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Studies using molecular modeling, genetic engineering, neurophysiology/pharmacology, and whole animals have advanced our understanding of where and how inhaled anesthetics act to produce immobility (minimum alveolar anesthetic concentration; MAC) by actions on the spinal cord. Numerous ligand- and voltage-gated channels might plausibly mediate MAC, and specific amino acid sites in certain receptors present likely candidates for mediation. However, in vivo studies to date suggest that several channels or receptors may not be mediators (e.g., gamma-aminobutyric acid A, acetylcholine, potassium, 5-hydroxytryptamine-3, opioids, and alpha(2)-adrenergic), whereas other receptors/channels (e.g., glycine, N-methyl-D-aspartate, and sodium) remain credible candidates.

Unlabelled: The in vivo potencies of anesthetics correlate with their capacity to suppress the reaction of luciferin with luciferase. In addition, luciferin has structural resemblances to etomidate. These observations raise the issues of whether luciferin, itself, might affect anesthetic requirement, and whether luciferase resembles the site of anesthetic action. Because the polar luciferin is unlikely to cross the blood-brain barrier (we found that the olive oil/water partition coefficient was 100 +/- 36 x 10(-7)), we studied these issues in rats by measuring the effect of infusion of luciferin in artificial cerebrospinal fluid into the lumbar subarachnoidal space and into the cerebral intraventricular space on the MAC (the minimum alveolar anesthetic concentration required to eliminate movement in response to a noxious stimulus in 50% of tested subjects) of isoflurane. MAC in rats given lumbar intrathecal doses of luciferin estimated to greatly exceed anesthetizing doses of etomidate, did not differ significantly from MAC in rats receiving only artificial cerebrospinal fluid into the lumbar intrathecal space. MAC slightly decreased when doses of luciferin estimated to greatly exceed anesthetizing doses of etomidate were infused intraventricularly (P < 0.05). In contrast to the absent or minimal effects of luciferin, intrathecal or intraventricular infusion of etomidate at similar or smaller doses significantly decreased isoflurane MAC. Luciferin did not affect +-aminobutyric acid type A or acetylcholine receptors expressed in Xenopus oocytes. These results suggest that luciferin has minimal or no anesthetic effects. It also suggests that luciferin/luciferase may not provide a good surrogate for the site at which anesthetics act, if this site is on the surface of neuronal cells., Implications: In proportion to their potencies, anesthetics inhibit luciferin's action on luciferase, and luciferin structurally resembles the anesthetic etomidate. However, in contrast to etomidate, luciferin given intrathecally or into the third cerebral ventricle does not have anesthetic actions, and it does not affect +-aminobutyric acid or acetylcholine receptors in vitro. Luciferase may not provide a good surrogate for the site at which anesthetics act.

Unlabelled: Meyer and Overton suggested that anesthetic potency correlates inversely with lipophilicity. Thus, MAC times the olive oil/gas partition coefficient equals an approximately constant value of 1.82 +/- 0.56 atm (mean +/- SD). MAC is the minimum alveolar concentration of anesthetic required to eliminate movement in response to a noxious stimulus in 50% of subjects. Although MAC times the olive oil/gas partition coefficient also equals an approximately constant value for normal alkanols from methanol through octanol, the value (0.156 +/- 0.072 atm) is 1/10th that found for conventional anesthetics. We hypothesized that substitution of sulfur for the oxygen in n-alkanols would decrease their saline/gas partition coefficients (i.e., decrease polarity) while sustaining lipid/gas partition coefficients. Further, we hypothesized that these changes would produce products of MAC times olive oil partition coefficients that approximate those of conventional anesthetics. To test these predictions, we measured MAC in rats, and saline and olive oil solubilities for the series H(CH(2))(n)SH, comparing the results with the series H(CH(2))(n)OH for compounds having three to six carbon atoms. As hypothesized, the alkanethiols had similar oil/gas partition coefficients, 1000-fold smaller saline gas partition coefficients, and MAC values 30 times greater than for comparable alkanols. Such findings are consistent with the notion that the greater potency of many alkanols (greater than would be predicted from conventional inhaled anesthetics and the Meyer-Overton hypothesis) results from their greater polarity., Implications: The in vivo anesthetic potency of alkanols and alkanethiols correlates with their lipophilicity and hydrophilicity.

Unlabelled: All series of volatile and gaseous compounds contain members that can produce anesthesia, as defined by the minimum alveolar anesthetic concentration (MAC) required to produce immobility in response to a noxious stimulus. For unhalogenated n-alkanes, cycloalkanes, aromatic compounds, and n-alkanols, potency (1 MAC) increases by two-to threefold with each carbon addition in the series (e.g., ethanol is twice as potent as methanol). Total fluorination (perfluorination) of n-alkanes essentially eliminates anesthetic potency: only CF4 is anesthetic (MAC = 66.5 atm), which indicates that fluorine atoms do not directly influence sites of anesthetic action. Fluorine may enhance the anesthetic action of other moieties, such as the hydrogen atom in CHF3 (MAC = 1.60 atm), but, consistent with the notion that the fluorine atoms do not directly influence sites of anesthetic action, adding -(CF2)n moieties does not further increase potency (e.g., CHF2-CF3 MAC = 1.51 atm). Similarly, adding -(CF2)n moieties to perfluorinated alkanols (CH2OH-[CF2]nF) does not increase potency. However, adding a second terminal hydrogen atom (e.g., CHF2-CHF2 or CH2OH-CHF2) produces series in which the addition of each -CF2- "spacer" in the middle of the molecule increases potency two- to threefold, as in each unhalogenated series. This parallel stops at four or five carbon atom chain lengths. Further increases in chain length (i.e., to CHF2[CF2]4CHF2 or CHF2[CF2]5CH2OH) decrease or abolish potency (i.e., a discontinuity arises). This leads to our hypothesis that the anesthetic moieties (-CHF2 and -CH2OH) interact with two distinct, spatially separate, sites. Both sites must be influenced concurrently to produce a maximal anesthetic (immobility) effect. We propose that the maximal potency (i.e., for CHF2[CF2]2CHF2 and CHF2[CF2]3CH2OH) results when the spacing between the anesthetic moieties most closely matches the distance between the two sites of action. This reasoning suggests that a distance equivalent to a four or five carbon atom chain, approximately 5 A, separates the two sites., Implications: Volatile anesthetics may produce immobility by a concurrent action on two sites five carbon atom lengths apart.

Unlabelled: Previous work demonstrates that various anesthetics enhance the effect of gamma-aminobutyric acid (GABA), and this enhancement has been proposed as an explanation for how anesthetics cause anesthesia. This explanation extends to both fluorinated and unfluorinated alkanols. In the present study, we tested the capacity of fluorinated alkanols to enhance the function of the GABA(A) receptors expressed in Xenopus oocytes. CF3CH2OH, CF3(CF2)2CH2OH and CF3(CF2)4CH2OH potentiated GABA(A) receptor function, but CF3(CF2)5CH2OH did not. The degree of potentiation decreased in proportion to the chain length of the alkanols. These findings were not specific for receptors expressed in oocytes, as similar results were obtained with muscimol-stimulated 36Cl- uptake using mouse brain membrane vesicles. Although CF3(CF2)5CH2OH has been reported to enhance the capacity of desflurane to produce immobility in vivo, in our in vitro studies, this compound reduced potentiation of GABA-gated response by anesthetics such as isoflurane, enflurane, and pentobarbital. CHF2(CF2)5CH2OH, which has in vivo anesthetic effects, also failed to potentiate GABA(A) receptor function. These results indicate that the GABA(A) receptor is not the only receptor affected by fluorinated alkanols and that other receptors contribute to the capacity of alkanols to produce immobility. In particular, CF3(CF2)5CH2OH and CF3CH2OH inhibited N-methyl-D-aspartate receptor-mediated responses, which raises the possibility that this receptor is important for actions of fluorinated alkanols., Implications: We find a consistent parallel between the immobilization produced by fluorinated alkanols and their actions on N-methyl-D-aspartate receptors but do not find a consistent parallel between immobilization and effects on gamma-aminobutyric acid type A receptors. Thus, we suggest that N-methyl-D-aspartate, but not gamma-aminobutyric acid type A, receptors may mediate the capacity of anesthetics to produce immobilization.

Unlabelled: The Meyer-Overton hypothesis predicts that the potency of conventional inhaled anesthetics correlates inversely with lipophilicity: minimum alveolar anesthetic concentration (MAC) x the olive oil/gas partition coefficient equals a constant of approximately 1.82 +/- 0.56 atm (mean +/- SD), whereas MAC x the octanol/gas partition coefficient equals a constant of approximately 2.55 +/- 0.65 atm. MAC is the minimum alveolar concentration of anesthetic required to eliminate movement in response to a noxious stimulus in 50% of subjects. Although MAC x the olive oil/gas partition coefficient also equals a constant for normal alkanols from methanol through octanol, the constant (0.156 +/- 0.072 atm) is one-tenth that found for conventional anesthetics, whereas the product for MAC x the octanol/gas partition coefficient (1.72 +/- 1.19) is similar to that for conventional anesthetics. These normal alkanols also have much greater affinities for water (saline/gas partition coefficients equaling 708 [octanol] to 3780 [methanol]) than do conventional anesthetics. In the present study, we examined whether fluorination lowers alkanol saline/gas partition coefficients (i.e., decreases polarity) while sustaining or increasing lipid/gas partition coefficients, and whether alkanols with lower saline/gas partition coefficients had products of MAC x olive oil or octanol/gas partition coefficients that approached or exceeded those of conventional anesthetics. Fluorination decreased saline/gas partition coefficients to as low as 0.60 +/- 0.08 (CF3[CF2]6CH2OH) and, as hypothesized, increased the product of MAC x the olive oil or octanol/gas partition coefficients to values equaling or exceeding those found for conventional anesthetics. We conclude that the greater potency of many alkanols (greater than would be predicted from conventional inhaled anesthetics and the Meyer-Overton hypothesis) is associated with their greater polarity., Implications: Inhaled anesthetic potency correlates with lipophilicity, but potency of common alkanols is greater than their lipophilicity indicates, in part because alkanols have a greater hydrophilicity--i.e., a greater polarity.

Unlabelled: How some noble and diatomic gases produce anesthesia remains unknown. Although these gases have apparently minimal capacities to interact with a putative anesthetic site, xenon is a clinical anesthetic, and argon, krypton, and nitrogen produce anesthesia at hyperbaric pressures. In contrast, neon, helium, and hydrogen do not cause anesthesia at partial pressures up to their convulsant thresholds. We propose that anesthetic sites influenced by noble or diatomic gases produce binding energies composed of London dispersion and charge-induced dipole energies that are sufficient to overcome the concurrent unfavorable decrease in entropy that occurs when a gas molecule occupies the site. To test this hypothesis, we used the x-ray diffraction model of the binding site for Xe in metmyoglobin. This site offers a positively charged moiety of histidine 93 that is 3.8 A from Xe. We simulated placement of He, Ne, Ar, Kr, Xe, H2, and N2 sequentially at this binding site and calculated the binding energies, as well as the repulsive entropy contribution. We used free energies obtained from tonometry experiments to validate the calculated binding energies. We used partial pressures of gases that prevent response to a noxious stimulus (minimum alveolar anesthetic concentration [MAC]) as the anesthetic endpoint. The calculated binding energies correlated with binding energies derived from the in vivo (ln) data (RTln[MAC], where R is the gas constant and T is absolute temperature) with a slope near 1.0, indicating a parallel between the Xe binding site in metmyoglobin and the anesthetic site of action of noble and diatomic gases. Nonimmobilizing gases (Ne, He, and H2) could be distinguished by an unfavorable balance between binding energies and the repulsive entropy contribution. These gases also differed in their inability to displace water from the cavity., Implications: The Xe binding site in metmyoglobin is a good model for the anesthetic sites of action of noble and diatomic gases. The additional binding energy provided by induction of a dipole in the gas by a charge at the binding site enhanced binding.

Unlabelled: We assessed the anesthetic properties of helium and neon at hyperbaric pressures by testing their capacity to decrease anesthetic requirement for desflurane using electrical stimulation of the tail as the anesthetic endpoint (i.e., the minimum alveolar anesthetic concentration [MAC]) in rats. Partial pressures of helium or neon near those predicted to produce anesthesia by the Meyer-Overton hypothesis (approximately 80-90 atm), tended to increase desflurane MAC, and these partial pressures of helium and neon produced convulsions when administered alone. In contrast, the noble gases argon, krypton, and xenon were anesthetic with mean MAC values of (+/- SD) of 27.0 +/- 2.6, 7.31 +/- 0.54, and 1.61 +/- 0.17 atm, respectively. Because the lethal partial pressures of nitrogen and sulfur hexafluoride overlapped their anesthetic partial pressures, MAC values were determined for these gases by additivity studies with desflurane. Nitrogen and sulfur hexafluoride MAC values were estimated to be 110 and 14.6 atm, respectively. Of the gases with anesthetic properties, nitrogen deviated the most from the Meyer-Overton hypothesis., Implications: It has been thought that the high pressures of helium and neon that might be needed to produce anesthesia antagonize their anesthetic properties (pressure reversal of anesthesia). We propose an alternative explanation: like other compounds with a low affinity to water, helium and neon are intrinsically without anesthetic effect.

Oil/saline partition coefficients for inhaled compounds often are defined by the ratio of the separately determined oil/gas and saline/gas partition coefficients. This approach assumes that the concurrent presence of oil with saline has no effect on the characteristics of either solvent. To test this assumption, we measured the oil/gas and saline/gas partition coefficients for CF3(CCIF)2CF3 and 1,2-dichloroperfluorocyclobutane (C4Cl2F6) separately and with the two phases mixed in a common container. We chose these compounds because they have radically different oil/gas and saline/gas partition coefficients and thus would provide a severe test of the assumption. For CF3(CCIF)2CF3, olive oil/saline partition coefficients were, respectively, 13,200 and 13,300 when measured separately and in mixed phases, and the octanol/saline partition coefficients were 19,200 and 18,100. Similarly, olive oil/saline partition coefficients for 1,2-dichloroperfluorocyclobutane were 3660 and 3500 when measured separately and in mixed phases, respectively, and the octanol/saline partition coefficients were 5140 and 4560. We conclude that differences between separate and mixed-phase determinations of ratios are small or nonexistent.

The authors are studying the molecular details of the process that begins with hepatic metabolism of halogenated inhalation anesthetics and ends with hepatic necrosis. In previous studies they have shown that the halothane-free radical produced by UV-irradiation is identical to that produced during reductive metabolism of halothane by hepatic cytochrome P-450. In the present study, the authors have examined a mechanism by which free radicals may propagate damage in the endoplasmic reticulum of liver cells. The 1-chloro-2,2,2-trifluoroethyl free radical produced by UV-irradiation of halothane can abstract a hydrogen radical from arachidonic acid to yield 2-chloro-1,1,1-trifluoroethane and an arachidonic acid-free radical. The arachidonic acid-free radical reacts with molecular oxygen to form 5- and 15-hydroperoxyeicosatetraenoic acid. There is considerable evidence that the peroxidation process that we studied in the model system will be similar when the arachidonic acid is an acyl chain on a membrane phospholipid and the free radicals are generated metabolically. The authors suggest that these hydroperoxides may be toxic by acting as intermediates in the pathway of leukotriene production as well as by direct oxidation of membrane components.

Nitrous oxide labeled with a stable heavy nitrogen isotope was used for in-vitro studies of nitrous oxide metabolism in man and rat. At 5 per cent oxygen tension, which is comparable to normal oxygen tension in the intestine in vivo, each gram of intestinal contents during a 16-hr in-vitro incubation produced 47 +/- 13 nmol of molecular nitrogen for the rat and 103 +/- 17 nmol for man. Active reductive metabolism of nitrous oxide by intestinal contents was significantly inhibited by antibiotics and by 20 per cent oxygen tension. It is suggested that the reduction of nitrous oxide to nitrogen may proceed through a single-electron transfer process with formation of free radicals. Under these circumstances, metabolism of nitrous oxide could produce toxic intermediates, even thought the end-metabolite is inert.

The molecular motion and distribution of the inhalation anesthetic halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) in a phospholipid bilayer model nerve membrane preparation was studied using fluorine nuclear magnetic resonance. Bilayers containing stable free radicals at known depths were studied to measure possible localization of halothane within certain areas of the bilayer. Bilayer suspensions containing manganese ions in the aqueous phase were used to test the partition of halothane between the aqueous and lipid phases. It was found that halothane rapidly achieves complete exchange throughout the bilayer and the surrounding aqueous phase. The results provide experimental evidence against the formation of anesthetic clathrates hypothesized by Pauling and Miller in their theories of anesthesia.

Binding of halothane metabolites to rat liver histones was investigated after in vivo administration of 14C-halothane. Animals were injected with either a mixture of triiodothyronine, glucagon and heparin (TGH) to stimulate liver growth or with saline as a control. Twenty-four hours later, animals were administered 14C-halothane and maintained at 8--10 per cent O2 for 6 hours. Detergent washed nuclei from liver homogenates were subfractionated to allow quantitative measurements of 14C-halothane binding to histones. Although our studies suggest that much of the previously reported binding of halothane metabolites to major cell fractions was a result of redistribution of endoplasmic reticulum components during isolation procedures, carefully controlled experiments demonstrated that the radioactivity associated with histones could not be due to residual microsomal lipid. Of the initial 132 mumol of 14C-halothane administered, 1.1 mumol remained as nonvolatile metabolites in the liver homogenate and 25 pmol were associated with purified histones. This corresponds to approximately one halothane moiety per 15,000 histone molecules. No significant binding to liver cell RNA or DNA was observed. With this low level of histone modification and lack of convincing evidence of halothane metabolite binding to hepatic DNA or RNA, it is unlikely that significant alteration of the genome occurs after exposure to halothane.

The presence of two volatile halothane metabolites, 2-chloro-1,1,1-trifluoroethane (CF3CH2Cl) and 2-chloro-1,1-difluoroethylene (CF2CHCl), and a metabolite-decomposition product, 2-bromo-2-chloro-1,1-difluoroethylene (CF2CBrCl), were identified by gas chromatography-mass spectrometry in exhaled gases of 16 patients anesthetized with halothane in nonrebreathing, semiclosed and totally closed anesthesia circuits. No significant differences in concentrations of CF3CH2Cl and CF2CHCl were found relative to the anesthesia circuits used. CF2CBrCl could not be identified in the expired gases of patients anesthetized with a nonrebreathing circuit (Bain), but was present in gases recovered from both semiclosed and totally closed circuits. Under totally closed-circuit rebreathing conditions, the concentration of CF2CBrCl increased to 4-5 ppm, indicating significant breakdown of halothane by the soda lime. Possible pathways for formation of the two metabolites and the metabolite-decomposition product are presented, as well as clinical implications of these findings.

The urinary metabolites of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) were investigated in five individuals given trace doses (25 muCi), and in three individuals given large doses (1 mCi) of radioactively labeled 14C-halothane. The latter were donor subjects for heart transplant operations. Separation of the nonvolatile urinary metabolites of halothane was accomplished by chemical extraction, electrophoresis, ion-exchange and high-pressure liquid chromatography, and gas chromatography. Identification of the individual metabolites was by nuclear magnetic resonance and mass spectrometry. Three major metabolites were identified: trifluoroacetic acid, N-trifluoroacetyl-2-aminoethanol, and N-acetyl-S-(2-bromo-2-chloro-1,1-difluoroethyl)-L-cysteine. Smaller unidentified radioactive peaks were also found. The presence of both ethanolamide and cysteine conjugates of halothane is of concern. These urinary products imply the presence of reactive intermediates. The conjugation of such intermediates to proteins and phospholipids may give rise to the high-molecular-weight covalently bound metabolites demonstrated to be present in the liver following halothane anesthesia. Elucidation of the structures of the urinary metabolites provides information important to an understanding of halothane metabolism and its potential hepatotoxicity.

Hypoxia, phenobarbital induction, and halothane anesthesia have been implicated in the pathogenesis of hepatotoxicity in the rat model. However, a controversy exists over the role of halothane in liver injury; does it act by reducing hepatic blood flow, thereby inducing hypoxia, or do its metabolites initiate the injury? These variables are difficult to separate during in vivo halothane exposure. In the present experiments, effects of halothane on hepatic perfusion were eliminated by exposing confluent monolayers of hepatocytes isolated from Fisher 344 rats livers, both with and without phenobarbital pretreatment, to 1.5% halothane or 2.0% isoflurane in 1%, 2%, or 4% (control) oxygen. Isoflurane exposure was included for a control of anesthetic effects on hepatocytes, because it is known to be metabolized minimally and probably is not associated with hepatic dysfunction. Oxygen levels were chosen to approximate those that may occur in the liver in vivo. Cell death was assayed via aspartate aminotransferase (AST) release, both immediately following a 2-h oxygen +/- anesthetic exposure and 6 h post-exposure. Per cent cell death data were analyzed using multiple regression techniques. Results obtained immediately, and 6 h after, exposure demonstrate that low oxygen levels, halothane, and phenobarbital were each highly significant factors (P less than .001) in relation to cell death, in agreement with the halothane-phenobarbital-hypoxia rat model. A toxic effect of isoflurane was not observed under identical experimental conditions. The results of the study clearly indicate that the origin of cell death in hepatocyte monolayers is multi-factorial; hypoxia, phenobarbital induction, and halothane exposure each contribute to the hepatocyte damage observed in our in vitro model.

This paper relates research on anesthetic effects on lipid membrane systems to mechanisms of neural function. A unitary theory of anesthesia based on anesthetic-induced changes in fluid-solid-phase separations in the lipid region of nerve membranes is presented. It is suggested that anesthetics act by fluidizing nerve membranes to a point where critical lipid regions no longer contain phase separations. As a consequence, the membranes are less able to facilitate the conformational changes in proteins that may be the basis for such membrane events as ion gating, synaptic transmitter release, and transmitter binding to receptors. It is proposed that the anesthetic-modified phase separation behavior of the membrane may alter neural function by a combination of the following effects: inhibition of conformational changes of intrinsic membrane proteins; prevention of the association of protein subunits to form polymeric ion channels; depression of transmitter release by preventing fusion of vesicles containing synaptic transmitter with the membrane of the presynaptic terminal.