METABOLISM OF SYNTHANE: COMPARISON WITH IN VIVO AND IN VITRO DEFLUORINATION OF OTHER HALOGENATED HYDROCARBON ANAESTHETICS

Br.J. Anaesth. (1979), 51, 839

METABOLISM OF SYNTHANE: COMPARISON WITH IN VIVO AND IN VITRO DEFLUORINATION OF OTHER HALOGENATED HYDROCARBON ANAESTHETICS R. I. MAZZE, W. J. BEPPU AND B. A. HITT SUMMARY

In vivo biotransformation of an inhalation anaesthetic agent was first demonstrated by Barrett and Johnston (1939) who recovered trichloroacetic acid from the urine of dogs anaesthetized with trichloroethylene. Metabolism of other inhalation anaesthetics was not reported until 1964 when Van Dyke, Chenoweth and Van Poznak, using radioactive labelled compounds demonstrated the in vivo metabolism of chloroform, diethyl ether, halothane and methoxyflurane. The first in vitro studies reporting metabolism of halogenated hydrocarbons were by Heppel and Porterfield (1948), who demonstrated enzymatic dehalogenation of a series of brominated and chlorinated methanes and ethanes by a halidase in rat liver. Van Dyke and Chenoweth (1965) applied these techniques to investigate biotransformation of fluorinated inhalation anaesthetic agents. Since then, all halogenated anaesthetics have been found to be biotransformed by animals and human beings. The present study examines the in vitro and in vivo defluorination of synthane (BAX3224; difluormethyl 1,2,2,3,3-pentafluoropropyl ether), a non-flammable investigational inhalation anaesthetic agent. The properties of synthane are: empirical formula, C 4 H 3 F,O; boiling point (101.3 kPa), 75 °C; liquid density (23 °C), 1.55 g ml- 1 ; vapour pressure (20 °C), 12kPa; RICHARD I. MAZZE, M.D.; WILLIAM J. BEPPU, M.D.; BEN A.

HITT, PH.D.; Department of Anesthesia, Stanford University School of Medicine, Stanford, CA 94305, U.S.A., and Veterans Administration Medical Center, Palo Alto, CA 94304, U.S.A. 0007-0912/79/090839-06 S01.00

water : gas partition coefficient (37 °C), 1.6; blood : gas partition coefficient (37 °C), 2.5; oil : gas partition coefficient (corn oil, 37 °C), 95; and MAC (canine), 1.2% (Travenol Laboratories, Chicago, Illinois: Summary of Information on BAX3224, a New General Anesthetic Agent, April 21, 1972). METHODS

In vitro studies Fourteen adult male Fischer 344 rats, weighing 338 ± 9 g (mean ± SEM), bedded on ground corn cob for 30 days were assigned randomly to two groups. One group drank plain tap water ad libitum. The other drank tap water which contained sodium phenobarbitone 1 mg ml" 1 for 7 days before sacrifice to induce the mixed-function oxidase system (Marshall and McLean, 1969). Animals were fasted overnight, then sacrificed by decapitation. Livers were excised and immediately placed in ice-cold Tris-HCl buffer 0.05 mol litre- 1 , pH 7.4, and microsomes prepared in standard fashion (Mazze, Hitt and Cousins, 1974). The microsomal pellet was suspended in buffer, so that the final protein concentration was 5 mg ml" 1 , and then assayed immediately. Optimal in vitro reaction conditions for defluorination of the various anaesthetic substrates were determined by varying protein concentration, amount of cofactors and reaction time. These conditions were then employed for the definitive experiments as noted below. Assays were carried out in 25-ml sealed reaction flasks which contained: microsomal protein © Macmillan Journals Ltd 1979

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Aerobic defluorination of the inhalation anaesthetic agent, synthane, was compared with that of methoxyflurane, enflurane and halothane and with two other anaesthetics, isoflurane and sevoflurane. In vitro, in microsomes prepared from phenobarbitone-induced and control livers, synthane and halothane were not defluorinated. The relative order of defluorination of the other anaesthetics was methoxyflurane > sevoflurane > enflurane > isoflurane. In vivo, following 4 h of 1.2% (MAC) synthane anaesthesia, urinary inorganic fluoride excretion was increased by only a trivial amount and only in phenobarbitone-treated rats; polyuria was not observed. Synthane is the least metabolized of the fluorinated ether anaesthetics; its administration will not result in inorganic fluoride nephropathy. An index of nephrotoxic potential for fluorinated anaesthetic agents was formulated utilizing in vitro fluoride production data and oil : gas partition coefficients.

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RESULTS

In vivo studies Ten adult male Fischer 344 rats, weighing 354 + 10 g, bedded on ground corn cob for 30 days were divided randomly into two treatment groups of equal size and placed in individual metabolic cages. After a 3-day adaptation period three control 24-h urine

In vitro Synthane and halothane were not defluorinated to a degree measurable by our methods in either control or phenobarbitone-induced hepatic microsomal preparations (table I). In control preparations (C), methoxyflurane and sevoflurane defluorination rates

TABLE I. Results of in vitro experiments, n = 7, all groups. SYN = synthane; HAL = halothane; ISO = isoflurane; ENF = enflurane; SEVO = sevoflurane; MOF = methoxyflurane. P<0.05: * v. ENF, SEVO and MOF; t v. MOF; % v. SEVO and MOF; § v. control, same agent; If v. all other groups F~ (nmol 30 min" 1 per mg protein) SYN

Control (C) mean ± SEM Induced (I) mean ± SEM I/C

mean ± SEM

HAL

ISO

0.51* + 0.09

1-51J§ ±0.25 3.60U + 0.80

ENF

1.311

+ 0.23 1.63J§ + 0.15 1.35U + 0.15

SEVO

MOF

1.82 + 0.30 3.22f § ±0.46 1.90U + 0.25

2.00 + 0.11 18.89 § + 2.40 9.75H + 1.50

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collections were obtained, following which one group 5 mg, NADP 25 fj.mol, glucose 6-phosphate 10 and glucose 6-phosphate dehydrogenase 1 unit in a of rats drank tap water ad libitum; the other group total volume of 5 ml. Reaction flasks were gassed with drank tap water containing sodium phenobarbitone 1 oxygen for 2 min at 1 litre min" 1 and then sealed. 1 mg ml" for 7 days. Both groups of rats were Reactions were started by adding one of the anaes- placed in a Plexiglass chamber of volume 100 litre thetics in liquid form to give a concentration of where synthane was administered for 4 h. In order to 1 mmol litre" 1 ; this corresponds to an in vivo blood conserve the drug a closed system was employed. anaesthetic partial pressure approximately equal to Soda-lime was placed on the floor of the anaesthetic the partial pressure at MAC. Aqueous phase concen- chamber to a depth of 12.7 mm and plastic wire mesh trations were verified by gas chromatography during was laid on top of it. After the rats were in the the last 5 min of the reaction. Two reactiion flasks and chamber, 10 ml of liquid synthane was sprayed on a two controlflaskswere prepared from each preparation gauze sponge and vaporized within the chamber. for each assay. The first control flask contained anaes- Ambient synthane concentration was measured by gas thetic but no microsomal protein and the second con- chromatography and 2-5-ml increments of drug were tained microsomal protein but no anaesthetic. All vaporized approximately every 15 min to maintain flasks were incubated for 30 min at 37 °C with cons- a concentration of 1.2% as determined by gas tant agitation; under these conditions defluorination chromatography. Oxygen was added to the chamber, was linear with time. Reactions were stopped by intermittently, to maintain an atmospheric concentraunsealing the flasks and heating to 60 °C. The mix- tion of 50%. After anaesthesia, rats received 100% tures were evaporated to dryness and resuspended in oxygen until awake, then were returned to their 1 ml of sodium acetate buffer 2.5 mol litre" 1 , pH 4.8. individual metabolic cages where 24-h urine collecInorganic fluoride activity was determined with an tions were obtained for the next 3 days. Urinary Orion ion specific electrode (Fry and Taves, 1970). output and inorganic fluoride excretion were deterInorganic fluoride reference standards were prepared mined for each rat for each day. by adding known amounts of sodium fluoride to reaction flasks containing all constituents of the Statistical analyses reaction mixture except the anaesthetic agent. Rates Mean values for each treatment group were deterof inorganic fluoride production were determined by mined. Comparisons were made using paired and subtracting the rate of fluoride production in the unpaired t tests. P<0.05 was considered statistically control flasks from the rate in the flasks with complete significant. reaction mixtures.

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ANAESTHETIC DEFLUORINATION 80-

were approximately the same and were greater than enflurane and isoflurane rates, respectively. For drugs with measurable rates of defluorination, phenobarbitone treatment (I) increased the rate of defluorination in all cases, but to different degrees, as shown by the values I/C (table I). The relative order of defluorination, however, remained the same.

J±SEM

40-

20-

MOF 10-

CONTRO

T

Pre

Days after anaesthesia

FIG. 1. Urinary inorganic fluoride excretion (Up —V) following administration of fluorinated hydrocarbon anaesthetic agents to non-induced rats. Anaesthetic exposure in each case was 4-5 MAC hours. The relative ease of defluorination was MOF > SEVO = ENF > ISO > SYN = HAL = control. Abbreviations: SYN = synthane: ISO = isoflurane; ENF = enflurane; SEVO = sevonurane; MOF = methoxyflurane; Pre = before anaesthesia.

barbitone-treated rats to approximately the same extent; phenobarbitone treatment increased urinary inorganic fluoride excretion compared with control when sevoflurane or methoxyflurane was administered subsequently. DISCUSSION

The results of the present study indicate that, under aerobic conditions, synthane is not significantly defluorinated in vitro and is only minimally defluorinated

TABLE II. Twenty-four hour urinary output and inorganicfluorideexcretion, before and after synthane anaesthesia; mean±SEM. * P<0.05 v. before anaesthesia {Pre) 1

Urinary output (ml day l)

Control (» = 5) Induced

Pre

Day 1

7.8 ±0.5 6.9 ±0.6

±0.6 7.3 ±0.7

7.1

Day 2

Day 3

7.9 ±0.6 7.2 ±0.5

8.0 + 0.7 6.7

±0.5

Urinary F~ excretion ((imol day"1) Pre 2.7

±0.1 2.7 ±0.1

Day 1 3.5

±0.5 4.6* ±0.2

Day 2

Day 3

3.4 ±0.4 4.1 ±0.2

3.0 ±0.3 3.5 ±0.5

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In vivo Rats were rapidly and smoothly anaesthetized following vaporization of synthane in the anaesthetic chamber. They recovered from anaesthesia quickly and without difficulty. After anaesthesia polyuria was not observed in either group (table II). Before anaesthesia, urinary inorganic fluoride excretion was 2.7 ±0.1 [xmol day" 1 in both control and phenobarbitone-treated rats (table II); after anaesthesia, urinary inorganic fluoride excretion increased significantly (P<0.05), but only in phenobarbitonetreated rats on day 1 after anaesthesia. The increase in inorganic fluoride excretion (fig. 1), however, was very small compared with that observed in previous studies from our laboratory with methoxyflurane, isoflurane, enflurane and sevoflurane (Mazze, Cousins and Kosek, 1972; Cousins et al., 1973; Barr et al., 1974; Cook et al., 1975a). The order of defluorination in the present study compared with the above experiments, in all of which non-induced rats received approximately the same anaesthetic exposure, 4-5 MAC hours, was: methoxyflurane > sevoflurane = enflurane > isoflurane > synthane = halothane = control. Figure 2 compares urinary inorganic fluoride excretion data from the present study with data from other experiments in which phenobarbitone-treated and control rats received identical exposures to the same agent, although anaesthetic dosage varied from agent to agent (Barr et al., 1974; Mazze, Hitt and Cousins, 1974; Cook et al., 1975b). Synthane was defluorinated by induced rats only; isoflurane and enflurane were defluorinated by control and pheno-

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| •

NIL

SYN

ISO

ENF SEVO

Day1 after anaesthesia

FIG. 2. Urinary inorganic fluoride excretion (UF_V) day 1 after anaesthesia, in which rats were either treated with phenobarbitone (I) or untreated (C). All rats received identical exposures to the same anaesthetic agent, although anaesthetic exposure from agent to agent may have varied. Prior phenobarbitone treatment increased (U F -V) only when sevofiurane or methoxyflurane was administered. SYN = synthane; ISO = isoflurane; ENF = enfiurane; SEVO = sevoflurane; MOF = methoxyflurane; NIL = no anaesthesia.

in vivo by Fischer 344 rats. These results are consistent with the findings of Sawyer and others (1975) who administered synthane to Macaca speciosa monkeys. Fluorine is the only halogen atom found in the synthane molecule, so that oxidation at any point will lead to the formation of unstable intermediates and ultimately to the liberation of free inorganic fluoride ions. Although a similar absence of inorganic fluoride was noted in experiments with halothane, the same conclusion concerning its biotransformation would not be correct. Oxidative metabolism of halothane to the organic fluoride metabolites, trifluoro-

TABLE III. Nephrotoxic potential of fluorinated anaesthetic agents. MOF = methoxyflurane; DOC = dioxychlorane; ENF = enflurane; SEVO = sevoflurane; ISO = isoflurane; HAL = halothane; SYN = synthane

F-

Agent MOF DOC ENF

SEVO ISO HAL SYN

(nmol 30 min" 1 per mg protein) 2.00 1.41 1.31 1.82 0.51 0 0

X

X

(oil : gas) 930 275 99 56 94 230 95

=

Defluorination index

Nephrotoxic potential

1860

1.00 0.23 0.07 0.05 0.03 0.00 0.00

387 129 101 48 0 0

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acetic acid and trifluoroethanolamide, without formation of inorganic fluoride, has been reported (Cohen et al., 1975). The present study compares in vivo and in vitro synthane defluorination with defluorination of several other inhalation anaesthetic agents both clinically available and under investigation. In vivo studies with Fischer 344 rats are of value in predicting the nephrotoxic potential of fluorinated inhalation agents in patients. In both man and rats, exposure to methoxyflurane leads to dose-related increases in serum inorganic fluoride with concentrations as great as 250 (i.mmol litre" 1 ; this is associated with vasopressinresistant, polyuric nephropathy (Mazze, Cousins and Kosek, 1972; Cousins and Mazze, 1973). In both species, prolonged enflurane exposure results in moderate increases of serum inorganic fluoride with concentrations of 35-50 [zmol litre" 1 occasionally noted; this is associated with a sub-clinical concentrating defect (Barr et al., 1974; Mazze, Calverley and Smith, 1977). Isoflurane and halothane administration do not cause inorganic fluoride concentrations in excess of 2-6 (xmol litre""1; this is not associated with impaired urinary concentrating ability (Cousins et al., 1973; Mazze, Cousins and Barr, 1974). While in vivo studies with Fischer 344 rats may be the most unequivocal approach apart from human investigations for examining anaesthetic-induced nephropathy, compared with in vitro studies they are costly and time consuming. Since the inhalation anaesthetics are nephrotoxic only as a consequence of their defluorination, a more convenient method of screening for nephrotoxic potential might be simply to measure in vitro inorganic fluoride formation. However, duration of the increase in serum inorganic fluoride bears on the development of nephrotoxicity. Assuming that exposure to the fluorinated anaesthetic agents is the same, the area under the serum inorganic

J ±SEM C = Control I = Induced

ANAESTHETIC DEFLUORINATION

ACKNOWLEDGEMENTS

This study was supported in part by Veterans Administration Hospital, Palo Alto, California and United States Public Health Services Grant No. GM22746. REFERENCES

Barr, G. A., Cousins, M. J., Mazze, R. I., Hitt, B. A., and Kosek, J. C. (1974). A comparison of the renal effects and metabolism of enflurane and methoxyflurane in Fischer 344 rats. J. Pharmacol. Exp. Ther., 188, 257. Barrett, H. M., and Johnston, J. H. (1939). The fate of trichloroethylene in the organism. J. Biol. Chem., 127, 765. Cohen, E. N., Trudell, J. R., Edmunds, H. N., and Watson, E. (1975). Urinary metabolites of halothane in man. Anesthesiology, 43, 392. Cook, T. L., Beppu, W. J., Hitt, B. A., Kosek, J. C , and Mazze, R. I. (1975a). Renal effects and metabolism of sevoflurane in Fischer 344 rats: an in vitro and in vivo comparison with methoxyflurane. Anesthesiology, 43, 70. (1975b). A comparison of renal effects and metabolism of sevoflurane and methoxyflurane in enzyme-induced rats. Anesth. Analg. (Cleve.), 54, 829. Cousins, M. J., and Mazze, R. I. (1973). Methoxyflurane nephrotoxicity: a study of dose-response in man. J.A.M.A., 225, 1611. Barr, G. A., and Kosek, J. C. (1973). A comparison of the renal effects of isoflurane and methoxyflurane in Fischer 344 rats. Anesthesiology, 38, 557.

Denson, D. D., Uyeno, E. T., Simon, R. L., and Peters, H. M. (1976). Preparation and physiological evaluation of some new fluorinated volatile anesthetics; in Biochemistry Involving Carbon Fluoride Bonds (ed. R. Filler). ACS Symposium Series No. 28, p. 190. Washington, D.C. Fry, B. W., and Taves, D. R. (1970). Serum inorganic fluoride analysis with fluoride electrode. J. Lab. Clin. Med., 75, 1020. Heppel, L. A., and Porterfield, V. T. (1948). Enzymatic dehalogenation of certain brominated and chlorinated compounds. J. Biol. Chem., 176, 763. Marshall, W. J., and McLean, A. E. M. (1969). The effect of oral phenobarbitone on hepatic microsomal cytochrome P-450 and demethylation activity in rats fed normal and low protein diets. Biochem. Pharmacol., 18, 153. Mazze, R. I., Calverley, R. K., and Smith, N. T. (1977). Inorganic fluoride nephrotoxicity: prolonged enflurane and halothane anesthesia in volunteers. Anesthesiology, 46, 265. Cousins, M. J., and Barr, G. A. (1974). Renal effects and metabolism of isoflurane anesthesia in man. Anesthesiology, 40, 536. Kosek, J. C. (1972). Dose-related methoxyflurane nephrotoxicity in rats: a biochemical and pathologic correlation. Anesthesiology, 36, 571. Hitt, B. A., and Cousins, M. J. (1974). Effect of enzyme induction with phenobarbital on the in vivo and in vitro defluorination of isoflurane and methoxyflurane. J. Pharmacol. Exp. Ther., 190, 523. Sawyer, D. C , Hartsfield, S. M., Howard, D. R., and Cummingham, J. G. (1975). Comparative evaluation of a new inhalation anesthetic BAX-3224 and halothane in Macaca speciosa. Anesth. Analg. (Cleve.), 54, 144. Van Dyke, R. A., and Chenoweth, M. B. (1965). Metabolism of volatile anesthetics II. In vitro metabolism of methoxyflurane and halothane in rat liver slices and cell fractions., Biochem. Pharmacol., 14, 603. Van Poznak, A. (1964). Metabolism of volatile anesthetics I. Conversion in vivo of several anesthetics to 14CO2 and chloride. Biochem. Pharmacol., 13, 1239. METABOLISME DU SYNTHANE: COMPARAISON DE LA DEFLUORATION IN VIVO ET IN VITRO AVEC LES AUTRES AGENTS ANESTHESIANTS A BASE D'HYDROCARBURES HALOGENES RESUME

La defluoration aerobie du synthane, agent anesthesiant par inhalation, a ete comparee a celle du methoxyflurane, de l'enflurane et de l'halothane de meme qu'a deux autres anesthesiques: l'isoflurane et la sevoflurane. In vitro, dans des microsomes prepares a partir de foies traites et controles par le phenobarbitone, le synthane et l'halothane n'ont pas ete defluores. L'ordre relatif de la defluoration des autres agents anesthesiants a ete methoxyflurane > sevoflurane > enflurane > isoflurane. In vivo, apres 4 h d'anesthesie au synthane a 1,2% (MAC), l'excretion urinaire de fluorure inorganique n'a augmente que d'une quantite negligeable et seulement dans le cas des rats traites au phenobarbitone; on n'a observe aucune polyurie. Le synthane est le moins metabolise des agents anesthesiants fluores a base d'ether; son administration ne

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fluoride or urinary inorganic fluoride excretion curves is related to the biological stability of the anaesthetic agent and the solubility of the drug in body tissues, particularly fat. Thus we have formulated a defluorination index which is the product of in vitro inorganic fluoride formation and the oil : gas partition coefficient of the anaesthetic agent (table III). The index for methoxyflurane, 1860, has been assigned a nephrotoxic potential of one. Other drugs have been given proportional values based on their defluorination index. The derived values appear to correlate well with the demonstrated nephrotoxicity of the fluorinated inhalation agents tested to date. Such an index also may have predictive value for new agents. We recently tested dioxychlorane (C 3 H 2 C1 2 F 2 O2)J an experimental inhalation anaesthetic agent of dioxalane ring structure (Denson et al., 1976), in our hepatic microsomal preparation. Inorganic fluoride production was 1.41 nmol per 30 min per milligram of protein. The oil : gas partition coefficient of dioxychlorane is 275 (table III). These data yielded a defluorination index of 387 and a nephrotoxic potential of 0.23, a value considerably greater than that calculated for enflurane, sevoflurane or isoflurane. Because of this, dioxychlorane was not synthesized commercially and in vivo confirmation in our laboratory of in vitro results was not possible.

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produira pas de nephropathie de fluorure inorganique. II a ete prepare un index du potentiel nephrotoxique des agents anesthesiants fluores a l'aide des donnees de production du fluorure obtenues in vitro et des coefficients de separation de l'huile et du gaz. SYNTHANSTOFFWECHSEL: VERGLEICH MIT IN VIVO UND IN VITRO DURCHGEFUHRTER DEFLUORINIERUNG ANDERER HALOGENIERTER KOHLENWASSERSTOFFNARKOTIKA

wurde ein Index des nierenschadigenden Potentials fiir fluorinierte Narkotika formuliert under Verwendung der in vitro erhaltenen Fluoriderzeugungsdaten und der Ol : Gas-Teilungskoeffizienten. METABOLISMO DE SINTANO: COMPARACION CON LA DEFLUORACION IN VIVO Y IN VITRO DE OTROS ANESTESICOS DE HIDROCARBURO HALOGENADO SUMARIO

ZUSAMMENFASSUNG

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Die aerobische Defluorinierung des Inhalationsnarkotikums Synthan wurde mit der von Methoxyfluran, Enfluran und Halothan und mit zwei anderen Narkotika, namlich Isofluran und Sevofluran, verglichen. Synthan und Halothan wurden in vitro, in aus mit Luminal behandelten und Kontrollebern praparierten Mikrosomen, nicht defluoriniert. Die Defluorinierung der anderen Narkotika betrug, in relativer GrOssenordnung: Methoxyfluran > Sevofluran > Enfluran > Isofluran. In vivo war nach 4 Stunden Synthannarkose (1,2%—hOchstzulassige Konzentration) die Urinausscheidung von anorganischem Fluorid nur unbedeutend hoher, und nur in mit Luminal behandelten Ratten; es wurde keine Polyurie beobachtet. Synthan ist das am wenigsten umgesetzte der fluorinierten Athernarkotika; seine Verabreichung hat kein, von anorganischem Fluorid herriihrendes Nierenleiden zur Folge. Es

Se comparo la defluoracion aerobia del agente anestesico inhalado sintano con la de metoxiflurano, enflurano y halotano y con dos otros anestesicos, isoflurano y sevoflurano. In vitro, en microsomas preparados con higados inducidos por fenobarbitona y otros de control, tanto el sintano como el halotano no fueron defluorados. El orden relativo de defluoracion de los demas anestesicos fue metoxiflurano > sevoflurano > enflurano > isoflurano. In vivo, tras 4 h de anestesia con 1,2% de sintano (MAC), la excrecion de fluoruro inorganico urinario aumento solamente en una cantidad trivial y unicamente en ratas tratadas con fenobarbitona; no se observo poliuria. El sintano es el menos metabolizado de los anestesicos de eter fluorado; su administraci6n no causara nefropatia de fluoruro inorganico. Se formulo un indice del potencial nefrotoxico de agentes anestesicos fluorados, utilizando datos de produccion de fluoruro in vitro y coeficientes de reparticion de aceite :gas.