Hiroshi Kamaya

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.

A solid-solution theory of anesthetic interaction with lipid membranes: temperature span of the main phase transition

Anesthetics (or any other small additives) depress the temperature of the main phase transition of phospholipid bilayers. Certain anesthetics widen the temperature span of the transition, whereas others do not. The widening in a first-order phase transition is intriguing. In this report, the effects of additive molecules on the temperature and its span were explained by the solid-solution theory. By assuming coexistence of the liquid-crystal and solid-gel phases of lipid membranes at phase transition, the phase boundary is determined from the distribution of anesthetic molecules between the liquid-crystal membrane versus water and between the solid-gel membrane versus water. The theory shows that when the lipid concentration is large or when the lipid solubility of the drug is large, the width of the transition temperature increases, and vice versa. Highly lipid-soluble molecules, such as long-chain alkanols and volatile anesthetics, increase the width of the transition temperature when the lipid:water ratio is large, whereas highly water-soluble molecules, such as methanol and ethanol, do not. The aqueous phase serves as the reservoir for anesthetics. Depletion of the additive molecules from the aqueous phase is the cause of the widening. When the reservoir capacity is large, the temperature width does not increase. The theory also predicts asymmetry of the specific heat profile at the transition.

The α-helix to β-sheet transition in poly(l-lysine): Effects of anesthetics and high pressure

Poly( l -lysine) exists in a random-coil formation at a low pH, α-helix at a pH above 10.6, and transforms into β-sheet when the α-helix polylysine is heated. Each conformation is clearly distinguishable in the amide-I band of the infrared spectrum. The thermotropic α-to-β transition was studied by using differential scanning calorimetry. At pH 10.6, the transition temperature was 43.5°C and the transition enthalpy was 170 cal/mol residue. At pH 11.85, the measurements were 36.7°C and 910 cal/mol residue, respectively. Volatile anesthetics (chloroform, halothane, isoflurane and enflurane) partially transformed α-helix polylysine into β-sheet. The transformation was reversed by the application of hydrostatic pressure in the range of 100–350 atm. Apparently, the α-to-β transition was induced by anesthetics through partial dehydration of the peptide side-chains (β-sheet surface is less hydrated than α-helix). High pressure reserved this process by re-hydrating the peptide. Because the membrane spanning domains of channel and receptor proteins are predominantly in the α-helix conformation, anesthetics may suppress the activity of excitable cells by transforming them into a less than optimal structure for electrogenic ion transport and neurotransmission. Proteins and lipid membranes maintain their structural integrity by interaction with water. That which attenuates the interaction will destabilize the structure. These data suggest that anesthetics alter macromolecular conformations essentially by a solvent effect, thereby destroying the solvation water shell surrounding macromolecules.

Do Anesthetics Fluidize Membranes

The so-called membrane fluidizing effect of anesthetics as a cause of anesthesia has been questioned, mainly because the magnitude of the increase in “fluidity” is insignificant at clinically relevant anesthetic pressures. However, the term “fluidity” has an unfortunate history of being misrepresented in membrane biology. It is often expressed as the ease of movement of probe molecules incorporated into the hydrophobic region of the membrane, thereby representing the property of the microenvironment where the probe molecules reside. In surface chemistry, “membrane fluidity” means inverse viscosity. Membrane viscosity is an integral property of a total membrane (not a part of membrane), and membrane molecules must dislocate and flow against resistance. The ease of motion of probe molecules, therefore, is not fluidity, and is now expressed by the order parameter. The present study measured the effect of halothane on surface viscosity of a phospholipid monolayer spread on a water surface by an oscillating pendulum surface viscometer. The results indicate a significant decrease of about 31% in the surface viscosity by the clinical pressure of halothane; anesthetics do fluidize membranes. Two factors contribute to the surface viscosity of the lipid monolayer; the property of the membrane proper (association between phospholipid molecules) and dragging of water (association between phospholipid and water molecules). The association between phospholipid molecules is in large part related to the order parameter. The fact that anesthetics show little effect on the order parameter, whereas halothane shows a significant effect on the membrane viscosity, indicates that halothane releases surface-bound water. It is postulated that the primary effect of anesthetics on membranes is to weaken the lipid-water interaction forces.

Fractal model for adsorption on activated carbon surfaces: Langmuir and Freundlich adsorption

Abstract Adsorption of organic compounds in aqueous media onto an activated carbon surface usually follows the empirically derived Freundlich equation W=KC1/n, where W is the mass of the adsorbed solute, C is the equilibrium solute concentration, and K and n are fitting constants. To analyze this equation, we propose a simple geometrical model for adsorption of organic compounds. Activated carbon surfaces are irregular, and the irregularity is similar at any magnification. Because of the self-similarity in raggedness at various resolutions, adsorption of a bulky organic molecule sequesters several neighboring sites from binding. Based on this model, a generalized equation was derived that encompasses the Langmuir and Freundlich equations. The Freundlich equation is shown to be a special case of the expanded Langmuir equation. The parameter n in the Freundlich equation is related to the number of binding sites wasted by the adsorbate binding; hence, it is related to the size of the adsorbate molecule. The experimental result that n is larger than unity supports the above model. The main force for the carbon surface adsorption is the tendency of the adsorbate molecule to be excluded from the aqueous phase. The larger the hydrophobic molecule, the greater the tendency to be excluded from the aqueous phase becomes. For this reason, n is related to the adsorbate affinity. The parameter K is related to the size of the adsorbing space, i.e. the binding capacity, and also to the adsorbate affinity. Furthermore, the relationship between n and K was derived and discussed.

TEMPERATURE-DEPENDENT EFFECTS OF HIGH PRESSURE ON THE BIOLUMINESCENCE OF FIREFLY LUCIFERASE

This study measured the effect of high pressure on the enzyme kinetics of firefly luciferase. When firefly luciferase is mixed with luciferin and ATP, a transient flash of light is produced, followed by a weak light, lasting hours. The first stage reaction produces an enzyme-luciferin-AMP complex and pyrophosphate. Addition of pyrophosphate to the reaction mixture decelerated the reaction rate, and the initial flash was prolonged to a plateau, showing a quasi-equilibrium state. The effects of temperature and pressure were analyzed at the plateau. The temperature scan showed that the maximum light intensity was observed at about 22.5 degrees C. When pressurized below the temperature optimum, pressure decreased the light intensity, while increasing it above the temperature optimum. According to the theory of absolute reaction rate, the following values were obtained for the bioluminescent reaction: delta V++ = 823.7 - 2.8 T cm3/mol and delta V = -280.47 + 0.94T cm3/mol, where T is the absolute temperature, delta V++ and delta V are, respectively, activation volume and the volume change due to thermal unfolding. The optimal temperature for the maximum light output occurs because the reaction rate increases with the temperature elevation at low temperature range, but the thermal unfolding of the enzyme decelerates the reaction velocity when the temperature exceeds a critical value. The intensity of luminescence is modified by the influence of pressure on both delta V++ and delta V. So long as the volume of the activated complex (V++) exceeds the average volume of the nonactivated complex (VN), pressure will slow down the reaction. At the point where the volumes become equal, there is no change in the rate under pressure. When the volume of the activated complex is less than that of the reactants, pressure will speed up the rate. This study showed that firefly luciferase is not exceptional to other enzymes in responding to high pressure.