The interactions of phospholipid monolayers (dipalmitoyl phosphatidyl choline; DPPC and dimyristoyl phosphatidyl choline; DMPC) with volatile anesthetic isoflurane were investigated using quartz crystal microbalance (QCM) and quartz crystal impedance (QCI) methods. The quartz crystal oscillator was attached horizontally on the surface of DPPC and DMPC monolayer formed on the water surface. Physisorption of isoflurane hydrate at the DPPC monolayer surface was monitored in terms of frequency and resistance change of quartz crystal on addition of anesthetics isoflurane. Both frequency and resistance change showed the elastic nature of DPPC monolayer. Measurement of DMPC monolayer-isoflurane hydrate revealed the expandable nature of DMPC monolayer. Variation of frequency and impedance of DPPC and DMPC monolayer on addition of isoflurane which proved a two-step change has occurred at monolayer surface at isoflurane concentration of ≤8 mM that has been attributed to isoflurane aggregation at monolayer/water interface. Isoflurane hydrates formed in the process have capability to affect the interfacial properties of monolayer such as existence of structured water.
Biomembranes have fluidic structures where proteins are buried in lipid bilayers. Lipids are composed of various kinds of phospholipids; a few of them are glycerol- and sphingophospholipids. Each protein and lipid has different functions for that they are called functional proteins and lipids. Investigation of function and structure reveals that intermolecular interactions such as: hydrogen bonding, hydrophobic, van der Waals interactions have an important role in performance of these biomolecules. Researchers [
In this study, we have investigated physisorption interactions between phospholipid monolayers (dipalmitoyl phosphatidyl choline, DPPC; dimyristoyl phosphatidyl choline, DMPC) as the model membrane and anesthetic isoflurane using our home designed and assembled high sensitive QCO apparatus [
DPPC (>99%) and DMPC (>99%) which were used as the model membrane lipids were purchased from Avanti polar lipids (AVT) Inc. and used without further purification. Volatile anesthetic isoflurane which has chemical formula, 1-Chloro-2,2,2-trifluoroethyl difluoromethyl ether, was purchased from Abbott Japan Corp. Ltd. The concentrations of isoflurane in aqueous solution were in the range of 1 - 8 mM. Spreading solvent for DPPC and DMPC monolayers was chloroform (99.0%, Wako Pure Chemical Industries Ltd.). Purified water (conductance < 0.07 μS/cm) for subphase was obtained using a Super Water Purifying System (WL-21P; Yamato Scientific Corp. Ltd.), and further boiled for 10 min and subsequently cooled to room temperature before use.
On the formation of each DPPC and DMPC monolayer, we adopted the dropping method at which solution droplets containing monolayer sample are dropped continuously on the water surface [
Details of the experimental apparatus used for QCM and QCI measurements have been reported previously [
For the QCM measurement, the frequency F of the QCO was measured by a Universal Counter (TR5822; Advantest Corp., Tokyo, Japan) and conducted at 1 min intervals. After the stabilization of F contacting the monolayer to within ±0.5 Hz, isoflurane was injected slowly at the rate 1 ml/s using a 100 μl microsyringe. The change in F was observed before and after the addition of isoflurane. For the QCI measurement, the resistance R in the QCO circuit was measured by an impedance-gain phase analyzer (SI1260; Solartron Analytical, Hampshire, UK) and conducted at 10 min intervals. R corresponds to the viscosity change at the QCO interface, and from the viewpoint of membrane fluidity we specially addressed the analysis of R [
In the case that the change in each ΔF and ΔR is observed, we decided to define the difference of the value
before and after the addition of isoflurane as respective Δf for QCM and Δr for QCI (inserted in
showed a second stage increase. At isoflurane 8 mM second saturation value of Δf reached to 3.8 Hz. These processes are similar to the Langmuir’s or BET’s adsorption isotherm and have similarity to isoflurane-DPPC monolayer isotherm as described above in this work. While on the QCI (
Many isotherm studies for DPPC monolayer by the compression method have shown that the DPPC
monolayer forms the following three types of molecular arrangements: liquid-expand (LE) state at low surface pressure, liquid-condensed (LC) state at higher surface pressure, and LE-LC transition state also known as plateau range at a surface pressure of approximately 8 mN/m. After LE-LC transition the pressure increased steeply up to 50 mN/m (solid line). The limiting molecular area (A0) is ca. 0.50 nm2/molecule [
The isotherm curve constructed by the dropping method in our measurement had a different shape compared to compression method. Surface pressure increased gradually at molecular area 1.7 nm2 where in compression method increase was at 1.0 nm2. After LE-LC transition state finishes at 12 mN/m, surface pressure surface pressure steeply increased up to 42 mN/m. A0 = 0.65 ± 0.05 nm2 and 30% larger than recorded by compression method.
In DPPC monolayer, two alkyl chains are all-trans type and also in rotational degree of freedom in each chain axis [
Many isotherm studies for DMPC monolayer by the compression method have shown that DMPC monolayer forms only one LE state. Surface pressure increases gradually from about 1.1 nm2 of molecular area and pressure increased monotonously up to 36 mN/m. The limiting molecular area A0 is 0.78 nm2/molecule [
The isotherm curve constructed by the dropping method in our experiment also presented a typical LE state. Surface pressure increases gradually at 1.4 nm2 molecular area and kept monotonously increased up to 41 mN/m. A0 was 0.80 ± 0.05 nm2/molecule and the measured value was quite similar to that recorded by other authors by compression method.
In the case of DMPC, two alkyl chains are disordered partially at 26˚C and the structure has gauche type conformation [
Isoflurane has chemical formula CF2H-O-CClH-CF3 and a typical inhalation anesthetic. It was synthesized by Terrell group in 1965. Clinical use was permitted at the USA in 1980. Since then anesthesia application has been spread widely [
1H-NMR spectroscopy has showed that anesthetics (halothane, enflurane, methoxyflurane, and chloroform) show different actions on the isothermal phase transition between the lipid core and the hydrophilic interface in a DPPC vesicle [
Based on the above-published reports the interactions between isoflurane and DPPC or DMPC have been explained as followings.
The present DPPC monolayer is an elasticity rich monolayer as mentioned in Section 4.1. There is possibility that the structural change in monolayer-water interface occurred by the physisorption of isoflurane hydrate. As described in
From reaching to first saturation values of both Δf (1.6 Hz) and Δr (0.14 Ω) at around 6 mM (
On the higher concentration at more than 6 mM (
The present DMPC monolayer is an expandability rich monolayer as mentioned in Section 4.1. Therefore, it would be hard to cause the structural change in the monolayer-water interface i.e. no change in Δr even if isoflurane hydrate physisorbed on the interface. It can be also explained as a simple physisorption model of isoflurane hydrate to the DMPC monolayer [
On the higher concentration at more than 5 mM (
In the present study, the interaction between two phospholipid monolayers DPPC and DMPC on the water surface and anesthetic isoflurane has been investigated using horizontally attached QCM and QCI devices. It was noticed that anesthetic isoflurane hydrate physisorbed on elasticity DPPC monolayer and changed its viscosity. While DMPC monolayer was expandability rich, no change in monolayer viscosity occurred. Process of physisorption showed a two-step change in the range of added isoflurane concentration, indicating that isoflurane aggregation was probably formed on each monolayer-water interface. Isoflurane hydrate physisorbed on the monolayer and brought changes in the monolayer properties. As investigated by several researchers [
This work was supported by a Grant-in-Aid from the Ministry of Science, Education, Sports and Culture (No. 15750118, 21750143 and 24550154), DVA Medical Research and Development Funds, and Sakakibara Memorial Hospital Incorporated Foundation.