Cysteine Conjugate β-Lyase-Dependent Metabolism of Compound A (2-[fluoromethoxy]-1,1,3,3,3-pentafluoro-1-propene) in Human Subjects Anesthetized with Sevoflurane and in Rats Given Compound A 

2-(Fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (compound 1) was provided by Abbott Laboratories (Abbott Park, IL). 1,1,1,3,3,3-Hexafluoro-2-propanol was obtained from Aldrich Chemical Co. (Milwaukee, WI). 2-(Fluoromethoxy)-3,3,3-trifluoropropanoic acid (compound 11) and 3,3,3-trifluorolactic acid (Figure 1, compound 12) were synthesized by previously reported procedures. [20] ¹⁹F Nuclear magnetic resonance (NMR) spectra were recorded with a Bruker 270-MHz spectrometer operating at 254 MHz for¹⁹F. Chemical shifts, delta, are reported in parts per million. Trifluoroacetamide (0.0 ppm) was used as the external standard for¹⁹F NMR spectra.

Male Fischer 344 rats (weight, 200–220 g; Charles River Laboratories, Wilmington, MA) were used after the protocol for animal use was reviewed and approved by the University Committee on Animal Resources. The rats were anesthetized with ether and killed by cardiac puncture.

The human studies were approved by the Human Subjects Review Boards of the Medical College of Wisconsin and the University of Arizona Medical Center. Participants provided informed consent and were enrolled if they had normal results of a medical history and physical examination and weighed more than 80 kg. None were taking prescription medications or illicit drugs. Subjects were instructed to fast for a minimum of 6 h and to abstain from using tobacco on the morning that anesthesia was administered. The details of the human volunteer studies can be found in our other article in this issue of the journal. [26] 

Thirteen human volunteers were anesthetized with sevoflurane at an end-tidal concentration of 3%(1.25 minimum alveolar concentration) for 8 h at a fresh-gas flow rate of 2 l/min. Urine samples were collected 24 h before and for three consecutive 24-h periods after anesthesia; the urine sample collected in the first 24-h period after anesthetization was used for metabolite analysis. Compound A concentrations in the anesthetic circuit were quantified by gas chromatography as described in our accompanying article. [26] 

Male Fischer 344 rats were given compound A (0.3 mmol/kg) in corn oil (2.5 ml/kg) intraperitoneally. Control animals were given corn oil alone. The rats were housed in metabolism cages, and urine was collected over ice for 24 h. For¹⁹F NMR spectroscopic analysis, urine (1 ml) was centrifuged, and the supernatant was mixed with 100 micro liter deuterium oxide and transferred to a 5-mm NMR tube. To confirm the presence of compound A metabolites, synthetic compounds 11 and 12 (0.2 mg/ml) were added to rat urine, and the samples were analyzed again by¹⁹F NMR spectroscopy.

Human urine samples (25–50 ml) were lyophilized, and the residue (about 200 mg) was suspended in 1 ml deuterium oxide. The suspension was centrifuged to remove insoluble material, and the supernatant was analyzed by¹⁹F NMR spectroscopy. Synthetic compounds 11 and 12 (0.2 mg/ml) were added to the urine samples, which were analyzed again by¹⁹F NMR spectroscopy.

For gas chromatography-mass spectrometry (GC/MS) analysis, human urine samples (25–50 ml) were lyophilized, and the residues (about 200 mg) were suspended in water (1 ml). The suspensions were brought to pH 1.4 with concentrated hydrochloric acid and extracted with ethyl acetate (3 x 2 ml). The organic layers were separated, dried over anhydrous magnesium sulfate, and evaporated to dryness. The dried samples were treated with excess diazomethane in ether, evaporated to dryness, and dissolved in dichloromethane (150 micro liter). The samples (1 micro liter) were analyzed by GC/MS with a Hewlett-Packard 5890 series II gas chromatograph (30 m x 0.25 mm, 0.5-micro meter film thickness, HP wax column; Hewlett-Packard, Wilmington, DE) coupled to a Hewlett-Packard 5972 series II mass selective detector; the injector and the transfer-line temperatures were 150 [degree sign] Celsius and 200 [degree sign] Celsius, respectively. The methyl esters of compounds 11 and 12 were analyzed with a temperature program of 32 [degree sign] Celsius for 1 min followed by a linear gradient of 10 [degree sign] Celsius/min to 200 [degree sign] Celsius.

Rat urine samples (5 ml) were lyophilized, and the residues were suspended in water (1 ml). The samples were prepared and analyzed by GC/MS as described before for human urine samples.

As noted in our accompanying article, the average and maximum inspired compound A concentrations were 27 +/- 7 and 34 +/- 6 ppm (mean +/- SD), respectively. [26],¹⁹F NMR spectroscopic examination of the urine of humans anesthetized with sevoflurane showed resonances assigned to S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-N-acetyl-L-cysteine (Figure 1, compound 6), (E)- and (Z)-S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cyst eine (Figure 1, compound 7), 2-(fluoromethoxy)-3,3,3-trifluoropropanoic acid (compound 11), 3,3,3-trifluorolactic acid (compound 12), and inorganic fluoride (Figure 2(B)). The 19F NMR resonances assigned to compounds 6, 7, 11, and 12 have been reported previously. [19,20]To confirm the presence of metabolites indicative of the metabolism of S-conjugates of compound A by cysteine conjugate beta-lyase, synthetic compounds 11 and 12 were added to the urine samples. Analysis by¹⁹F NMR spectroscopy showed that the resonances assigned to compounds 11 and 12 coresonated with synthetic compounds 11 and 12 (Figure 2(C)). Metabolites formed by beta-lyase-dependent metabolism of S-conjugates of compound A were observed in all human urine samples (n = 13) analyzed by¹⁹F NMR spectroscopy but were not detected in urine collected from humans before administration of sevoflurane (Figure 2(A)).

The presence of compounds 11 and 12 in the urine of humans was confirmed by GC/MS (Figure 3). The total ion chromatograph of the human urine samples after derivatization with diazomethane showed peaks for the methyl esters of compounds 11 (t^R= 9.26 min) and 12 (t^R= 10.90 min;Figure 3(A)). The retention times of the methyl esters of compounds 11 and 12 were identical to the methyl esters of synthetic compounds 11 and 12 (data not shown). Compounds 11 and 12 were observed in all human urine samples (n = 13) analyzed by GC/MS but were not detected in urine collected from humans before administration of sevoflurane. Selected ion monitoring of the methyl ester of compound 11 showed diagnostic ions and fragments; that is, m/z 142 (M - OCH²F) sup +, 131 (M - COOCH³) sup +, and 112 (C³H³OF³) sup +(Figure 3(B)). Similarly, selected ion monitoring of the methyl ester of compound 12 also showed diagnostic ions and fragments; that is, m/z 99 (M - COOCH³) sup +, 91 (C³H⁷O³) sup +, and 80 (C²H²OF²) sup +, as shown in Figure 3(C).

sup 19 F NMR spectroscopic analysis of the urine of rats given compound A also showed the presence of compounds 6, 7, 11, 12, and inorganic fluoride (Figure 4(B)). The presence of compounds 11 and 12 in the urine of rats given compound A was confirmed by adding synthetic compounds 11 and 12 and analysis by¹⁹F NMR spectroscopy, which showed coresonance of metabolically formed and synthetic compounds 11 and 12 (Figure 4(C)). Compound A metabolites were not detected in urine samples of control rats given corn oil alone (n = 3;Figure 4(A)). The presence of compounds 11 and 12 in the urine of rats given compound A was also confirmed by GC/MS (data not shown).

1,1,1,3,3,3-Hexafluoroisopropyl glucuronide, which is formed by the cytochrome P450-dependent oxidation of sevoflurane to 1,1,1,3,3,3-hexafluoroisopropanol followed by glucuronide conjugation, [27–29]was also detected by¹⁹F NMR spectroscopy in the urine of humans exposed to sevoflurane (data not shown). Hydrolysis of the glucuronide with beta-glucuronidase resulted in the release of 1,1,1,3,3,3-hexafluoroisopropanol, which was identified by GC/MS and¹⁹F NMR spectroscopy (data not shown).

The objective of the present experiments was to determine whether compound A undergoes metabolism by the beta-lyase pathway in humans. Accordingly, human volunteers were anesthetized with sevoflurane in a circular system with a carbon dioxide absorbent and thereby exposed to compound A. The formation of compound A was quantified by gas-chromatographic analysis. Urine from humans anesthetized with sevoflurane was analyzed by¹⁹F NMR spectroscopy. To compare the fate of compound A in humans and rats, rats were given compound A, and its metabolic fate was studied by¹⁹F NMR spectroscopy.

sup 19 F NMR spectroscopic examination of urine from humans anesthetized with sevoflurane showed the presence of compounds 6, 7, 11, 12, and inorganic fluoride (Figure 2). The presence of compounds 11 and 12 was confirmed by GC/MS (Figure 3). The same pattern of metabolites was detected in the urine of rats given compound A by intraperitoneal injection (Figure 4), indicating that the metabolic fate of compound A is qualitatively similar in both species. Furthermore, the pattern of metabolites of compound A was the same in rats given compound A by either intraperitoneal injection or inhalation exposure.*

The observed fate of compound A in humans and in rats indicates that it undergoes metabolism by the beta-lyase pathway (Figure 1). The beta-lyase pathway is a well-established bioactivation pathway for a range of nephrotoxic fluorinated alkenes, including chlorotrifluoroethylene, tetrafluoroethylene, hexafluoropropene, and 2-bromo-2-chloro-1,1-difluoroethylene, which is a degradation product of the anesthetic halothane. [30]The beta-lyase pathway involves glutathione S-conjugate formation, hydrolysis of the glutathione S-conjugates to the corresponding cysteine S-conjugates, and bioactivation by renal cysteine conjugate beta-lyase. The formation of compound A-derived mercapturates (compounds 6 and 7) indicates that compound A undergoes glutathione S-conjugate formation to give diasteriomeric S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]glutathione (Figure 1, compound 2) and (E)- and (Z)-S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]glutathione (Figure 1, compound 3). Compounds 2 and 3 have been detected in the bile of rats given compound A. [18]Gamma-glutamyltransferase- and dipeptidase-catalyzed hydrolysis of the compound A-derived glutathione S-conjugates (compounds 2 and 3) would yield diasteriomeric S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl-]-L-cysteine (compound 4) and (E)- and (Z)-S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-L-cysteine (Figure 1, compound 5), respectively. Enzymatic N-acetylation of compounds 4 and 5 would yield the observed mercapturates (compounds 6 and 7), respectively. Compounds 6 and 7 have been detected in the urine of rats given compound A. [19]2-(Fluoromethoxy)-3,3,3-trifluoropropanoic acid (compound 11) is formed by the cysteine conjugate beta-lyase-dependent metabolism of compound 4 to 2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropylthiolate (Figure 1, compound 8), which loses fluoride to give a thioacyl fluoride (Figure 1, compound 10); hydrolysis of compound 10 affords compound 11. The compound A-derived cysteine S-conjugate (compound 5) may undergo beta-lyase-dependent metabolism to 2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenethiolate (Figure 1, compound 9), which may also give compound 10. Again, hydrolysis of compound 10 affords observed compound 11. Compound A-derived cysteine S-conjugates (compounds 4 and 5) are substrates for human renal cytosolic and mitochondrial beta-lyase. [25]Compound 11 has been identified in the urine of rats given compound A and as an in vitro metabolite of compound 4. [19,20]The formation of compound 12 as a degradation product of compound 11 has been reported previously. [20]Inorganic fluoride may arise by reaction of glutathione with compound A to yield compound 3, by the conversion of compound 8 to compound 10, by the hydrolysis of compound 10, and by the conversion of compound 11 to compound 12. In addition, the cytochrome P450-dependent biotransformation of sevoflurane and compound A yields inorganic fluoride. [29,31,32] 

The present findings establish conclusively that compound A undergoes beta-lyase-dependent metabolism in humans. This is the first evidence for the beta-lyase-dependent metabolism of any haloalkene in humans exposed under actual-use conditions and the first evidence for the beta-lyase-dependent metabolism of compound A in humans.

A range of nephrotoxic haloalkenes undergo beta-lyase-dependent bioactivation. [30]Similarly, the fluoroalkene compound A is nephrotoxic when given to rats by inhalation or intraperitoneally. [4,11–17,33]In addition, the established fate of compound A in rats indicates the involvement of the beta-lyase pathway, [15,18–20]and the present study confirms a role for the beta-lyase-dependent metabolism of compound A in rats. Several observations support a role for the beta-lyase pathway in the nephrotoxicity of compound A: Compound A-derived cysteine S-conjugates (compounds 4 and 5) are substrates for rat, human, and nonhuman primate beta-lyase, [25]and the corresponding glutathione S-conjugates (compounds 2 and 3) and cysteine S-conjugate (compound 4) are nephrotoxic in rats. [34]Further, (aminooxy)acetic acid, a beta-lyase inhibitor, and probenecid, an anion transport inhibitor, reduce the toxicity of compound A in rats. [15,16]Although these findings implicate the beta-lyase pathway in the nephrotoxicity of compound A, a report that purports to show that the beta-lyase pathway is not involved has been presented. [33] 

Conclusive evidence for compound A-related renal impairment has not been observed in the human clinical use of sevoflurane. [6,7,26,35–41]The low human renal cytosolic and mitochondrial beta-lyase activities, compared with the rat, may limit the bioactivation of compound A-derived cysteine S-conjugates in humans. [22–25]Increased excretion of some biochemical markers indicative of renal impairment has, however, been reported in human volunteers anesthetized with sevoflurane. [42,43] 

In summary, these studies show that compound A formed as a degradation product of sevoflurane in the anesthesia circuit undergoes beta-lyase-dependent metabolism in humans and that products of the beta-lyase-dependent metabolism of compound A in the rat are qualitatively similar.

The authors thank Scott E. Morgan for collecting and preparing human urine samples and Sandra E. Morgan for preparing the manuscript.

*Male Wistar rats were exposed to 150 ppm compound A for 3 h (Z Tong, RA Iyer, MW Anders, unpublished observations).