoroethanol (TFE; and its beta-glucuronide), trifluoroacetaldehyde (TFAA; as its hydrate and urea adduct), and trifluoroacetic acid (TFA) as fluorine-containing metabolites of HCFC133a. Of the total amount of metabolite eliminated in urine, 83% was excreted within 24 hr postdose, establishing a rapid elimination of metabolites by this route. TFAA, an established testicular toxicant, was the major metabolite accounting for 57% of the total fluorinated metabolites eliminated in urine, whereas TFA and TFE accounted for 29% and 14%, respectively. The presence of these metabolites in urine is consistent with an oxidative route of metabolism of this fluorocarbon.

Structure-mutagenicity and structure-cytotoxicity studies on bromine-containing cysteine S-conjugates and related compounds.

Glutathione and cysteine S-conjugates of several haloalkenes are nephrotoxic and cytotoxic. Chloroalkene-derived S-(1-chloroalkenyl)-L-cysteine conjugates, but not fluoroalkene-derived S-(2,2-dihalo-1,1-difluorethyl)-L-cysteine conjugates, are mutagenic in the Ames test, although both types of S-conjugates are cytotoxic and nephrotoxic. Recent studies showed that bromine-containing S-(2,2-dihalo-1,1-difluoroethyl)-L-cysteine conjugates are mutagenic in the Ames test, thus challenging the generalization that S-(2,2-dihalo-1,1-difluoroethyl)-L-cysteine conjugates are not mutagenic. Hence a series of bromine-containing and bromine-lacking S-(2,2-dihalo-1,1-difluoroethyl)-L-cysteine conjugates was prepared, and their mutagenicity was assessed in the Ames test with Salmonella typhimurium TA2638 as the test strain. In addition, several indices of cytotoxicity, including cytotoxicity in LLC-PK1 cells, induction of Ca2+ release from pig kidney mitochondria, and DNA double-strand breaks in LLC-PK1 cells, were measured. The bromine-containing S-conjugates S-(2-bromo-2-chloro-1,1-difluoroethyl)-L- cysteine (BCD-FC), S-(2-bromo-1,1,2-trifluoroethyl)-L-cysteine (BTFC), and S-(2,2-dibromo-1,1-difluoroethyl)-L-cysteine (DBDFC) were mutagenic in the Ames test, whereas S-(2-chloro-1,1,2-trifluorethyl)-L-cysteine (CTFC), S-(2,2-dichloro-1,1-difluoroethyl)-L-cysteine (DCDFC), and S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFC), which lack bromine, were not. BCDFC, BTFC, CTFC, DBDFC, and TFC were cytotoxic in LLC-PK1 cells, and their cytotoxicity was blocked by the cysteine conjugate beta-lyase inhibitor (aminooxy)acetic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of fluoroethane chlorofluorocarbon (CFC) substitutes with microsomal cytochrome P450. Stimulation of P450 activity and chlorodifluoroethene metabolism.

The abilities of halothane and the fluoroethane chlorofluorocarbon (CFC) substitutes, FC-123, FC-133a, FC-124, FC-134a and FC-125, to stimulate cytochrome P450 activities and 2-chloro-1,1-difluoroethene (CDE) defluorination in hepatic microsomes from phenobarbital-treated rabbits were compared. At 1% (v/v) each, halothane and FC-123 similarly increased the consumption of NADPH and O2 by 300 and 100%, respectively, over that in microsomes without substrate. FC-133a and FC-124 were less effective, increasing NADPH and O2 consumption by 150-200 and 70%. FC-134a and FC-125 were the least effective, increasing NADPH and O2 consumption by only 70 and 50%, respectively. No metabolism of any fluoroethane could be detected under the incubation conditions used. Halothane and FC-123 were most effective in stimulating CDE metabolism with increases of CDE defluorination ranging from 1.5- to 2-fold. FC-133a and FC-124 enhanced CDE oxidation 89 and 74%, respectively, and FC-134a and FC-125 had no effect. While CDE metabolism was enhanced in the presence of the fluoroethanes, no additional NADPH or O2 was consumed when halothane or FC-124 was incubated with CDE compared with incubations containing only halothane or FC-124. Log-log plots of NADPH consumption and CDE metabolism with the olive oil/gas partition coefficients of each fluoroethane showed linear relationships. These data demonstrate that the activity of the fluoroethanes in stimulating P450 activity and CDE metabolism is a function of their lipid solubility, and fluoroethane-enhanced CDE metabolism is related to the ability of these compounds to increase uncoupled P450 activity.

Nephrotoxicity of the glutathione and cysteine conjugates of 2-bromo-2-chloro-1,1-difluoroethene.

The mercapturate S-(2-bromo-2-chloro-1,1-difluoroethyl)-N-acetyl-L-cysteine, which is apparently derived from the halothane degradation product 2-bromo-2-chloro-1,1-difluoroethene, is excreted in urine. S-(2-Bromo-2-chloro-1,1-difluoroethyl)glutathione (BCDFG) and S-(2-bromo-2-chloro-1,1-difluoroethyl)-L-cysteine (BCDFC) are putative intermediates in the metabolism of 2-bromo-2-chloro- 1,1-difluoroethene and are analogs of nephrotoxic and cytotoxic S-haloalkyl glutathione and cysteine conjugates. The objective of the research was to study the nephrotoxicity and cytotoxicity of 2-bromo-2-chloro-1,1-difluoroethene-derived S-conjugates. BCDFG and BCDFC were nephrotoxic in Fischer 344 rats and caused diuresis, increases in urine glucose and protein concentrations, in blood urea nitrogen concentrations, in kidney/body weight percentages and in serum glutamate-pyruvate transaminase activities. Both S-conjugates also produced severe morphological changes in the kidneys, especially in the proximal tubules. Morphological changes indicative of hepatotoxicity were seen in some animals given BCDFG and BCDFC. Both BCDFG and BCDFC were cytotoxic to LLC-PK1 cells, as shown by lactate dehydrogenase release into the medium. The cytotoxicity of BCDFG was blocked by the gamma-glutamyltransferase inhibitor acivicin, and the cytotoxicity of both BCDFG and BCDFC was blocked by the cysteine conjugate beta-lyase inhibitor aminooxyacetic acid. Also, S-(2-bromo-2-chloro-1,1-difluoroethyl)-DL-alpha-methylcysteine, which can not be metabolized by beta-lyase, was not toxic to LLC-PK1 cells. These in vivo and in vitro data provide evidence that BCDFG and BCDFC are nephrotoxic and that their toxicity is dependent on renal bioactivation by cysteine conjugate beta-lyase.

Halothane reductive metabolism in an adult surgical population.

The reductive metabolism of halothane was studied in 34 adult patients undergoing routine surgery. Reductive biotransformation of halothane was more extensive in females than males and was also enhanced in two patients treated preoperatively with phenytoin, an enzyme-inducing drug. Tobacco, ethanol and the patient's age, body weight and previous exposure to halothane did not influence reductive metabolism of halothane.

Effects of piperonyl butoxide on halothane hepatotoxicity and metabolism in the hyperthyroid rat.

A series of experiments were conducted to examine the potential role of phase I metabolism in halothane-induced liver injury in the hyperthyroid rat. The metabolism of halothane was determined in both hyperthyroid (triiodothyronine, 3 mg/kg per day, for 6 days) and euthyroid rats and in animals pre-treated with the cytochrome P-450 inhibitor piperonyl butoxide (75-100 mg/kg, i.p.). It was found that the hyperthyroid state, which is associated with a substantial increase in sensitivity to the hepatotoxic effects of halothane, decreases both oxidative and reductive routes of halothane metabolism in the rat. The production of trifluoroacetic acid (TFA), an oxidative metabolite, as well as that of chlorodifluoroethylene (CDF) and chlorotrifluoroethane (CTF), 2 reductive metabolites, was significantly reduced in hyperthyroid animals. Consistent with these findings serum and urinary bromide levels resulting from the formation of TFA, CDF or CTF were significantly reduced. The only route of halothane metabolism significantly increased by the hyperthyroid condition was the defluorination of halothane. Piperonyl butoxide administration did not render euthyroid animals sensitive to the halothane-induced hepatotoxicity and had no effect on the defluorination of halothane in euthyroid animals. However, piperonyl butoxide markedly increased the hepatotoxicity of halothane in hyperthyroid rats and, except for a modest increase in debromination reactions, decreased all measured indices of halothane metabolism including the defluorination of halothane. Thus, none of the observed changes in halothane metabolism produced by triiodothyronine or piperonyl butoxide treatment could be consistently correlated to the increases in hepatotoxicity linked to these 2 treatments. Based on these studies we suggest that the halothane hepatotoxicity induced in the hyperthyroid rat results from effects produced by either the parent compound or an as yet unidentified metabolite. In addition, these studies further demonstrate that considerable mechanistic differences exist for halothane-induced hepatotoxicity when comparing euthyroid and hyperthyroid animal models.

Metabolic activation of the halothane metabolite, [14C]2-chloro-1,1-difluoroethene, in hepatic microsomes.

Halothane is reduced to 2-chloro-1,1,1-trifluoroethane (CTE) and 2-chloro-1,1-difluoroethene (CDE) by cytochrome P-450. These compounds may potentially undergo secondary metabolism in vivo, but their capacity to undergo metabolic activation and bind to macromolecules is unknown. This study, therefore, compared the abilities of CDE and CTE to bind to microsomal components in relation to that of halothane in hepatic microsomes. The results show that CDE, in addition to halothane, binds to microsomes under conditions of cytochrome P-450 activity. While halothane bound predominantly to lipids under nitrogen, CDE bound mainly to protein under oxygen. No CTE binding under any conditions could be detected. On an equimolar basis, CDE binding to protein was approximately one-third of that of halothane under oxidative conditions, however, CDE binding was enhanced in the presence of halothane. The results support the hypothesis that CDE metabolism may contribute to the metabolic binding due to halothane exposures.

Modulation of the reductive metabolism of halothane by microsomal cytochrome b5 in rat liver.

To study the modulation of the reductive metabolism of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) by microsomal cytochrome b5, formation of 2-chloro-1,1,1-trifluoroethane (CTE) and 2-chloro-1,1-difluoroethylene (CDE), major reduced metabolites of halothane, was analyzed in vivo and in vitro. Rats were pretreated with both malotilate (diisopropyl-1,3-dithiol-2-ylidenemalonate) and sodium phenobarbital (malotilate-treated rats) or only with sodium phenobarbital (control rats). The microsomes of malotilate-treated rats had significantly more cytochrome b5 than the controls, whereas the cytochrome P-450 content was not different between the two groups. At the end of 2-h exposure to 1% halothane in 14% oxygen, the ratio of CDE to CTE in arterial blood was significantly higher in malotilate-treated rats than in the controls. Under anaerobic conditions, the formation of CDE and the ratio of CDE to CTE were significantly greater in microsomal preparations of malotilate-treated rats than those of the controls. In a reconstituted system containing cytochrome P-450PB purified from rabbit liver, addition of cytochrome b5 to the system enhanced the formation of CDE and increased the ratio of CDE to CTE. These results suggested that cytochrome b5 enhances the formation ratio of CDE to CTE by stimulating the supply of a second electron to cytochrome P-450, which might reduce radical reactions in the reductive metabolism of halothane.

Metabolism of halothane in children having repeated halothane anaesthetics.

Metabolism of halothane was studied in nine children receiving daily halothane anaesthetics (from 10 up to a maximum of 31) over periods of two to seven weeks. Serum bromide concentrations never exceeded 3.5 mmol/l, a concentration below the toxic threshold. Repeated halothane anaesthetics at short intervals did not induce the reductive metabolism of halothane as assessed by 2-chloro-1,1,1-trifluoroethane (CTF) in the expired breath. One patient developed viral hepatitis A during the course of anaesthetic administration; this patient was the only one whose serum bromide concentrations fell substantially and whose exhaled CTF concentration increased as more anaesthetics were administered.

Effects of combined extradural blockade and general anaesthesia on indocyanine green clearance and halothane metabolism.

Cardiovascular effects, indocyanine green clearance and the reductive metabolism of halothane were compared in 10 patients having peripheral surgery who received either general anaesthesia or general anaesthesia plus extradural blockade. Bupivacaine or saline was given extradurally 1 h before anaesthesia with nitrous oxide:oxygen (70:30) and halothane (end-tidal concentration 0.35%). Indocyanine green clearance was measured before (stage I) and after (stage II) bupivacaine or saline, during anaesthesia before surgery (stage III), and during surgery (stage IV). Reductive metabolism of halothane was assessed by measurement of exhaled 2-chloro-1,1,1-trifluoroethane. In the general anaesthesia plus extradural blockade group, arterial pressure was decreased to 58% (SD = 6) of stage I values over stages II-IV, whereas no changes were observed in the general anaesthesia group. There was no significant difference in indocyanine green clearance or the reductive metabolism of halothane, despite the occurrence of significant decreases in arterial pressure.

Reductive halothane metabolite formation and halothane binding in rat hepatic microsomes.

The production of the reductive [14C]halothane metabolites, 2-chloro-1, 1,1-trifluoroethane ( CTE ) and 2-chloro-1,1- difluoroethylene (CDE), was determined in anaerobic microsomal incubations by high pressure liquid chromatography (HPLC). The HPLC technique used allowed accurate measurements of low levels of [14C]halothane metabolites. Comparisons of metabolic profiles and halothane binding in microsomes reduced with NADPH and sodium dithionite show that dithionite stimulates CDE production and total halothane degradation, but inhibits CTE formation and [14C]halothane binding. Similarly, the addition of isoflurane, but not enflurane, to microsomes increases CDE production and decreases CTE formation and [14C]halothane-lipid binding. Measurement of fluoride in similar incubations show that fluoride release from halothane correlates with the formation of CDE and not CTE . The results demonstrate that the relative production of CTE and CDE may not remain constant in microsomal preparations, and that halothane binding correlates with CTE formation and not CDE and fluoride production.