NMR IN BIOMEDICINE, VOL. 5 , 101-lo6 (1992)

NMR Studies of Enflurane Elimination and Metabolism 19F

N. E. Preece* Hunterian Institutc. The Royal College of Surgeons of England. 35-43 Lincoln's Inn Fields. London WC2A 3PN, U K

J. Challands Department of Anaesthetics, T h e Royal London Hospital, Whitechapel, London El IBB, U K

S. C. R. Williams U L l R S NMR Imaging Facility, Department of Chemistry, Queen Mary and Westfield College, London El 4NS. UK

The elimination and metabolism of enflurane, a fluorinated ether anaesthetic, was studied by '% NMR in uiuo in both rat liver and brain as well as human body fluids. In the liver of thiobarbitone-anaesthetized rats the half-life for enflurane following exposure to 0.15% (vlv) for 30 min was 76 min but this could be decreased to 39 min by pretreatment of the animals with isoniazid (0.1% in the drinking water for 7 days), an agent known to enhance enflurane metabolism. In these animals the major organic metabolite difluoromethoxy difluoroacetate (DFMDFA) was also detected by 'v NMR in uiuo. This metabolite was detected along with fluoride ion in rat and human urine and plasma by high resolution I9F NMR. Human urine also contained signals from a probable DFMDFA conjugate and unexpectedly from trifluoroacetate.

INTRODUCTION Most routine surgery relies on the use of fluorinated anaesthetic agents. However, the mode of action of these drugs is still poorly understood. In addition severe liver and kidney toxicity still occurs in a small number of patients as a result of the (largely hepatic) metabolism of these agents. Halothane and the fluorinated ethers (Fig. 1 ) have proved eminently suitable to study by non-invasive I9F NMR spectroscopy and imaging.' The fluorine nucleus like the proton, has a high intrinsic sensitivity (83% of 'H) yet unlike 'H NMR studies of metabolism, IyF NMR of animal tissues does not suffer from endogenous background signal.* Fortuitously these drugs are necessarily given in high doses as NMR is an inherently insensitive technique. Many of these I9FNMR studies have concentrated on halothane (Fig. 1). The three fluorine atoms of its trifluoromethyl (TFM) group coresonate which increases the sensitivity of detection. The major organic metabolite (-40% of a given dose) of halothane is trifluoroacetic acid' (TFA). Similarly the major metabolite of methoxyflurane which is also extensively metabolized is methoxydifluoroacetate. The signals from the parent and metabolite difluoromethyl (DFM) moieties are well resolved in ~ i u o . ~ Methoxyflurane has been withdrawn from clinical use as a result of its fluoride-associated nephrotoxicity. Following phenobarbitone pretreatment, enhanced metabolism of halothane and methoxyflurane can be observed by I9FNMR in uiu0.3.~ Isoflurane and enflurane were more recently intro-

'Author to whom correspondence should be addressed. Abbreviations used: TFM. trifluoromethyl; TFA, trifluoroacetic

acid; DFM, difluoromethyl; TSP, sodium 3trimethylsilyl-(2,2.3,3,-d,)propionate; DFMDFA, ditluoromethoxy difluoroacetate.

0952-3480/92/020 101-06 $05 .OO 01992 by John Wiley & Sons. Ltd.

duced to clinical practice for reasons which include their limited metabolism. Enflurane metabolism, though limited compared to halothane (3-9%), is not as limited as that of isoflurane ( < I % ) and can be further induced by isoniazid pretreatment.s In these studies we have investigated: (i) enflurane elimination from liver and brain of anaesthetized rat by "F NMR in uiuo; and (ii) metabolite production in rat and human biofluids, principally urine, by high resolution NMR.

METHODS Animal pretreatment. Male Sprague-Dawley rats (200250 g body wt) were fed standard rat chow ad libitum

and given either tap water or 0.1% isoniazid:HCI (pH7.4) to drink for 7 days. This isoniazid pretreatment protocol has previously been shown to induce an increase in the rate of rat liver microsomal para-

F H \ \ F -C-C-Br / I F- Cl

CI F H I \ \ H-C-C-0-CH I H

Halothane

Methoxyf lurane

F\ H FI \ F-C -c-0-CH I c'l F

FI F\ F\ H-C-C-0- CH

lsoflurane

Enflurane

i

dl ;

66

I

F

Figure 1. Structures of h a l o t h a n e a n d t h e fluorinated e t h e r

anaesthetics (see Christ e t a/.23.24 for metabolism). Receioed 1.3 June 1991 Accepted (revised) 9 September 1991

102

N. E. PREECE, J . CHALLANDS AND S. C. R. WILLIAMS

hydroxylation of aniline compared with microsomes prepared from control rats.6 This assay is believed to be a reliable if not a specific indicator of cytochrome P450IIEI isozyme induction.’

receiving isoniazid treatment. Approval by the ethical committee was obtained before collection of any samples and all patients gave informed consent. Sample preparation. Urine, plasma and neutralized

Anaesthetic exposure. The animals were sealed individu-

ally within a 2.5 L glass exposure chamber with an associated atmosphere recirculating system (Teflon/ stainless steel). Carbon dioxide was absorbed with a soda-lime trap, and oxygen was monitored and maintained at 20.9% with an oxygen electrode and electrically activated gas servo valve, respectively.’ After the animals had adapted to the enclosed environment (5 min) enflurane (0.15% v/v) was injected into the system up stream of a Teflon-laminated pump which vaporized and mixed the anaesthetic within the system. Preliminary experiments indicated that the dose of enflurane subsequently inhaled was just sufficient to induce anaesthesia within the chamber under these conditions. (The minimum alveolar concentration of enflurane required to anaesthetize Sprague-Dawley rats is 2 . 2 % . ) The rats were removed following a 30 min exposure period and then rapidly given saline or 0.23 mmol sodium thiopentobarbitone/kg i.p. before recovery from the enflurane anaesthesia occurred. In uioo NMR. Enflurane

exposed thiobarbitoneanaesthetised rats were placed individually in a 4 . 7 T SISCO horizontal magnet and surrounded by cotton wool to maintain body heat. A 15 mm i d . surface coil was placed over the abdomen immediately posterior to the sternum. Alternatively it was placed on the back of the head equidistant from the eyes and ears an estimated 0-4mm posterior to the bregma. Initially the magnetic field homogeneity was optimized by observing the proton signals of water and fat at 200 MHz. The water signal linewidth was decreased to a minimum and the fat signal was reduced as much as possible (-10% the area of the water peak). After 30 min the coil was retuned to 188MHz and “F NMR spectra were recorded from 1000 signal averaged FIDs following multiplication by a left-shifted sine bell to simultaneously enhance sensitivity and eliminate broad signal components emanating from the coil’s insulation material (total repetition time = 0.6 s; 25 ps pulse; sweep width 32 kHz; 4000 data points). These spectra were acquired over 10 min intervals for up to 3 h. Sample collection. Alternatively individual rats were

housed in unsealed ‘Metabowls’ for urine collection over 24 h post-exposure to the anaesthetic(s) and given food and tap water ad libitum. Some animals were killed between 2 h and 6 h after exposure to obtain tissue and plasma samples. Patient studies. Plasma (2 and 6 h) and urine (up to 24 h) samples were collected post-operatively from eight male (aged 40-85 years) and four female subjects (aged 31-58 years) who had been anaesthetized with enflurane (0.5-4%) for periods ranging from 20 to 65 min following thiobarbitone induction for various minor operations and were otherwise considered relatively fit and healthy. Samples from patients with hepatic complications or renal insufficiency were excluded from observation. None of the subjects in the study were

perchloric acid (12%) tissue extract samples were lyophilized and reconstituted in DzO (100%) in order to concentrate them prior to rapid NMR analysis, suppress the water peak for proton NMR and to provide an internal field frequency lock. Selected samples were freeze dried or reconstituted in Hz0(80%)/D,0(20X) with essentially similar results. All samples except those from plasma were ultimately treated with chelating resin (Chelex 100; Sigma, St Louis, MO, USA) and centrifuged (15 000 X g) with the aim of removing transition metals and residual proteidlipid which can broaden I9F NMR signals of interest (particularly that of fluoride). Sodium 3-trimethylsilyl(2,2,3,3-d4)propionate (TSP) and TFA (Sigma) were subsequently added to some of the samples to provide an internal chemical shift/signal intensity reference of known concentration for proton and ”F NMR quantitation, respectively. Spectra were recorded on Bruker WH spectrometers operating at 5.9 and 9.4 T.

RESULTS

In vivo N M R Following exposure to enflurane, liver and brain ”F NMR spectra of the anaesthetic were reproducibly obtained with adequate S/N in a few minutes. The high resolution spectrum of enflurane is relatively complex (Fig. 2(a)), however four of the five fluorine atoms appeared to co-resonate in uiuo (4.7T) producing a major peak (set to 0.0 ppm). The fifth terminal fluorine atom (on the chlorine-bearing carbon) gave rise to a minor peak at -70.1 ppm with respect to the major peak (Fig. 2(b)). On integration the ratio of the peak areas in the first 10 min was ca 4 : 1 in all rats irrespective of isoniazid pretreatment. In control rats (n = 4) the intensity of the major and minor peaks decreased exponentially from liver spectra recorded at 10 min intervals over the next 2.5 h, at which point the experiment was terminated. During this time the peaks maintained the 4: 1 ratio. In those rats which had received the isoniazid pretreatment (n = 4) the intensity of the peaks also decreased with time in an exponential fashion; however the ratio of the peaks had increased to between 5: 1 and 6 : 1 by 1.5 h. By this time the intensity of the minor peak had become inadequate for accurate integration of the area so these experiments were usually terminated earlier than the controls. These data were normalized and calculated as a fraction of the initial total area for each animal and grouped (Fig. 3(a)). The isoniazid pretreated rats had consistently eliminated the enflurane more rapidly than the control rats from their livers. These data fitted single first-order decays with correlation coefficients of >0.98 (Fig. 3(b)). On summing three blocks of loo0 FIDS (30 min acquisition) it became evident that a third novel peak was also present 6.8ppm to lower field of the major

"F NMR STUDIES OF ENFLURANE

peak in the livers of isoniazid-pretreated rats (Fig. 2(c)). The presence of this novel peak was established within 30 min, after which its intensity remained constant, possessing an area 5 6 % of the initial area of the major enflurane peak. The novel peak was also present in the brains of isoniazid-pretreated rats at a similar

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Time (minl Figure 3. (a) Decrease in the normalized areas of the major and minor liver peaks of control (M, +)and isoniazid-pretreated rats (0,A )with time expressed as a fraction (%) of the initial total enflurane signal observed. Points and bars represent the mean +SEM of four rats. (b) Same data as in (a) drawn with log scale to demonstrate the exponential nature of the decays and respective half-lives.

m , l . , , , l . , , , , , , . , l " , . , , , . 10 0 -10 -20 -30 - 4 0 -50 -60 -70 -80

PPm

Figure 2. (a) High resolution 'F NMR spectrum (expanded) of enflurane at 9.4 T. The two triplets around -70 ppm are assigned to the terminal fluorine atom and those around 0.1 ppm to the difluoromethoxy moiety after Koehler et (b) ''F NMR in vivo spectrum of enflurane from the liver of an anaesthetized rat maintained on tap water recorded in the first 10 min following 30 min set-up and shimming period. Spectrum recorded from 1000 signal averaged FlDs following multiplication by a left-shifted sine bell to simultaneously enhance sensitivity and eliminate broad signal components emanating from the coil's insulation material (total repetition time = 0.6 s; 25 ps pulse; sweep width 32 kHz at 4.7 T; 4000 data points). (c) As for (b) except the rat received isoniazid pretreatment and 3000 FlDs were signal averaged from the liver for 0.5-1 h following shimming. (d) As for (c) except 10 000 FlDs were signal averaged from the brain of an isoniazid-pretreated rat. As shimming was usually finer in brain tissue in favourable cases the fluorine atoms on the middle carbon of enflurane could also be distinguished as a shoulder to higher field of the major peak.

intensity relative to enflurane but required more signal averaging in order to be observed. We believe this signal emanates from the fluorine atoms on the difluor, l . ~ acetate moiety of difluoromethoxy difluoroacetate (DFMDFA; Fig. 4(a)), the major organic metabolite of enflurane.' The other signals from DFMDFA i.e., the DFM moiety, are not resolved from the major enflurane peak but their presence may explain why the ratio of the major to minor peaks increased from the expected 4 : 1 ratio with time in the isoniazid-pretreated rats. The rate of enflurane elimination from brain was similar to that seen in liver for each group. I n uiuo liver or brain '"F NMR spectra contained no peaks which could be attributed to "F NMR observable fluoride ion. High resolution N M R

"F NMR spectra of urine samples collected from rats (n = 16) or patients (n= 12) exposed to enflurane were invariably found to contain three triplets (J = 4.2 Hz)

N . E. PKEEC'E. J . CHALLANDS AND S. C. R. W I I ~ I J A M S

104

with chemical shifts at 6.77, 0.46 and 0.25ppm (Fig. 4(a)) and an intense singlet or a broad hump at 33; 43 ppm with respect to an external TFA standard. On integration the triplets were found to have arcas approximately in the ratio 2 : 1 : 1. If broad-band filtered proton irradiation was applied to the sample the two smaller triplets were found to collapse into a singlet triplet at 0.36 ppm ("F-lH J = 70.4 Hz) identical to the triplet at 6.77ppm. They were in fact a doublet of triplets split by heteronuclear coupling. We believe the triplets arise from cxcreted DFMDFA.' Thc multiplicity is a consequence of "F-'"F coupling across the ether bond to t h e adjacent pair of fluorine atoms. 'We note that Selinsky et u1.' reported that the signal from methoxydifluoroacetate the equivalent metabolite of methoxyfluranc resonated at a similar chemical shift. On treating the samples with chelation resin or the addition of excess hydroxide ion the broad hump at 33-43 ppm when present is replaced 42

HI

a

I

Table 1. Total DFMDFA excretion over 24 h by rats exposed to enflurane (0.1So/~) for 30 min DMFDFA (~mollprnol creatininel

Animal treatment

n

None 2.1 k0.5 8 lsoniazid 3.7 It_ 1.3 4 Thiobarbitone 1.6 2 0.8 4 Rats received either tap water or 0.1% isoniazid (pH 7.4; HCI) to drink for 7 days before enflurane exposure or tap water and then 0.23 mmol sodium thiopentobarbitone/kg i.p. immediately after enflurane exposure.

with the intense singlet at 41.5 ppm which was vcrified as fluoride ion by standard addition of NaF. Rat liver and brain tissue extracts ( n = 3 ) , and rat (n = 5 ) or human (12 = 6) plasma samples contained DFMDFA, but at much lower concentrations than were present in urine necessitating prolonged signal averaging to obtain adequate S/N. In some of the patient urines the three DFMDFA triplets were all shadowed to slightly higher field (0.05-0.37 ppm) by equivalent species at lower concentration in the same 2 : 1 : 1 ratio. The area of these species ranged from 6 to 12% of their DFMDFA partners. In addition some patient urines also contained a singlet at 9.62 pprn which was verified as trifluoroacetate by standard addition (Fig. 4(b)). Urine from rats pretreated with isoniazid ( n = 4) had a greater "F NMR observable fluoride content than urine from control rats ( n = 8 ) . DFMDFA was also present at higher concentration with respect to the urinary creatinine content when compared to urine from controls rats (Table 1). Those rats which received thiobarbitone in addition to enflurane (n = 4) appeared to excrete less enflurane metabolites than those receiving only enflurane. Patient urines contained 0.5-2.7 pmol DFMDFA/ pmol of creatinine and considerably more "F NMR observable fluoride ion following chelation resin treatment. The ' H NMR spectra (Y.4T) of patient urine were also observed but were not dissimilar to spectra of control urine samples. "'

DISCUSSION ~

8

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PPm Figure 4. (a) ''F NMR high resolution spectrum of lyophilized D,O-reconstituted rat urine collected over 24 h from an animal which received 0.15% enflurane for 30 min showing a triplet and a doublet of triplets from DFMDFA (6.77, 0.46 and 0.25). the major organic metabolite of enflurane. Spectrum recorded from 144 signal averaged FlDs following low frequency filtering (total repetition time=3.1 s; 90" pulse, 10 ps; sweep width 2.6 kHz at 5.9 T; 16000 data points). (b) 'F NMR high resolution spectrum of lyophilized D,O-reconstituted human urine collected over 24 h from a 56-year-old male patient after receiving 2.5% enflurane for 30 min showing DFMDFA plus trifluoroacetate (9.62 ppm) and a possible DFMDFA conjugate. The regions around 6.7 and 0.2ppm have been expanded to show the additional signals from the conjugate. Fine couplings are not resolved. Spectrum recorded from 4000 signal averaged FlDs following low frequency filtering (total repetition time = 1.3 s; 90" pulse, 12 ps; sweep width 25 kHz at 9.4 T; 4000 data points).

~

~~

Enflurane is specifically metabolized by a form of rat liver cytochrome P450 which can be induced by isoniazid.5.11 We were able to detect increased rates of enflurane elimination in oiuo in rats pretreated with isoniazid. We believe this was due to increased oxidative metabolism of enflurane particularly as it was associated with observation of DFMDFA, the organic metabolite of enflurane, and fluoride ion. Induction of increased anaesthetic metabolism in the liver has previously been observed by in uiuo "F NMR. Selinsky er al. found that pretreatment of rats with phenobarbitone decreased the half-life of halothane elimination from 3.5 to 2.5 h in liver while the concentration of TFA reached a steady-state level not seen without phenobarbitone pretreatment.3 The value of our and other in uiuo animal experiments may be impaired by the necessity to anaesthetize

“’F NMR STUDIES OF ENFLURANE

the rat with an alternative non-fluorinated anaesthetic agent so as to observe the elimination of enflurane by in oioo I’ F NMR. The trend towards decreased urinary excretion of DFMDFA in those rats administered thiobarbitone in addition to enflurane in our studies (Table 1) implies the former may have altered the latter’s pharmacokinetics in oioo. Similar complications can arise during in uioo 3 1 P NMR studies as a result of impaired rat liver metabolism.” Presumably this problem would not occur in a similar human study of the lower abdomen as suitable volunteers could be asked to remain immobile when conscious. The animal study reported here demonstrates that although measurement of the enflurane clearance rate by in uioo ”F NMR is probably compromised by thiobarbitone anaesthesia, despite this the effects of isoniazidinduction are still manifest. I9F NMR studies have proved useful in elucidating the mechanism of anaesthetic action on the central nervous system in uiuo. Studies by Evers et a/.” have demonstrated that T? relaxation correlates with anaesthetic potency in brain tissue. Other studies by these w o r k e r ~ ’indicate ~ there are two distinct sites for halothane in rat brain with different relaxation characteristics. Similarly studies by Wyrwicz et al. also described two sites for halothane15 and two sites for isoflurane“ in rabbit brain with different half-lives. However Mills et al.” have demonstrated that when a large surface coil ( 3 cm diameter) is used to maximize signal the half-life of isoflurane in rabbit brain cannot be easily distinguished from the longer half-life of the anaesthetic in peripheral cranial fat where it is more abundant. Studying the relaxation properties of the major enflurane peak in uioo will no doubt be even more complicated because of its complex unresolved multiplicity and the likely contributions to the signal from DFMDFA. In addition some recent studiesIx imply the molecular orientation of enflurane in lipid membranes may be atypical. We have assumed the I9F NMR signals we obtained in these studies emanated from enflurane and its metabolite DFMDFA in the liver of the anaesthetized rats. Experience with in oiuo ” P NMR suggest applying an equivalent pulse from a coil of this diameter (15mm i.d.) penetrates through abdominal muscle and fat and receives a signal the majority of which arises from underlying liver tissue. In contrast we cannot locate the enflurane signals detected in the head with similar confidence as emanating from the brain without using imaging techniques. Nevertheless the wash-out times measured by NMR are consistent with a combination of rapid respiratory elimination and relocation to fat stores resulting in the early termination of anaesthesia seen in the clinical use of enflurane for minor operations. Some I9F NMR imaging studies have shown that the distribution of halothane varies in rat brain” and is primarily detected in lipophilic regions of the

10s

rabbit head.”’ We have not concurrently investigated tissue energy status and pH by ”P NMR at 4 . 7 T but other similar studies we have performed at 8.5 T suggest no major changes are observed under anaesthesia alone (results not shown). The signals shadowing the DFMDFA signals in human urine are so similar as to suggest they emanate from a DFMDFA-like structure (Fig. 4(b)). We believe it is a minor conjugate formed in uioo, the nature of which we have not yet elucidated. The defluorination of enflurane in rats and man has been previously investigated.”,” Following these studies Burke et d.” identified DFMDFA as the organic metabolite of enflurane by G U M S but their method necessarily required extraction and conversion of DFMDFA to an ethanolamine derivative before detection. Identification of the free compound in biofluids by ‘“F NMR is more practical if less sensitive. In addition an unstable DFMDFA conjugate seen by NMR in urine might be hydrolysed to DFMDFA in such a derivatization procedure. Cytochrome P450 mediated oxidation at the chlorine-bearing carbon is the only imporant metabolic transformation of enflurane. The resultant acid halide would be rapidly hydrolysed predominantly to DFMDFA. A small fraction however would be expected to react with nucleophilic species on the surfaces of tissue macromolecules. Once labelled with DFMDFA-derived antigenic groups critical tissue macromolecules such as proteins, even if they still functioned, might elicit a potentially lethal autoimmune response in susceptible individuals. The presence of TFA, which has only been reported as a metabolite of halothane and isoflurane, in the urine of some patients anaesthetized with enflurane is most interesting. Particularly as antibodies have been detected in the sera of patients displaying enflurane hepatitis which possess cross-reactivity to antigenic material produced during halothane metabolism.” ?‘ It seems reasonable that an antibody to a TFM species could recognize a DFM group considering the similarity between the proton and fluorine atom’s radii and hydrogen-bonding potential. Indeed it would require an unlikely intramolecular rearrangement for TFA to be produced during the metabolism of enflurane or a DFMDFA moiety. An alternative explanation to the presence of TFA in the patients’ urine might be prior contamination of the anaesthetic apparatus with halothane which probably occurs in clinical practice more often than is expected.

Acknowledgements The authors arc grateful lor the use 0 1 ULIKS NMR facilities at Queen Mary and Westfield College and Kings College. London. Dr Precce was supported by the Wcllcorne Trust.

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2. Malet-Martino, M. C. and Martino, R. The application of nuclear magnetic resonance spectroscopy to drug metabolism studies. Xenobiot. 19, 583-607 (1989). 3. Selinsky, 6. S., Thompson, M. and London, R. E.

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N. E. PREECE, J . CHALLANDS AND S. C. R . WILLIAMS Measurement of in vivo hepatic halothane metabolism in rats using ''F NMR spectroscopy. Biochem. Pharmacol. 36, 413-416 (1987). Selinsky, B. S., Perlrnan, M. E. and London, R. E. In vivo NMR studies of hepatic methoxyflurane metabolism. I. Verification and quantification of methoxydifluoroacetate. 11. A reevaluation of hepatic metabolic pathways. Mol. Pharmacol. 33, 559-566, 567-573 (1988). Rice, S. A. and Talcott, R. E. Effect of isoniazid treatment on selected hepatic mixed-function oxidases. Drug Metab. D ~ s ~ o7, s .260-262 (1979). Morgan, E. T., Koop, D. R. and Coon, M. J. Catalytic activity of cytochrome P-450 isozyme 3a isolated from liver microsomes of ethanol-treated rabbits. J. Biol. Chem. 257, 1395113957 (1982). Nebert. D. W., Adesnik, M., Coon, M. J., Estabrook, R. W., Gonzalez, F. J., Guengerich, F. P., Gunsalus, I. C., Johnson, E. F., Kemper, 6.. Levin, W., Phillips, I. R., Sato, R. and Waterman, M. R. The P-450 gene superfamily: recommended nomenclature. DNA 6, 1-11 (1987). Preece, N. E., Evans, P. F., Howarth, J. A,, King, L. J. arid Parke, D. V. The induction of autoxidative tissue damage by iron nitrilotriacetate in rats and mice. Tox. Appl. Pharmacol. 93, 89-100 (1988). Burke, T. R., Branchflower, R. V., Lees, D. E. and Pohl, L. R. Mechanism of defluorination of enflurane. Identification of an organic metabolite in rat and man. Drug Metab. Dispos. 9, 19-24 (1981). Bales, J. R., Higham, D. P., Howe, I., Nicholson, J. K. and Sadler, P. J. High-resolution proton NMR spectroscopy for rapid multi-component analysis of urine. Clin. Chem. 30, 426-432 (1984). Ryan, D. E., Ramanathan, L., lida, S., Thomas, P. E., Haniu, M., Shively, J. E., Lieber, C. S. and Levin, W. Characterisation of a major form of rat hepatic microsomal cytochrome P-450 induced by isoniazid. J. Biol. Chem. 260, 6385-6393 (1985). Preece, N. E., Ghatineh, S. and Timbrell, J. A. Course of ATP depletion in hydrazine hepatotoxicity. Arch. Toxicol. 64,4953 (1990). Evers, A. S., Haycock, J. C. and d'Avignon, D. A. The potency of fluorinated ether anesthetics correlates with their "F spin-spin relaxation times in brain tissue. Biochem. Biophys. Res. Commun. 151, 1039-1045 (1988).

14. Evers, A. S., Berkowitz, B. A. and d'Avignon, D. A. Correlation between the anaesthetic effect of halothane and saturable binding in brain. Nature328, 157-160 (1987); 341, 766 (1990). 15. Wyrwicz, A. C., Conboy, C. 6..Ryback, K. R., Nichols, B. G. and Eisele, P. In vivo "F-NMR study of isoflurane elimination from brain. Biochim. Biophys. Acta 927, 86-91 (1987). 16. Wyrwicz, A. C., Conboy, C. B., Nichols, 6.G., Ryback, K. R. and Eisele, P. In vivo "F-NMR study of halothane distribution in brain. Biochim. Biophys. Acta 929, 271-277 (1987). 17. Mills, P., Sessler, D. I., Moseley, M., Chew, W., Pereira, 6.. James, T. L. and Litt, L. An in vivo ''F-NMR study of isoflurane elimination from the rabbit brain. Anesthesiol. 67, 169-173 (1987). 18. Yoshida, T., Takahashi, K. and Ueda, 1. Molecular orientation of volatile anesthetics at the bonding surface. 'H- and ''F NMR studies of submolecular affinity. Biochim. Biophys. Acfa 985, 331-333 (1989). 19. Wyrwicz, A. C. and Conboy, C. 6. Determination of halothane distribution in the rat head using "F-NMR technique. Magn. Res. Med. 9, 219-228 (1989). 20. Chew, W. M., Moseley, M. E., Mills, P. A,, Sessler, D., Gonzales-Mendez, R., James, T. L. and Litt, L. Spin-echo fluorine magnetic resonance imaging at 2 T: in vivo spatial distribution of halothane in the rabbit head. Magn. Reson. h a g . 5, 51-56 (1987). 21. Chase, R. E., Holaday, D. A., Fiserovas-Bergerova, V., Saidman, L. J. and Mack, F. E. The biotransformation of ethrane in man. Anesfhiol. 35, 262-267 (1971). 22. Hitt, B. A., Mazze, R. I., Beppu, W. J., Stevens, W. C. and Eger, E. I. Enflurane metabolism in rats and man. J. Pharmacol. Exp. Ther. 203, 193-202 (1977). 23. Christ, D. D., Kenna, J. G., Kammerer, W., Satoh, H. and Pohl, L. R. Enflurane metabolism produces covalently bound liver adducts recognised by antibodies from patients with halothane hepatitis, Anaesthesiol. 69, 833-838 (1988). 24. Christ, D. D., Satoh, H., Kenna, J. G. and Pohl, L. R. Potential metabolic basis for enflurane hepatitis and the apparent cross-sensitization between enflurane and halothane. Drug. Metab. Dispos. 16, 135- 140 (1988). 25. Koehler, K. A., Elwood, E. S., Shelton, R. A,, Jarnagin, F., Koehler, L. S. and Fossel, E. T. Interaction of fluorinated ether anaesthetics. J. Magn. Reson. 30, 75-84 (1978).

19F NMR studies of enflurane elimination and metabolism.

The elimination and metabolism of enflurane, a fluorinated ether anaesthetic, was studied by 19F NMR in vivo in both rat liver and brain as well as hu...
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