Article pubs.acs.org/Langmuir
3‑Hydroxybutyric Acid Interacts with Lipid Monolayers at Concentrations That Impair Consciousness Tienyi T. Hsu,† Danielle L. Leiske,† Liat Rosenfeld,† James M. Sonner,‡ and Gerald G. Fuller*,† †
The Department of Chemical Engineering, Stanford University, Stanford, California 94305, United States The Department of Anesthesia and Perioperative Care, University of California, San Francisco, California 94143, United States
‡
ABSTRACT: 3-Hydroxybutyric acid (also referred to as βhydroxybutyric acid or BHB), a small molecule metabolite whose concentration is elevated in type I diabetes and diabetic coma, was found to modulate the properties of 1,2-dipalmitoylsn-glycero-3-phosphocholine (DPPC) monolayers when added to the subphase at clinical concentrations. This is a key piece of evidence supporting the hypothesis that the anesthetic actions of BHB are due to the metabolite’s abilities to alter physical properties of cell membranes, leading to indirect effects on membrane protein function. Pressure−area isotherms show that BHB changes the compressibility of the monolayer and decrease the size of the two-phase coexistence region. Epi-fluorescent microscopy further reveals that the reduction of the coexistence region is due to the significant reduction in morphology of the liquid condensed domains in the two-phase coexistence region. These changes in monolayer morphology are associated with the diminished interfacial viscosity of the monolayers (measured using an interfacial stress rheometer), which gives insight as to how changes in phase and structure may contribute to membrane function.
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INTRODUCTION Acquired metabolic diseases are a group of conditions that occur when organs that regulate the blood concentration of metabolites fail. They include end stage kidney disease, fulminant liver failure, and type I diabetes, the latter occurring when the insulinproducing cells of the pancreas fail.1 Among the most devastating symptoms in these diseases are impairments in consciousness that range from difficulty concentrating to inattention, lethargy, and coma in severe disease.1−3 Yet, these neurologic symptoms raise a puzzling question: why do diseases of abdominal organs affect the brain? One hypothesis is that the metabolites that are elevated in these diseases1,4 act as drugs (anesthetics) to impair consciousness and that they do so like anesthetics by acting on the brain’s ion channel receptors.5,6 Clinical support for this idea comes from treatments that reduce the levels of metabolites and reverse impairments in consciousness in these diseases: dialysis in end stage kidney disease, reversal of ketoacidosis through the administration of insulin in type I diabetes, and restoration of liver function by transplantation in fulminant hepatic failure.1 Experimental support for this hypothesis comes from application of metabolites to anesthetic-sensitive ion channels, which demonstrates that the same metabolites that depress behavior can modulate the function of particular ion channels in a manner that is qualitatively similar to anesthetics.5,7,8 But how do so many dissimilar metabolites, which differ in size, shape, and charge distribution, modulate the same ion channel receptors? Ammonia, for example, which is elevated in fulminant liver failure, is a cation at physiological pH. Methylmalonic, isovaleric, and propionic acids are elevated in the organic acidurias, a group of genetic metabolic diseases.8 They are anions © 2013 American Chemical Society
at physiological pH. In type I diabetes, the level of acetone, an uncharged compound, is elevated.7 The presumed existence of interactions between such diverse metabolites and ion channels is difficult to reconcile with functional binding sites on a protein, since ligand binding sites are usually highly specific and only interact with a small number of compounds. Recent work suggests that the activity of transmembrane proteins can be changed in predictable ways not just by ligand binding but also when interfacially active chemical compounds are incorporated in lipid membranes.8−10 In the case of metabolic brain diseases, it has been proposed that metabolites that are elevated to high concentrations in the blood alter physical properties of neuronal membranes, which are coupled to the function of membrane-embedded ion channels.8 An example of such a property is the distribution of stresses in membranes.11 An indirect effect on protein function by altering such a material property of lipid membranes could explain why chemically dissimilar metabolites lead to the same effects. In the work reported here, we investigated the hypothesis that 3-hydroxybutyric acid (also known as β-hydroxybutyric acid or BHB), a metabolite that is elevated in type I diabetes, can modulate the properties of membrane lipids. BHB has previously been reported to be an anesthetic in animals,7 and like the metabolites mentioned previously,8 it too modulates the function of human glycine receptor ion channels.7 Of all the metabolites elevated in disease, BHB reaches the highest concentration, with Received: November 27, 2012 Revised: January 16, 2013 Published: January 22, 2013 1948
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The illumination source was a p-polarized light at 10 mW and 632.8 nm from a HeNE laser (model 1125P, Uniphase, Manteca, CA). A CCD camera (model C2400, Hamamatsu, Bridgewater, NJ) was used as the detector. The resolution of the instrument was 10 μm, and the field of view was 1.1 mm in the focal plane. Fluorescence Microscopy. The fluorescence microscopy images were obtained using an epi-fluorescence microscope (Zeiss, Germany) with a 10× objective. A handmade Teflon trough was used to image the structures at the interface. It was equipped with only one barrier, so the compression was not symmetric as in the BAM, pressure−area isotherm, and interfacial stress rheometry (ISR) (see below) experiments. The trough and barriers were soaked in piranha solution (a 3:1 mixture of concentrated sulfuric acid and 30% hydrogen peroxide) and washed copiously with ultrapure water distilled in glass. The dye Texas Red 1,2dihexadecanoyl-sn-glycero-3-phosphoethanolamine triethylammonium (Texas Red-DHPE) was purchased from Molecular Probes (now Invitrogen, Carlsbad, CA) and mixed with DPPC in chloroform to a final concentration of 0.2 mol % to form a 1 mg/mL solution. With this particular fluorophore, the Texas Red-DHPE is excluded from more ordered phases so that ordered domains appear dark.21,22 The trough was filled with ultrapure water. Images were acquired during compression of the pure DPPC monolayer and then again following injection of BHB into the subphase to achieve a final concentration of 20 mM. The subphase was gently pipetted to facilitate mixing after injection, and 20 min were allowed for the system to achieve equilibrium. An independent experiment where water was pipetted in the subphase of a DPPC monolayer in the same manner as injection of BHB was performed, to ensure that the injection process did not alter the domain morphology observed. The sizes of the domains were measured by imaging a 1 mm glass scale bar on the microscopy setup. Interfacial Stress Rheometry. An interfacial stress rheometer was used to obtain the oscillatory shear rheology data. The validation details of this technique have been described elsewhere.23,24 In brief, the rheometer is equipped with two Helmholtz coils, which apply an oscillating magnetic field to move a magnetized Teflon-coated rod floating at the air−water interface in a KSV Langmuir trough (KSV Instruments, Helsinki, Finland). The rod was contained in a rectangular quartz channel to maintain the lateral position of the rod and produce a well-defined flow field during the experiments. Barriers were compressed at a rate of 1.5 cm2/min while the interfacial rheology was monitored. The oscillating magnetic field moved the magnetic rod along its major axis at sinusoidal strains, causing it to glide on the interface and shear the monolayer of interest. The rod position was monitored using a CCD camera (Basler Electric Company, Highland, IL), and the applied strain was determined from the images. Experiments were performed for pure DPPC films and DPPC films with BHB (prepared in the same manner as the pressure−area isotherms) at a frequency of 1 Hz with a strain amplitude of 0.029, which was found to be in the linear viscoelastic regime of DPPC.
blood concentrations between 20 and 30 mM in the most severe form of diabetes12 compared to 100 μM in health.13 To model interactions of BHB with a cell membrane, we used monolayers of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), a phospholipid that is a major constituent in cell membranes.14,15 This particular system is well studied, and its monolayer phase diagram has been extensively documented. 16 We made equilibrium measurements on DPPC monolayers with and without BHB added in clinical concentrations to the aqueous subphase using a Langmuir film balance to measure surface pressure−area isotherms.17 We also used epi-fluorescence microscopy and Brewster angle microscopy to reveal the mesoscale structure of phase-separated domains. We found that BHB was able to interact with DPPC from the subphase, which was demonstrated by increased surface pressures at equivalent mean molecular areas of DPPC. In the liquidexpanded (LE)−liquid-condensed (LC) coexistence region for DPPC, BHB−DPPC interactions produced LC domains that were atypical in both size and shape, in addition to decreasing the area range over which this phase transition occurred. In addition, BHB diminished the interfacial viscosity of DPPC monolayers, which gives some insight as to how these changes in phase and structure may contribute to membrane function.
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MATERIALS AND METHODS
Pressure−Area Isotherms. Pressure−area (π−A) isotherms were measured using a KSV Langmuir−Blodgett minitrough (KSV Instruments, Helsinki, Finland). The surface pressure balance used a platinum Wilhelmy plate, which was cleaned with ethanol and then heated in a flame before use. The resolution of the balance was 4 μN/m. The Teflon trough and Delrin barriers were cleaned prior to use with HPLC grade butyl acetate followed by ethanol and Milli-Q water. Milli-Q water, with or without added BHB, was used for the subphase. The purity of the subphase was confirmed by ensuring no change in surface pressure during compression of the barriers prior to spreading the monolayer. Experiments were performed at a temperature of 23.1 °C. The spreading agent was HPLC grade chloroform. DPPC was purchased as a powder (Avanti Polar Lipids, Alabaster, AL). A stock solution of 1 mg/mL in chloroform was applied to the surface by touching microdrops of the solution to the subphase using a Hamilton syringe. Afterward, the monolayer was allowed to remain undisturbed for 15−20 min to allow the spreading agent to fully evaporate and the system to achieve equilibrium. Then, the monolayer was compressed at a rate of approximately 2 Å2/molecule/min. The Wilhelmy balance recorded the resulting surface pressure every second. Two pressure− area isotherms were obtained for each concentration of BHB, averaged, and compared to a reference isotherm for pure DPPC. Data Analysis for Pressure−Area Isotherms. Compressibility was calculated by first smoothing the data by applying a three-point moving average and then numerically differentiating surface pressure π with respect to mean molecular area A. From this, compressibility was calculated as −(∂A/∂π)/A.18 Surface pressures were initially constant during compression of the monolayer and then rose above baseline values. This liftoff from the baseline was calculated as the mean molecular area at which surface pressure increased by 0.05 mN/m when the mean molecular area was changed by 0.5 Å2. The surface pressure at a DPPC mean molecular area of 60 Å2 (similar to that found in bilayers19) was determined for each concentration of BHB. Linear regression was performed to determine if there was an increase in the mean molecular area at liftoff or in surface pressure at 60 Å2 with increasing BHB. P < 0.05 was considered significant. Brewster Angle Microscopy. The lipid films were deposited on the same minitrough where the pressure−area isotherms experiments were performed and imaged using a home-built Brewster angle microscopy (BAM) setup. The principle of BAM has been described elsewhere.20
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RESULTS BHB changed the shape and position of the DPPC pressure− area isotherm, as seen in Figure 1a. The monolayer is initially compressed from high mean molecular areas where DPPC exists as a two-dimensional gas and the surface pressure is zero. With further compression, the monolayer enters the disordered LE state during which surface pressure rises. At 6 mN/m, DPPC enters a two-phase coexistence region in which both LE and LC phases are present and the surface pressure is constant. Additional compression results in an ordered, rigid, LC phase. This general profile found for the pure DPPC monolayer was similar to those published in literature,25,26 which exhibited liftoff around 90−100 A2/molecule and the characteristic plateau at ∼7 mN/m attributed to the first-order LE to LC phase transition. However, as the concentration of BHB was increased, the mean molecular area range of the LE−LC plateau is reduced and becomes less horizontal. The pressure at which liftoff from the baseline occurred increased linearly with increasing BHB 1949
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Figure 3. Monolayer compressibility. Compressibility = −(∂mean molecular area/∂surface pressure)/mean molecular area, (−1/A)(dA/ dπ).
shows that BHB shifted the compressibility peak toward higher surface pressures (from ∼6 to ∼8−10 mN/m). Therefore, the monolayer must be compressed to a higher surface pressure to induce the phase transition to the LC phase, indicating the monolayer is more expanded when BHB is present in the subphase. In Figure 4, BAM images of a DPPC monolayer with and without 20 mM BHB in the subphase at comparable surface pressures are presented. The pure DPPC monolayer showed domains at pressures of 5−8 mN/m, corresponding to the LE− LC phase transition. At higher surface pressures, the monolayer became homogeneous. Similar results were reported by Nishimura et al.25 for DPPC. However, the BAM images for DPPC with BHB in the subphase did not show the scalelike domains. This is consistent with the pressure−area isotherm results that showed major reduction in the LE−LC coexistence plateau that was present in the pure DPPC monolayers. Figure 5 presents fluorescence microscopy images of DPPC monolayers doped with Texas Red. Images of the monolayer without (left panel) and with BHB at 20 mM in the water subphase (right panel) at comparable surface pressures are shown. For the pure DPPC monolayer, the LC domains started to appear at ∼6 mN/m. Upon further compression of the monolayer to ∼9 mN/m, the domains were approximately 10 μm in diameter and acquired irregular “clover” shapes distinct to DPPC, as reported in previous studies of fluorescence microscopy of DPPC monolayers.19 The domain size was homogeneous throughout the interface. As the monolayer is further compressed, the monolayer moves from the LE−LC coexistence phase and approached a uniform LC phase, and the domains begin to blur and disappear again (as seen in Figure 5d) where the surface pressure is at 12 mN/m. When BHB was injected to the subphase, however, no visible domains were present at surface pressure below 9 mN/m. The domains for the monolayer with BHB in the subphase were significantly smaller, about 2 μm in diameter. The domains were also rounder compared to the sharp-edged pure DPPC domains. Unlike the pure DPPC monolayer, the domains were still present at 12 mN/ m and persisted until a surface pressure of about 15 mN/m. The presence of homogeneous but altered appearance of the DPPC phase coexistence domains shown by fluorescence micrographs and BAM images suggests that BHB does not form a separate phase but rather it interacts with DPPC to create a homogeneous monolayer. In contrast, previous studies on the interaction between DPPC monolayers and macromolecules (polyethylene
Figure 1. (a) Pressure−area isotherm for pure DPPC and after addition of BHB. Surface pressure (total lateral pressure) is expressed in units of mN/m on the y axis. Mean molecular area is in units of Å2 on the x axis. (b) Mean molecular area at which the DPPC pressure−area isotherm lifts off the baseline.
concentration (Figure 1b). This reflects incorporation of BHB into the monolayer. For this regression, the slope equals 1.60 ± 0.16 Å/mM (p = 0.001), the intercept equals 94.70 ± 1.51 Å (p < 0.001), and r equals 0.98. In addition, at a mean molecular area of 60 Å2, which is approximately that found in lipid bilayers, the surface pressure was elevated compared to pure DPPC at all BHB concentrations (Figure 2). It is important to note here that BHB
Figure 2. Effect of BHB on surface pressure at a physiological lipid packing density of approximately 60 Å2. The line is present to guide the eye.
itself does not appear to be surface active at the concentration range explored here; when the barriers were compressed without depositing the DPPC monolayer, the measured surface pressure did not increase above baseline values. At BHB concentrations of 3 mM and above, the compressibility of the monolayer after liftoff changed (Figure 3): no peak in compressibility was seen when BHB was present. Figure 3 also 1950
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Figure 4. BAM images of pure DPPC monolayers (left) or DPPC monolayers with 20 mM BHB (right) in the subphase. The surface pressure for each set of images is as follows: (a) 5; (b) 7; (c) 8; and (d) 10 mN/m.
oxide−polybutylene oxide block copolymers) showed that, at surface pressure above 8 mN/m, DPPC and the copolymer phase separate, exhibiting an inhomogenous monolayer with two types of domains differing in shape and sizes.26 The interfacial rheology for the DPPC monolayers as measured by ISR is presented in Figure 6. At surface pressures below 10 mN/m, the rheological properties were below the sensitivity level of the instrument. For pure DPPC, as the monolayer was further compressed and surface pressure was increased, the interfacial viscous modulus became detectable and increased with increasing surface pressure. However, until the surface pressure was increased to near 40 mN/m, no interfacial elastic modulus was detected, showing that the monolayer was dominated by viscous behavior with no measurable elasticity. Beyond 40 mN/m, the interfacial elastic modulus became measurable, and the interfacial moduli increased rapidly with
compression. However, the interfacial viscous modulus was higher than the interfacial elastic modulus throughout the entire range of surface pressure tested in the experiment, up to 45 mN/ m. This shows that the monolayer remained predominantly fluid. When BHB was added to the subphase, the surface viscosity and viscous modulus were reduced. The interfacial elastic modulus became measurable at a lower surface pressure than the pure DPPC monolayer (around 34 instead of 40 mN/m), but the interfacial moduli increase upon compression was not as rapid. The reduction of both interfacial elastic and viscous moduli was consistent with the overall reduction in surface viscosity (Figure 6a).
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DISCUSSION Diabetes, kidney disease, and liver disease are among the leading causes of morbidity and mortality in the developed world.27 1951
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Figure 5. Fluorescence microscopy images of pure DPPC monolayers (left) or DPPC monolayers with 20 mM BHB (right) in the subphase. The DPPC monolayer was stained with 0.2% Texas Red. The surface pressure for each set of images is as follows: (a) 5.9; (b) 9.1; (c) 9.9; and (d) 12 mN/m.
in modeling one leaflet of the membrane bilayer16 and because a wider variety of thermodynamic, structural, and mechanical experiments can be performed on lipid monolayers compared to bilayers. DPPC was chosen as the model cell membrane because it is a major constituent in cell membranes.14 DPPC monolayers undergo a distinctive first-order phase transition from a LE to a LC state.28 The addition of BHB to the subphase decreased the plateau region of the room temperature pressure−area isotherms and increased the pressure at which it
Impairment in consciousness is a debilitating complication of these diseases.1 It has been shown that the small molecule metabolite BHB, which is dramatically elevated in diabetic ketoacidosis, modulates ion channel function and depresses behavior.7 Here, we examined whether BHB interacts with cell membrane lipids using lipid monolayers as a model system. We encompassed the entire pathophysiological concentration range of BHB, which extends up to approximately 30 mM,12,13 in these studies. We used monolayers because of their history of success 1952
dx.doi.org/10.1021/la304712f | Langmuir 2013, 29, 1948−1955
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In the coexistence region between the LE and LC states, epifluorescence microscopy revealed rigid domains existing within a continuous background of a fluid, LE material; the shapes and sizes of the domains were characteristic for DPPC. BHB had a noticeable effect on the DPPC monolayers: the coexistence region was strongly attenuated in the range of mean molecular area and the LC domains were diminished in size and became more circular. BAM confirmed that, at similar surface pressures, the domain structures of the monolayer in the two-phase coexistence region were altered in the presence of BHB. These changes in monolayer morphology were associated with changes in the mechanical properties of the monolayers. The ISR results showed that, generally, the interaction of BHB with the DPPC monolayer resulted in a reduction of mechanical strength of the monolayer. This is presumably due to changes in DPPC packing, as illustrated by the fluorescent and BAM images. It is worth noting that, while the fluorescent and BAM images were only able to provide information on monolayer properties at the LC−LE coexistence phase, the ISR result provided information at higher surface pressures, above the coexistence region. At surface pressures of 30 mN/m or above, ISR demonstrates that the surface viscosity of the monolayer was reduced by interaction with BHB in the subphase. These combined results showed that BHB interacted with the DPPC monolayer and affected the monolayer’s properties at both the macroscopic and microscopic levels at a wide range of surface pressures and BHB concentrations. To our knowledge, this is the first study to report that small endogenous molecules (i.e., other than macromolecules such as lysozyme) can influence both the morphology and interfacial mechanical properties of a lipid monolayer. The effect of BHB on the monolayer remained even up to 45 mN/m, showing that the BHB molecules did not simply insert themselves into the monolayer at low surface pressures and become “squeezed out” or ejected from the monolayer at high surface pressures, as has been reported with block copolymers.26,29 These findings demonstrate that, as hypothesized, BHB can interact with a lipid aggregate and it does so at physiological concentrations and over a large range of surface pressures. The correlation between the concentrations at which physical effects of BHB on monolayers occur and the concentrations encountered in disease supports the hypothesis that BHB can indirectly exert biological effects on transmembrane proteins through its interactions with lipid membranes. This observation is also consistent with the hypothesis that BHB impairs consciousness by acting as an anesthetic, several of which have been shown to increase the surface pressure of monolayers at constant area32,33 and have been proposed to have mechanistically important effects on bilayers.11 This hypothesis has only been proposed recently,7 even though diabetic ketoacidosis has been known since antiquity1 and anesthesia has been used for surgery since the 1840s.34 Indeed, Banting and Best showed in the 1920s35 that the unresponsiveness in “diabetic coma” was a reversible state of unresponsiveness. This defines the state of anesthesia. Our explorations so far have been restricted to DPPC monolayers at room temperature, since this is a well-documented system that has been extensively studied using a variety of microscopy and interfacial rheology techniques. However, the next step would be to choose a monolayer that is more physiologically relevant and more robust than DPPC, whose surface viscosity would be too low to measure at physiological temperatures.
Figure 6. Interfacial rheology of DPPC monolayers with and without BHB in the subphase, as measured using ISR. (a) Surface viscosity of the films as functions of surface pressure. (b) Interfacial elastic (G′) and viscous (G″) moduli of the films as functions of surface pressure.
occurred (Figure 1a). BAM and fluorescent micrographs demonstrated that, although BHB altered the phase behavior of DPPC, the monolayers were homogeneous over large areas. This indicates that BHB was either incorporated into the DPPC monolayers or interacted with DPPC headgroups from the subphase, rather than inserting into the monolayer as a separate phase. If they were phase separated into distinct domains, the shape of the pressure−area isotherm would have remained unchanged and would only shift laterally. In addition, the surface pressure of the phase transitions would not have changed. For example, amphiphilic block copolymers, such as poloxamers, incorporate into DPPC monolayers but remain in a separate, distinct phase from DPPC, resulting in inhomogeneous monolayers.26,29 At all concentrations studied, with increasing BHB concentration, there was an increase in the mean molecular area at which the surface pressure rose above baseline (liftoff, Figure 1a). There was also a marked difference in compressibility of the monolayer when BHB was present, even at relatively low concentrations that are produced by the ketogenic diets that are used to treat seizures30,31 (Figure 2). At lipid packing densities that are typical of membrane bilayers, 60 A2 /molecule, clinical concentrations of BHB more than doubled the surface pressure (Figure 3). 1953
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Our work does not tell us which properties of membrane bilayers might be affected by BHB but rather demonstrates that BHB is capable of interacting with lipids and altering phase behavior. It has been proposed that anesthetics, and other small interfacially active solutes like BHB, may modulate ion channel function by altering the distribution of stresses in the membrane bilayer.11 This in turn changes the work done in transitioning between the open and closed conformation of embedded membrane proteins. This hypothesis is consistent with the present work. The intricate relationship between the lipid order in biological membranes and the embedded receptor proteins and that structural changes in one could lead to changes in the other has recently been demonstrated.36 Work using X-ray diffraction showed that cholera toxins, by specific binding to membrane-embedded protein receptors, perturbed the structural order of the surrounding lipid bilayers. The perturbed lipid order was transferred from the receptor laden exterior membrane leaflet to the inner leaflet, which may allow proteins and other exogenous molecules to affect cell signaling. If BHB modulates ion channel function via an action that depends in part on interactions with the neuronal membranes, then the kinetics of that process should depend on the adsorption and desorption rate constants to the bilayer and should not saturate until very high BHB concentrations, when adsorption to the bilayer saturates. This suggests a next possible step in examining the physical basis for the modulation of ion channel function by BHB: a chemical kinetic analysis of the time course of currents through an ion channel that is sensitive to BHB (e.g., the glycine receptor7), from which the membrane adsorption and desorption rate constants might be extracted and compared to those determined by physical methods.37 A scheme for performing such a kinetic analysis has recently been published.38 Such an analysis should would either support or falsify a connection between the physical effects of BHB on membranes and its functional effects on ion channels.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors would like to thank S. Nishimura and R. Cantor for useful discussions. This work was supported in part by NIGMS R01 GM069379.
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REFERENCES
(1) Warrell, D.; Cox, T.; Firth, J.; Benz, E. Oxford Textbook of Medicine, 4th ed.; Oxford University Press: Oxford, U.K., 2003; Vol. 2. (2) Sass, D. A.; Shakil, A. O. Fulminant hepatic failure. Liver Transplant. 2005, 11 (6), 594−605. (3) Brouns, R.; De Deyn, P. P. Neurological complications in renal failure: a review. Clin. Neurol. Neurosurg. 2004, 107, 1−16. (4) Yavuz, A.; Tetta, C.; Ersoy, F. F.; D’Intini, V.; Ratanarat, R.; De Cal, M.; Bonello, M.; Bordoni, V.; Salvatori, G.; Andrikos, E.; Yakupoglu, G.; Levin, N. W.; Ronco, C. Uremic toxins: a new focus on an old subject. Semin. Dial. 2005, 18, 203−11. (5) Brosnan, R. J.; Yang, L.; Milutinovic, P. S.; Zhao, J.; Laster, M. J.; Eger, E. I., II; Sonner, J. M. Ammonia has anesthetic properties. Anesth. Analg. 2007, 104 (6), 1430−3. (6) Sonner, J. M. A hypothesis on the origin and evolution of the response to inhaled anesthetics. Anesth. Analg. 2008, 107, 849−54. (7) Yang, L.; Zhao, J.; Milutinovic, P. S.; Brosnan, R. J.; Eger, E. I., II; Sonner, J. M. Anesthetic properties of the ketone bodies betahydroxybutyric acid and acetone. Anesth. Analg. 2007, 105, 673−9. (8) Weng, Y.; Hsu, T. T.; Zhao, J.; Nishimura, S.; Fuller, G. G.; Sonner, J. M. Isovaleric, methylmalonic, and propionic acid decrease anesthetic EC50 in tadpoles, modulate glycine receptor function, and interact with the lipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. Anesth. Analg. 2009, 108, 1538−45. (9) Yang, L.; Sonner, J. M. Anesthetic-like modulation of receptor function by surfactants: a test of the interfacial theory of anesthesia. Anesth. Analg. 2008, 107, 868−74. (10) Cantor, R. S. Breaking the Meyer-Overton rule: predicted effects of varying stiffness and interfacial activity on the intrinsic potency of anesthetics. Biophys. J. 2001, 80, 2284−97. (11) Cantor, R. The lateral pressure profile in membranes: a physical mechanism of general anesthesia. Biochemistry 1997, 36, 2339−44. (12) Marlchoff, C. D.; Pohl, S. L.; Kaiser, D. L.; Carey, R. M. Determinants of glucose and ketoacid concentrations in acutely hyperglycemic diabetic patients. Am. J. Med. 1984, 77, 275−85. (13) Mitchell, G. A.; Wang, S. P.; Ashmarina, L.; Robert, M. G.; Bouchard, G.; Laurin, N.; Kassovska-Bratinova, S.; Boukatane, Y. Inborn errors of ketogenesis. Biochem. Soc. Trans. 1998, 26, 136−40. (14) Li, Z.; Agellon, L. B.; Allen, T. M.; Umeda, M.; Jewell, L.; Mason, A.; Vance, D. E. The ratio of phosphatidylcholine to phosphatidylethanolamine influences membrane integrity and steatohepatitis. Cell Metab. 2006, 3, 321−331. (15) Takamori, S.; Holt, M.; Stenius, K.; Lemke, E. A.; Gronborg, M.; Riedel, D.; Urlaub, H.; Schenck, S.; Brugger, B.; Ringler, P.; Muller, S. A.; Rammner, B.; Grater, F.; Hub, J. S.; De Groot, B. L.; Mieskes, G.; Moriyama, Y.; Klingauf, J.; Grubmuller, H.; Heuser, J.; Wieland, F.; Jahn, R. Molecular anatomy of a trafficking organelle. Cell 2006, 127, 831−46. (16) McConnell, H. M.; Vrljic, M. Liquid-liquid immiscibility in membranes. Annu. Rev. Biophys. Biomol. Struct. 2003, 32, 469−92. (17) Petty, M. C. Langmuir-Blodgett Films; Cambridge University Press: Cambridge, U.K., 1996; p 234. (18) Keller, S. Miscibility transitions and lateral compressibility in liquid phase of lipid monolayers. Langmuir 2003, 19, 1451−6. (19) White, S. H.; King, G. I. Molecular packing and area compressibility of lipid bilayers. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 6532−6536.
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SUMMARY In this work, we demonstrate that the small molecule metabolite BHB is capable of interacting with lipids (modeled using a DPPC monolayer) and altering phase behavior at clinical concentrations. Increased surface pressures at equivalent mean molecular areas were observed for the Langmuir pressure−area isotherms, as well as changes to the mesoscale structure of phaseseparated domains in Brewster angle and fluorescent microscopy images. BHB also diminished the interfacial viscosity of DPPC monolayers, as measured using an interfacial stress rheometer. The effect of BHB on the monolayer was present over a wide range of mean molecular area and surface pressures. At lower surface pressures (∼30 mN/m or below), the shift in compressibility peak toward higher surface pressures suggests that the monolayer is becomes more fluidized and expanded with the presence of BHB. At higher surface pressures (∼30 mN/m or above), BHB was found to decrease the viscosity and mechanical strength of the monolayer. This shows the BHB molecules did not simply insert themselves into the monolayer at low surface pressures and become “squeezed out” or ejected from the monolayer at high surface pressures. These findings are consistent with the proposed mechanism that anesthetics and other small interfacially active solutes may modulate ion channel function by altering the distribution of stresses in the membrane bilayer. 1954
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dx.doi.org/10.1021/la304712f | Langmuir 2013, 29, 1948−1955
