Issaku Ueda

Partition equilibrium of inhalation anesthetics and alcohols between water and membranes of phospholipids with varying acyl chain-lengths

From the depression of the phase-transition temperature of phospholipid membranes, the partition coefficients of inhalation anesthetics (methoxyflurane, halothane, enflurane, chloroform and diethyl ether) and alcohols (benzyl alcohol and homologous n-alcohols up to C = 7) between phospholipid vesicle membranes and water were determined. The phospholipids used were dimyristoyl-, dipalmitoyl- and distearoylphosphatidylcholines. It was found that the difference in the acyl chain length of the three phospholipids did not affect the partition coefficients of the inhalation anesthetics and benzyl alcohol. The actions of these drugs are apparently directed mainly to the interfacial region. In contrast, n-alcohols tend to bind more tightly to the phospholipid vesicles with longer acyl chains. The absolute values of the transfer free energies of n-alcohols increased with the increase of the length of the alkyl chain of the alcohols. The increment was 3.43 kJ per each carbon atom. The numerical values of the partition coefficients are not identical when different expressions for solute concentrations (mole fraction, molality and molarity) are employed. The conversion factors among these values were estimated from the molecular weights and the partial molal volumes of the phospholipids in aqueous solution determined by oscillation densimetry.

Kinetic and Thermodynamic Aspects of the Mechanism of General Anesthesia in a Model System of Firefly Luminescence in Vitro

Cell-free firefly-tail extract emits light when ATP is added. The flash intensity follows first-order kinetics. Methoxyflurane, chloroform, halothane, enflurane, and fluroxene inhibited this reaction. Tensions which inhibited flash intensity 50 per cent were 2.1 X 10-3 atm, 9.0 X 10-4 atm, 1.04 X 10-2 atm, 2.0 X 10-2 atm, and 5.1 X 10-2 atm, respectively. These values correlate better with oil/gas partition coefficients than with hydrate-dissociation pressures. Thermodynamic analysis showed that the inhibition mechanism of anestheties is identical to reversible thermal inactivation of the enzyme. In this reaction the origital “folded” enzyme became inactive by transformation into the “unfolded” or expanded type. Inhibition was accompanied by a high heat of reaction (ΔH: −80,000 to −89,900 cal/mol) and a large entropy change (ΔS: −277 to −320 entropy units), and was non-competitive with ATP binding. The magnitudes of the heat of reaction and the entropy change support the theory that anesthetics act at a hydrophobic site of luciferase, inducing a structural change of the enzyme to form the “unfolded” type. The correlation between the ED20s of the anesthetics and their oil/gas partition coefficients together with the enzyme kinetic data indicates that the site of anesthetic action may be hydrophobic.

Dissociation constants of local anesthetics and their temperature dependence.

Dissociation constants are generally obtained by potentiometric titration. If the dissociated and undissociated molecules show differences in the optical absorption wavelength, photometric methods may be used also. Although aromatic amine local anesthetics show UV absorption from the aromatic moiety, the anesthetics fail to show a detectable spectral change at different pH values, perhaps because of the remoteness of the benzene ring from the dissociating amine moiety. The main problem in obtaining an accurate dissociation constant for local anesthetics by potentiometric methods is the low solubility of the uncharged species in water. One way to circumvent the problem is to titrate at a sufficiently dilute drug concentration, so that precipitation does not occur. This demands high precision in potentiometric instrumentation, and automatic titration apparatus generally lack the precision required for the determination. Aside from the above technical difficulty, estimation of dissociation constants is complicated further by theoretical problems caused by indiscriminate use of activity and concentration of the components (see Appendix). In the present paper, we report the dissociation constants (based on concentrations) of seven local anesthetics that are commonly used in clinical anesthesia. The temperature effect on the dissociation constants is reported also.

Transfer of anesthetics and alcohols into ionic surfactant micelles in relation to depression of krafft point and critical micelle concentration, and interfacial action of anesthetics

Abstract The Krafft points of sodium dodecyl sulfate (SDS) and sodium tetradecyl sulfate (STS) decreased linearly with the increase of the concentration of added alcohols (1-butanol, 1-pentanol, 1-hexanol, and 1-heptanol). On the basis of a theory which treats the Krafft point as the melting point of the hydrated solid surfactant, the partition coefficients, K , of alcohols between the aqueous and the micellar phases were calculated from the Krafft-point depressions by the aid of thermodynamics. The values of K obtained by the present method were in good agreement with those measured by other methods. The transfer free energies of alcohols from the bulk solution to the SDS and STS micelles, ΔG p o = − RT In K , decreased linearly with the increase of the carbon number of the alcohol. ΔG p o per methylene group was almost the same for both SDS and STS micelles: −2.45 kJ mol −1 for the SDS micelle and −2.51 kJ mol −1 for the STS micelle. However, the values of ΔG p o of the alcohols into the STS micelle were more negative compared with those into the SDS micelle. This means that the more hydrophobic micelles are favored for the transfer of alcohol. The method presented here is generally applicable to determine the micelle/water partition coefficients of solutes which form mixed micelle. The partition coefficients of inhalation anesthetics between the aqueous and SDS micellar phases were methoxyflurane 1320, halothane 1140, and enflurane 990. These values are in the same order of their clinical potencies. The critical micelle concentration (CMC) of SDS was measured by the conductivity method as a function of the added anesthetics. The CMC decreased linearly with the increase of the anesthetic concentration. The decrease of the CMC by anesthetics was correlated to the micelle/water partition coefficient of these anesthetics by means of the relation proposed by Shirahama and Kashiwabara ( J. Colloid Interface Sci. 36 , 65 (1971)) , and was ascribed mainly to the increase in entropy of mixing in the micelle due to the solubilization of the inhalation anesthetics. The decrease of CMC by anesthetics was accompanied by a small but unequivocal release of the counterions from the micellar surfaces which indicates the tendency of anesthetics to dehydrate the interface.

Thermodynamics of pressure—anesthetic antagonism on the phase transition of lipid membranes: Displacement of anesthetic molecules

Abstract From the depression of the phase-transition temperature of dipalmitoylphosphatidylcholine vesicle membranes by inhalation anesthetics (halothane, methoxyflurane, enflurane, and chloroform), the apparent partition coefficient, K app , of these drugs between the lipid membrane and water was estimated and the effect of hydrostatic pressure upon the partition was examined. By assuming nonzero partition of anesthetics into the solid-gel membrane, K app is defined as (1 - k ) K , where K is the partition coefficient between the liquid-crystalline membrane and water, and k is the partition coefficient between the liquid-crystalline membrane and the solid-gel membrane. The value of K app was little affected by the change of temperature but was significantly decreased by the high pressure. The high pressure squeezed out the anesthetic molecules from the liquid-crystalline membrane, as evidenced by the decrease of the partition coefficient. The decrement of the number of the anesthetic molecules from that adsorbed at ambient pressure was halothane 10.4 × 10 −2 , chloroform 6.42 × 10 −2 , enflurane 9.58 × 10 −2 , and methoxyflurane 8.45 × 10 −2 % per 1 bar. The volume change due to the transfer of anesthetics from the aqueous phase to the lipid membrane, calculated from the pressure dependence of the apparent partition coefficient, was found to show a large positive value of about 15% of their molal volume. The magnitude of the volume increase is rather large and is difficult to ascribe to the breakage of anesthetic-water contact alone. The volume increase may be caused by the following factors: the structural change of the membrane, the change of the interaction forces between membrane and water due to anesthetic adsorption, the change of interaction between the anesthetics and water, etc. The positive sign indicates that anesthetics must be translocated from the lipid membrane into the aqueous phase by high pressure. Although pressure reversal of anesthesia may be caused mainly by restoration of order in the membrane and by enhancement of the cooperativity of the phase transition, displacement of anesthetics from the binding sites may also contribute to the phenomenon.

Is There a Specific Receptor for Anesthetics? Contrary Effects of Alcohols and Fatty Acids on Phase Transition and Bioluminescence of Firefly Luciferase

Firefly luciferase emits a burst of light when mixed with ATP and luciferin (L) in the presence of oxygen. This study compared the effects of long-chain n-alcohols (1-decanol to 1-octadecanol) and fatty acids (decanoic to octadecanoic acids) on firefly luciferase. Fatty acids were stronger inhibitors of firefly luciferase than n-alcohols. Myristyl alcohol inhibited the light intensity by 50% (IC50) at 13.6 microM, whereas the IC50 of myristic acid was 0.68 microM. According to the Meyer-Overton rule, fatty acids are approximately 12,000-fold stronger inhibitors than corresponding alcohols. The Lineweaver-Burk plot showed that myristic acid inhibited firefly luciferase in competition with luciferin, whereas myristyl alcohol inhibited it noncompetitively. The differential scanning calorimetry (DSC) showed that an irreversible thermal transition occurred at approximately 39 degrees C with a transition DeltaHcal of 1.57 cal g-1. The ligand effects on the transition were evaluated by the temperature where the irreversible change is half completed. Alcohols decreased whereas fatty acids increased the thermal transition temperature of firefly luciferase. Koshlands transition-state theory (Science. 1963. 142:1533-1541) states that ligands that bind to the substrate-recognition sites induce the enzyme at a transition state, which is more stabilized than the native state against thermal perturbation. The long-chain fatty acids bound to the luciferin recognition site and stabilized the protein conformation at the transition state, which resisted thermal denaturation. Eyrings unfolding theory (Science. 1966. 154:1609-1613) postulates that anesthetics and alcohols bind nonspecifically to interfacial areas of proteins and reversibly unfold the conformation. The present results showed that alcohols do not compete with luciferin and inhibit firefly luciferase nonspecifically by unfolding the protein. Fatty acids are receptor binders and stabilize the protein conformation at the transition state.

Interfacial preference of anesthetic action upon the phase transition of phospholipid bilayers and partition equilibrium of inhalation anesthetics between membrane and deuterium oxide

The half-height linewidth (v 1/2) of the 1H-NMR spectra of dipalmitoylphosphatidylcholine vesicles changes abruptly at the phase transition temperature. In the absence of inhalation anesthetics, proton signals from the choline head group (hydrophilic interface) and acyl-chain tails (lipid core) change at the same temperature of 39.6 degrees C. The present study compared the effect of four inhalation anesthetics, i.e., methoxyflurane, chloroform, halothane and enflurane, upon the ligand-induced phase transition of phosphatidylcholine vesicle membranes at 37 degrees C. The anesthetics showed differential action upon the phase transition of the phospholipid vesicle membranes between the lipid core and the hydrophilic interface. The concentrations of anesthetics which induced the phase transition of the lipid core were about 2-fold greater than those required for the phase transition of the interfacial choline head groups. From the area under the proton signals of inhalation anesthetics in the NMR spectra, the maximum solubilities of methoxyflurane, chloroform and halothane in 2H2O at 37 degrees C were determined to be 0.671 . 10(-4), 2.637 . 10(-4) and 1.398 . 10(-4) (expressed as mole fractions), or 3.35, 13.17 and 6.98 mmol/1000 g 2H2O, respectively. The solubilities of the anesthetic vapor in 2H2O expressed as mole fractions according to Henrys law ere 9.586 . 10(-4), 6.432 . 10(-4) and 2.311 10(-4)/atm (1.013 . 10(5) Pa) partial pressure, respectively. The presence of phospholipid vesicles in 2H2O increased the solubility of the inhalation anesthetics. From difference between solubility in 2H2O and a dipalmitoylphosphatidylcholine vesicle suspension, the partition coefficients of methoxyflurane, chloroform and halothane between the phospholipid vesicle membranes and 2H2O were estimated. These values, calculated from the mole fractions, were 3364, 1660 and 3850, respectively at 37 degrees C.

Anesthetic Interaction with a Model Cell Membrane: Expansion, Phase Transition, and Melting of the Lecithin Monolayer

A synthetic L-α-dipalmitoyl lecithin monolayer at an air-water interface was used as a model to study the effects of volatile anesthetics on cell membranes. Methoxyflurane, chloroform, halothane, enflurane and fluroxene were used in this study. When the surface pressure of the monolayer was kept constant, anesthetics at clinically effective tensions expaned the area 0.5 per cent When the area of the monolayer was kept constant, the surface pressure was increased about 1.0 dyn/cm by anesthetics. This increase of the surface energy was caused by the addition of 2.70 x 1013 anesthetic molecules to one square cm of the interface. The surface pressure-surface area curve shows a discontinuity, and a transition from liquid-expanded phase to liquid-condensed phase occurs at this point The liquid-expanded phase is regarded as the “melted” state of the monolayer. Anesthetics shifted the phase transition point towards a more condensed region, indicating melting of the monolayer membrane. Anesthetics decreased the latent heat and entropy change of the phase transition, implying that anesthetics facilitate the melting of the membrane. The compressional modulus, a measure of the rigidity of the monolayer, was decreased by anesthetics. This decrease of rigidity, or increase of fluidity, was also disclosed by analysis of the hysteresis curve (surface pressure-surface area) obtained by compression and expansion of the monolayer. The results support the unfolding theory of anesthesia which postulates that disordering and

400 MHz two-dimensional nuclear Overhauser spectroscopy on anesthetic interaction with lipid bilayer

Interaction between a volatile anesthetic, methoxyflurane, and dipalmitoylphosphatidylcholine (DPPC) vesicle membrane was analyzed by nuclear Overhauser effect (NOE) difference spectroscopy and two-dimensional nuclear Overhauser spectroscopy (NOESY). The NOE difference spectra were obtained by selectively irradiating methoxy protons (hydrophobic end) of the anesthetic: a negative nuclear Overhauser effect of -2.94% was observed with the choline methyl protons of DPPC. The NOESY spectra revealed a cross-peak between the anesthetic methoxy protons and the choline methyl protons. A dipole-dipole interaction exists between the hydrophobic end of the anesthetic and the hydrophilic head group of DPPC. No other cross-peaks were observed. The anesthetic orients itself at the membrane/water interface by interacting with the hydrophilic surface of the DPPC membrane, leaving the hydrophilic end of the anesthetic molecule in the aqueous phase. The preferred residence site of dipolar volatile anesthetics is the membrane/water interface.

Unisotropic solubilization of an inhalation anesthetic, methoxyflurane, into the interfacial region of cationic surfactant micelles.

Abstract Proton-NMR shows that methoxyflurane (HCCl2-CF2-O-CH3) binds hexadecyltrimethylammonium bromide micelles only at the interfacial regions and does not mix with the lipid core isotropically. The protons of the -O-CH3 end is oriented into the hydrophobic interior, while the proton of the HCCl2-end stays at the interfacial region in the close vicinity of the aqueous phase.