Kohyu Fujii

Reaction of sevoflurane and its degradation products with soda lime : toxicity of the byproducts

Sevoflurane previously has been reported to undergo extensive degradation in the presence of soda lime. To more completely characterize the extent and significnce of this reaction, we studied degradation of sevoflurane with and without soda lime, as well as the toxicity and mutagenicity of the degradation products. Two degradation products detected were CF2 = C(CF3)OCH2F (compound A) and CH3OCF2CH(CF3)OCH2F (compound B). During circulation of 1%, 2%, and 3% sevoflurance in a closed anesthesia circuit for 8 h, peak concentrations of compound A were 13.3 +/- 0.27, 30.2 +/- 0.10, and 42.1 +/- 1.07 ppm at 2 h, respectively. The concentrations of compound B did not exceed 2 ppm. The temperature of the soda lime was 43.3 +/- 2.8 degrees C at 1 h and increased gradually to 47.9 +/- 1.5 degrees C after 8 h. In closed flasks with soda lime, the magnitude of the decrease in sevoflurance concentrations (3%) and of the increase in compound A concentrations was temperature dependent. The peak concentrations of compound A at 23 degrees C, 37 degrees C, and 54 degrees C were 32.8 +/- 6.8 at 2 h, 46.6 +/- 1.0 at 0.5 h, and 78.5 +/- 2.3 ppm at 0.5 h, respectively. The LC50 (50% lethal concentration) of compound A in Wistar rats was 1,090 ppm in males and 1,050 ppm in females exposed for 1 h. The LC50 was 420 ppm in males and 400 ppm in females exposed for 3 h. The chronic toxicity of compound A in Wistar rats was studied by exposing rats 24 times, for 3 h each, to initial concentrations of 30, 60, or 120 ppm in a ventilated chamber. At all concentrations, there were no apparent effects other than a loss of body weight in females (120 ppm) on the final day (P < 0.01). Compound A did not induce mutation on the reverse (Ames) test at less than 2,500 micrograms/dish (culture medium 2.7 ml) with activation by S-9 mixture, and below 1,250 micrograms/dish (culture medium 2.7 ml) without activation, in four strains of S. typhimurium and in 1 strain of E. coli. Exposure of fibroblasts to 7,500 ppm of compound A for 1 h, compound A did not induce structural change. In a study of acute toxicity of compound B, there was no toxicity in Wistar rats after 3 h of exposure at 2,400 ppm. The reverse (Ames) test for compound B was negative at 625-1,250 micrograms/dish.(ABSTRACT TRUNCATED AT 400 WORDS)

Volatile Metabolites of Halothane in the Rabbit

To date, carbon dioxide is the only volatile metabolite that has been identified to result from the biotransformation of halothane. This study was undertaken to determine whether other volatile metabolites might be formed. Expiratory gas from four rabbits given halothane by inhalation and from three rabbits into which the halothane was injected intraperitoneally was analyzed by gas chromatography. Qualitative analysis of the metabolites was made by injecting 50–70 µ1 of the expired halothane condensed in an ultralow-temperature device (−80 C) attached to the mass spectrometer. Gas chromatography revealed two volatile metabolites between the air peak and the halothane peak. They were identified by mass spectra to be CF2:CHCl and CF3CH2Cl. These volatile metabolites appeared immediately after the beginning of anesthesia. The present investigation suggests the possible existence of a previously unknown metabolic pathway of defluorination and debromination occurring in the early stage of halothane biotransformation. These volatile metabolites may be toxic, highly reactive intermediates that undergo further biotransformation.

In vivo spin-trap study on anaerobic dehalogenation of halothane

Radical formation in vivo by anaerobic dehalogenation of halothane is described in this paper. The radicals were stabilized by spin-trapping and assayed by electron spin resonance spectrometry. The radical adducts were formed by inhalation of halothane in vivo and increased with decrease in inspired oxygen concentration. Following administration of the spin-trap, the expired concentration of CF2CHCl and CF3CH2Cl which are the anaerobic metabolites of halothane decreased, but bilious trifluoroacetate which are aerobic did not change. These results strongly suggest that radical intermediates are produced in anaerobic dehalogenation of halothane to CF2CHCl and CF3CH2Cl.

A possible role of cytochrome P450 in anaerobic dehalogenation of halothane

Abstract NADPH reduced rabbit liver microsomal enzymes catalyzed anaerobic dehalogenation of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) to produce CF2CHCl and CF3CH2Cl. Anaerobic dehalogenation was optimal at pH7.4 and was blocked by either oxygen or carbon monoxide. The degree of inhibition of anaerobic dehalogenation by carbon monoxide was closely correlated to the proportion of carbon monoxide complex of cytochrome P450. Anaerobic dehalogenation was enhanced by pretreatment of the animals with phenobarbital but not with methylcholanthrene.

Reaction between imidazolineoxil N-oxide (carboxy-ptio) and nitric oxide released from cultured endothelial cells: Quantitative measurement of nitric oxide by ESR spectrometry

The performance of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide for the monitoring agent of nitric oxide was investigated. The agent (125-500 microM) was mixed with equal volume of nitric oxide solution, and aliquots of the mixture were applied to ESR spectroscopy. ESR spectra of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl, a product of the agent reacted with nitric oxide, were observed. A linear relationship was observed between the amplitude of the signal and concentrations of nitric oxide up to 80 microM. Endothelial cells cultured on microcarriers were packed in a column, perfused with Krebs solutions and the effluent was mixed to the agent. The same ESR spectra were obtained and amplitude of the signal was increased by bradykinin (3-300 nM), decreased by preincubation of NG-monomethyl-L-arginine (3-100 microM) and reversed by following incubation of L-arginine (100 microM).

Inhibitory effect of sevoflurane on nitric oxide release from cultured endothelial cells

We investigated the effect of sevoflurane (fluoromethyl-2,2,2-trifluoro-1-(trifluoromethyl) ethylether) on intracellular calcium concentration ([Ca2+]i) and nitric oxide (NO) release from cultured porcine aortic endothelial cells using fura-2 fluorometry, and direct (ESR spectrometry with NO-trapping by 2-(4-carboxyphenyl)-4,4,5,5-tetramiethylimidazoline-1-oxyl 3-oxide) or indirect (nitrite accumulation measured by Greiss reaction) NO measurement. Sevoflurane alone did not change resting [Ca2+]i, but diminished bradykinin-induced transient increase in [Ca2+]i in a concentration-dependent manner. The inhibitory effect of sevoflurane on bradykinin-induced transient rise in [Ca2+]i was larger than that of a non-selective Ca2+ channel blocker (CO2+). Application of sevoflurane following bradykinin-evoked [Ca2+]i transient diminished [Ca2+]i significantly, while bradykinin B2 receptor antagonist (D-Arg-[Hyp3, Thi5,8, D-Phe7] bradykinin) or CO2+ abolished it. Sevoflurane impaired nitrite accumulation stimulated by bradykinin, and reduced the amount of NO released from endothelial cells. Our results indicate that the negative effect of sevoflurane appears to be due to the inhibition of bradykinin-induced Ca2+ efflux from endoplasmic stores and Ca2+ influx through membrane Ca2+ channels.

Urinary excretion of hexafluoroisopropanol glucuronide and fluoride in patients after sevoflurane anaesthesia.

Abstract— The excretion of sevoflurane metabolites in the urine collected every 12 h after sevoflurane anaesthesia was measured by ion exchange chromatography. A metabolite, which was converted on incubation with glucuronidase to hexafluoroisopropanol was detected in the urine. The maximum excretion was found in the first 12 h after anaesthesia, none was found in the last collection 3 days after anaesthesia. The excretion half‐life for the metabolite was calculated to be 55 h. A significant increase in the urinary excretion of organic and inorganic fluoride was also observed during the first 12 h after anaesthesia. The cumulative organic and inorganic fluoride excretion in the 3 days after sevoflurane anaesthesia was 1588 and 856 μmol, respectively (ratio = 1·85). The excreted half‐lives for organic and inorganic fluoride were calculated to be 4028 and 2069 min, respectively. Our study showed that a hexafluoroisopropanol glucuronide is excreted in the urine, and the major part of urinary metabolites of sevoflurane, organic and inorganic fluoride, are excreted within 2 days of sevoflurane inhalation in man.

Self-limiting enhancement by nitric oxide of oxygen free radical-induced endothelial cell injury: evidence against the dual action of NO as hydroxyl radical donor/scavenger

1 The effects of oxygen free radical scavengers and endothelial cell‐derived nitric oxide (EDNO) on the death of porcine cultured aortic endothelial cells exposed to exogenous superoxide‐ [xanthine (0.4 mM)/xanthine oxidase (0.04 unit ml−1) + diethylenetriaminepentaacetic acid (DTPA, 10 μm)] or hydroxyl radical‐generating system(s) [superoxide generating system + ferric iron (Fe3+, 0.1 mM) or peroxynitrite (0–100 μm)] have been evaluated. 2 Spin trapping studies using 5,5‐dimethyl‐1‐pyrroline‐N‐oxide (DMPO) with electron paramagnetic resonance spectrometry were also conducted to determine qualitatively the oxidant species generated by the oxidant generating systems. 3 Endothelial cell injury provoked by the exogenous superoxide generating system was inhibited by catalase, DTPA and a hydroxyl radical scavenger (dimethyl sulphoxide, DMSO), but not by superoxide dismutase (SOD). Addition of Fe3+ to the superoxide generating system enhanced the cell injury. These suggested that the direct cytotoxicity of exogenous superoxide is limited, and that endogenous transition metal‐dependent hydroxyl radical formation is involved in the cell injury. 4 An inhibitor of the constitutive NO‐pathway, NG‐monomethyl‐L‐arginine, did not influence cell injury induced by the superoxide generating system, suggesting that basal NO production is not responsible for the cytotoxicity. 5 Stimulation of endothelial cells with bradykinin enhanced cell injury provoked by the exogenous superoxide generating system, but not by the exogenous hydroxyl radical generating system. The enhancement by bradykinin was inhibited by NG‐monomethyl‐L‐arginine and bradykinin B2‐receptor antagonist, D‐Arg‐[Hyp3, Thi5, 8, D‐Phe7] bradykinin, suggesting that an interaction of NO with superoxide is involved in the enhanced cytotoxicity. A possible intermediate of this reaction, peroxynitrite, also caused endothelial cell injury in a concentration‐dependent manner. 6 The modulatory effects of NO on hydroxyl radical‐like activity (= formaldehyde production) from the superoxide generating system was also evaluated in a cell‐free superoxide/NO generating system, consisting of xanthine/xanthine oxidase, DTPA, DMSO, and various amounts of a spontaneous NO generator, sodium nitroprusside (SNP) and were compared with those of Fe3+. At doses up to 10 μm, SNP concentration‐dependently increased the formaldehyde production while the higher concentrations of SNP decreased. The maximum amount of formaldehyde produced by SNP was 5 fold less than that produced by Fe3+ (0.1 mM). Peroxynitrite‐induced formaldehyde formation was concentration‐dependently inhibited by SNP. 7 We conclude that agonist‐stimulated but not basal NO production acts as cytotoxic hydroxyl radical donor as well as the endogenous transition metal when endothelial cells are exposed to exogenous superoxide anion, while the modulatory effect of EDNO is limited by a secondary reaction with hydroxyl radicals.

Anaerobic dehalogenation of halothane by reconstituted liver microsomal cytochrome P-450 enzyme system☆

Abstract Cytochrome P-450 from liver microsomes of phenobarbital-treated rabbits catalyzed anaerobic dehalogenation of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) when combined with NADPH and NADPH-cytochrome P-450 reductase. Cytochromes P-450B1 and P-448 from liver microsomes of untreated rabbits were less active. Triton X-100 accelerated the reaction. Unlike anaerobic dehalogenation of halothane in microsomes, the major product was 2-chloro-1,1,1-trifluoroethane and 2-chloro-1,1-difluoroethylene was negligible. These products were not detected under aerobic conditions, and dehalogenation activity was inhibited by carbon monoxide, phenyl isocyanide and metyrapone.

Biotransformation of isoflurane: urinary and serum fluoride ion and organic fluorine

The serum and urinary concentrations of fluorinated metabolites of isoflurane after inhalation of three different concentrations of isoflurane were studied in 18 ASA physical status 1 or 2 patients, scheduled for orthopedic or otolaryngeal surgery. Isoflurane was administered for 60 min during fentanyl-nitrous oxide-oxygen, and its end-tidal concentration was maintained at 0.3, 0.6, or 1.15% (groups I, II, and III). The organic fluorine was determined by combustion and fluoride ions were analyzed by ion chromatography. The amounts were expressed in terms of fluoride ion. The concentrations of serum fluoride ion and organic fluorine increased significantly 15 min after the onset of inhalation of isofluorane. The mean peak values of fluoride ions were 3.8 ± 1.1, 3.9 ± 1.4, and 4.2 ± 0.9 mole/1 (M ± SD) in patients in groups I, II, and III, respectively. The half-lives of fluoride ion and of organic fluorine as metabolites of isoflurane, calculated from the amounts excreted in urine, were 36 h and 41 h, respectively. The cumulative amounts of fluoride ion and organic fluorine excreted up to the 6th postoperative day were 548 ± 230 and 785 ± 452 moles in group I, 594 ± 138 and 1,378 ± 807 moles in group II, and 1,302 ± 496 and 728 ± 265 moles (M ± SD) in group III, respectively. The urinary excreted fluoride ion increased in proportion to the dose of isoflurane and approximately 1.3 mmol was excreted per 1 MAC X hour inhalation of isoflurane. The authors concluded that isoflurane might be biotransformed to a greater extent than reported previously, although the serum fluoride ion level was found to be low.