Article pubs.acs.org/Langmuir
Influence of pH on Sphingomyelin Monolayer at Air/Aqueous Solution Interface Aneta Dorota Petelska,*,† Monika Naumowicz,† and Zbigniew Artur Figaszewski†,‡ †
Institute of Chemistry, University in Bialystok, Al. J. Pilsudskiego 11/4, 15-443 Bialystok, Poland Laboratory of Electrochemical Power Sources, Faculty of Chemistry, University of Warsaw, Pasteur St. 1, 02-093 Warsaw, Poland
‡
ABSTRACT: The pH dependence of the interfacial tension is an important factor in the behavior of sphingomyelin (SM) monolayers. We developed a theoretical model to describe this dependence in which the interfacial tension and molecular area contributions of each sphingomyelin form were additive and dependent on pH. The interfacial tension values and the molecular areas values for the SMH+ and SMOH− forms of sphingomyelin were calculated and the proposed model was experimentally verified. The theoretical predictions agreed with the experimental results for pH values ranging from 2 to 12.
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INTRODUCTION
with identical charges have different binding constants and can exhibit very different effects on membrane properties.13 Sphingomyelin is a neutral, zwitterionic sphingolipid with an amphiphilic character. The surface pressure−area per molecule (π−A) curves of SM have been previously described.14,15 While it is generally believed that the pH of the subphase usually does not affect the structure of sphingomyelin monolayers, a recent study of SM bilayers indicated that the interfacial tension reaches a maximum at a particular pH,16 suggesting that further careful study is warranted. Since changes in interfacial tension induce changes in area per molecule, it is important in the context of biological membranes to determine the pH dependence of the molecular packing. This paper describes the properties of mono- and bilayer lipid membranes formed from sphingomyelin as a continuation of the physicochemical investigations undertaken by Figaszewski and co-workers.15−19 The SM molecule is zwitterionic and participates in equilibria with H+ as well as OH−. A model of the ion−monolayer interactions was developed using equations derived from the π−A curves.
Natural membranes are principally composed of lipids and proteins. A significant mass fraction of the lipid portion in eukaryotic cells is sphingomyelin (SM), 1,2 a type of sphingolipid first isolated from brain tissue in the 1880s by Johann L.W. Thudicum.3 In contrast to lipids with more passive roles, sphingolipids are actively involved in cellular processes.4 Analyses in 1927 revealed that SM is N-acyl-sphingosine-1phosphorylcholine.5 In most mammalian tissues, the SM content ranges from 2% to 15% of the total organ phospholipid content.2 Even higher levels of SM are found in erythrocytes, the lens of the eye, peripheral nerve tissue, and the brain.2,6 SM functions as a structural component in biological membranes together with other phospholipids, glycolipids, cholesterol, and integral membrane proteins. In addition to its structural role, SM also participates in cell signaling.7 The effect of pH on the behavior of amphiphilic substances at air/water interfaces was first investigated at the beginning of the last century,8,9 in the form of interfacial tension measurements and surface potential investigations at fixed values of area/molecule. The membrane surface charge and the pH and ionic composition of the aqueous medium are important parameters influencing membrane organization. For example, the structure of aqueous dispersions of negatively charged phosphatidylserine depends markedly on pH and salt concentration (particularly the concentration of divalent cations) which determine whether the molecules will arrange in lamellar or nonlamellar phases.10 It is well-known that ions interact with charged lipids, but they may also bind to zwitterionic lipids, as indicated by the electrophoretic mobility of zwitterionic lipid vesicles dispersed in salt solutions11 and by NMR measurements.12 Moreover, adsorption of ions on lipid bilayers appears to be highly ion-specific, since different ions © 2012 American Chemical Society
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THEORY Since the sphingomyelin molecule (SM) possesses both positively and negatively charged groups, it can participate in equilibrium reactions with both hydrogen and hydroxyl anions. SM + H+ ⇔ SMH+
(1)
SM + OH− ⇔ SMOH−
(2)
SM + HOH ⇔ SMHOH
(3)
Received: July 16, 2012 Revised: August 17, 2012 Published: August 27, 2012 13331
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The associations described in eqs 1−3 may be considered as adsorption processes. Following adsorption of H+ and OH− ions on the surface of a sphingomyelin layer, the SM molecules exist in one of four forms: SMH+ with H+ adsorbed, SMOH− with OH− adsorbed, SMHOH with both H+ and OH− adsorbed, and free sphingomyelin with no ions adsorbed. The relative contribution of each form is pH-dependent. Equations 1−3 may be rewritten as equilibria, from which the activity of each sphingomyelin form may be calculated:9 aSMH+ = KSMH+aSMa H+
(4)
aSMOH− = KSMOH−aSMaOH−
(5)
aSMHOH = KSMHOHaSM
(6)
concentration. The equation obtained after multiplication by aH+ is in a form in which the negative terms may be considered negligible and the equation becomes linear. For higher H+ concentrations (i.e., when aH+ → ∞), eq 9 assumes the following form:9 A − A 2 ASMH+ a H+ = ASMH+a H+ + 1 s KSMH+
Equation 9 may be treated in an analogous way following substitution of H+ concentration by OH− concentration. For higher OH− concentrations (i.e., when aOH− → ∞), the equation is reduced to9 aOH− A − A 2 ASMOH− = ASMOH−aOH− + 1 s KSMOH−
where aSM, aSMH+, aSMOH−, and aSMHOH are the surface concentrations of SM, SMH+, SMOH−, and SMHOH forms of sphingomyelin (mol m−2); aH+,aOH− are the concentrations of ions in the subphase (mol m−3); KSMH+, KSMOH−, and KSMHOH are the equilibrium constants of adsorption process of H+ or OH− ions on sphingomyelin (m3 mol−1). The sum of the surface concentrations of all sphingomyelin forms at the air/water interface must be equal to the total surface concentration of sphingomyelin (s). This concentration may be easily determined from the π−A isotherms. Moreover, the sum of the area fractions of the four sphingomyelin forms should be unity. These relationships are mathematically expressed as aSM + aSMH+ + aSMHOH + aSMOH− = s (7)
Using these relationships, it is possible to calculate ASMH and ASMOH− by regression in regions of higher H+ or OH− concentrations. Equation 12 may then be used to compare the calculated and experimental values obtained on the basis of eqs 10 and 11. Good agreement indicates that the system is well described by the above equations. To perform the comparison, eq 9 is presented in the form9 1 = s
A1 KSMOH−
K
+
+ a H+ASMH+ K SMH − + aOH−ASMOH− SMOH
A2 KSMOH−
K
+
+ a H+ K SMH − + aOH− SMOH
(12)
The equations necessary to evaluate (KSMH+)/(KSMOH−) may be obtained from eqs 4 and 5 using the values of aSMH+ and aSMOH− at the isoelectric point. The interfacial tension may be calculated using the model provided that the interfacial tension of the sphingomyelin layer is the sum of the contributions from all forms, i.e., that ideal mixing of the different forms of sphingomyelin occurs in the layer. As was mentioned above, the molecular areas of the sphingomyelin forms influence the interfacial tension. The surface concentrations of the forms are the same as those described by eqs 4−6. Equations 13 and 14 describe further dependencies in the system:
(8)
where s is the total surface concentration of sphingomyelin based on π−A isotherms (mol m−2) and ASM, ASMH+, ASMOH−, and ASMHOH are the molecular areas of SM, SMH+, SMOH−, and SMHOH (Å2 molecule−1). Equations 4−8 quantitatively describe the influence of subphase pH on the sphingomyelin monolayer. Monolayers assembled from a single SM form would have different stability constants depending on the form of SM that was present. The surface concentration of each sphingomyelin form affects the molecular packing of the head groups, which subsequently influences the interfacial tension of the lipid monolayer. The surface area per molecule and surface concentration are therefore dependent on the pH of the subphase, and monolayers composed of various relative amounts of each SM form will exhibit different areas per molecule. Elimination of the aSM, aSMH+, aSMHOH, and aSMOH− terms from eqs 4−8 yields9 A + a H+ASMH+KSMH+ + aOH−ASMOH−KSMOH− 1 = 1 s A 2 + a H+KSMH+ + aOH−KSMOH−
(11) +
aSMASM + aSMH+ASMH+ + aSMHOHASMHOH + aSMOH− ASMOH− = 1
(10)
Ai =
γi0 γ ·s
(13)
0 0 0 0 γ = γSMH + γSM + γSMOH + + γ − SMHOH
(14)
Ai is the area occupied by 1 mol of adequate form of sphingomyelin (SM, SMH+; SMOH−; SMHOH (Å2 molecule−1), γ0i is the interfacial tension of the specified form of sphingomyelin (mN m−1), and γ is the measured interfacial tension obtained from π−A isotherms. As the interfacial tension may be treated as the interfacial energy concentrated at the interfaces, we assume based on the additivity rule that the interfacial tension of the sphingomyelin layer is the sum of the interfacial tensions of the SM forms. The relationship between the surface concentration, the total surface concentration s, and the interfacial tension becomes aSMHOH aSMH+ aSMOH− 0 0 0 γ = γSMHOH + γSMH + γSMOH + − s s s 0 aSM + γSM (15) s
(9)
where A1 = ASM + ASMHOHKSMHOH A 2 = KSMHOH + 1
The direct form of eq 9 is not convenient for calculations, but after substitution of the OH− concentration with the quotient of KH2O and H+ concentration, the numerator of the polynomial may be divided by the denominator, resulting in a series of terms containing decreasing powers of H +
After substitution of eqs 4−6 into eq 15 we obtain9 13332
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Langmuir γ=
Article
0 0 − γ1 + a H+γSMH K +K SMH+ + aSMOH−γ SMOH− SMOH
a H+KSMH+ + aOH−KSMOH−
The surface concentrations were calculated from measurements of the surface area occupied by sphingomyelin molecules using the equation
(16)
where
s=
0 0 γ1 = γSM + γSMHOH KSMHOH
in which s is the surface concentration of sphingomyelin (mol m ), A is the surface area occupied by a phospholipid molecule (m2 molecule−1), and NA is Avogadro’s constant. The experimental results were highly reproducible, and the standard deviation for the surface area measurements was less than 1%. The reported values represent the average of at least five experiments. Reagents. Sphingomyelin from chicken egg yolk (98%) was purchased from Fluka and used as received. The molecular weight of the sphingomyelin was approximately 731.1 g mol−1. The 1-chloropropane solvent (>98% pure) was supplied by Aldrich. Solutions were prepared by dissolving appropriate amounts of each material in 1-chloropropane at a concentration of 1 mg cm−3 and were stored at 4 °C. The water used in the experiments was prepared by triple distillation (the second distillation was performed over KMnO4 and KOH to remove organic impurities). Buffers (pH 2−12) were prepared21 by adding 0.2 M sodium hydroxide to 100 mL of solution containing 0.04 M 80% acetic acid (Polish Chemical Reagents, POCh), 0.04 M phosphoric acid (POCh), and 0.04 M boric acid (POCh). The pH of the buffer was adjusted using sodium hydroxide. The Britton and Robinson21 buffer formulation was selected for the experiments, because it is a standard buffer in biochemical experiments, because of its applicability to a wide pH range (2−12), and because it does not interact with biological membranes.
0 γa H+ = γSMH +a H+ 0 0 0 0 KSMHOH(γSMHOH − γSMH − γSMH +) + (γ +) SM
KSMH+ (17)
This approximation enables calculation of the interfacial tension value of sphingomyelin form with adsorbed H+. For basic solutions as aOH− → ∞9 0 γaOH− = γSMOH −aOH−
+
0 0 0 0 KSMHOH(γSMHOH ) − γSMOH − γSMOH −) + (γ SM
KSMOH− (18)
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The accuracy of the assumed model and/the additivity of the sphingomyelin forms may be verified using eq 19.9 γ1
γ=
KSMOH−
KSMH+ 0 0 + a H+γSMH + aSMOH−γSMOH + − KSMOH− γ2 K + + a H+ K SMH − + aOH− KSMOH− SMOH
RESULTS AND DISCUSSION Measurements of interfacial tension in lipid monolayers are useful in determining the surface area per molecule. The pH dependence of various membrane properties is of interest in both medical and biological applications. The dependence of the surface area per sphingomyelin molecule on the pH of the subphase may be investigated using π−A isotherms. Figure 1 contains π−A isotherms of sphingomyelin in Britton and Robinson buffer at pH 2.5 (denoted by 1), pH 5 (denoted
(19)
where 0 0 γ1 = γSM + γSMHOH KSMHOH
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(20) −2
Similar to the equations describing the area per molecule, applying reasonable approximations to the polynomial eq 16 yields the following forms depending on the conditions For higher H+ concentrations (i.e., when aH+→ ∞)9
+
1 NA ·A
γ2 = KSMHOH + 1
EXPERIMENTAL SECTION
Measuring Apparatus and Measuring Procedures. Surface tension measurements were obtained using a previously described homemade computer-controlled apparatus.17 The measurements were carried out at an air/water interface at 22 °C, and the results were expressed as surface pressure−area per molecule (π−A) isotherms. Before each trial, the Teflon trough (size 648 cm2) was washed and rinsed with purified water. The subphase surface was cleaned just prior to each measurement by suction with a vacuum pump until the surface tension was constant and equal to the surface tension of pure water at 22 °C (approximately 72 mN m−1). All glassware in contact with the samples was cleaned with chromic acid and repeatedly rinsed with purified water before use. The system was enclosed in an acrylic box to minimize water evaporation, to ensure constant humidity, and to avoid contamination of the system. Triple-distilled water was used as the subphase in all experiments. The monolayers were prepared by spreading a defined volume of lipid dissolved in 1-chloropropane on the aqueous subphase using a Hamilton microsyringe. Ten minutes were allowed before each experiment for solvent evaporation and monolayer equilibration. The monolayer was continuously compressed using a glass barrier to obtain the π−A isotherms. The Nima ST9002 computer program was used to calculate the surface pressure of the monolayer π as a function of surface area per molecule A: π = γ − γ0 = f(A), where γ is the surface tension of the lipid-covered surface and γ0 is the surface tension of the bare air/water interface.17
Figure 1. π−A isotherms of sphingomyelin monolayers: Britton and Robinson buffer at pH = 2.5 (1), pH = 5 (2), pH = 8 (3), and pH = 11 (4).
by 2), pH 8 (denoted by 3), and pH 11 (denoted by 4) subphases. The sphingomyelin isotherms were in satisfactory agreement with previous reports.15,20 Sphingomyelin monolayers are an example of liquid-expanded membranes, with the hydrophilic head groups located in the aqueous subphase and the hydrophobic fatty acid tails oriented toward the air. The surface area per lipid molecule assumes various values depending on the length, conformation, and degree of unsaturation of the hydrocarbon chains. The surface areas for the sphingomyelin molecule in Britton and Robinson buffers were determined experimentally by extrapolating the isotherm 13333
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to π = 0. The molecular surface areas in pH 2.5, pH 5, pH 8, and pH 11 buffers were 35 Å2, 37 Å2, 38 Å2, and 39 Å2. Figure 2 presents the inverse values of the measured sphingomyelin surface concentrations as a function of subphase
phospholipid bilayers is assumed to be negligible. In addition, the interfacial tension at the interface between the phospholipid headgroups and the subphase are the same for both monolayers and bilayers. As a result, the difference between the monolayer interfacial tension obtained experimentally and the interfacial tension of the bilayer is equal to the interfacial tension of the hydrophobic layer−air interface. We proceeded in a way similar to Jähning,23 who approximated the hydrophobic layer−air interfacial tension by substituting the interfacial tension of an nalkane−air interface. Since the interfacial tension of the hydrophobic chain−air interface is predominant, these values were applied to the system and used in subsequent calculations.24 In Figure 4, the points denote the calculated interfacial tension of lipid monolayers for the air−sphingomyelin
Figure 2. Inverse of sphingomyelin surface concentration as a function of pH subphase (the experimental values are indicated by points and the theoretical values by the curve).
pH. The experimental values are depicted as points, and the solid curve was calculated using eq 12. When the subphase was acidic (pH 2.0), the surface concentration s was equal to 4.88 × 10−6 mol/m2. The value of s increased sharply, reaching a maximum near the isoelectric point of sphingomyelin. It is noteworthy that this maximum was not obtained at the isoelectric point (4.01) determined for SM bilayers.16 In the case of monolayers, increasing ionization caused by the increasing H+ concentration resulted in a maximum value of 2.77 × 105 m2/mol at pH 4.05. When the pH of the subphase was further increased, the surface concentration decreased sharply between pH 4.05−5.00, then remained relatively constant between pH 5−12 (Figure 2). Using eq 10, the area of the SMH+ form was calculated to be 2.05 × 105 m2 mol−1 (34 Å2 molecule−1). Since extrapolation from only a few experimental points can produce unreliable results, we also calculated the value of the SMH+ form by fitting the experimental curve using a least-squares estimation algorithm, obtaining a value of 2.29 × 105 m2 mol−1 (38 Å2 molecule−1). The relative stability from pH 5 to 12 permits the use of the extrapolated SMOH− surface concentration for similar calculations to obtain a surface concentration for the SMOH− form of 2.28 × 105 m2 mol−1 (∼38 Å2 molecule−1). Figure 3 presents the interfacial tension values measured for the sphingomyelin monolayer. The interfacial tension changed
Figure 4. Air/hydrophobic chain interfacial tension of sphingomyelin monolayer as function of pH (the experimental values are indicated by points and the theoretical values by the curve).
interface. The values were determined from the difference between the interfacial tension of monolayers and bilayers composed of the same form of sphingomyelin.16 The calculated values obtained from eq 19 are denoted in the figure by a solid line. It is worth emphasizing that the interfacial tension of the hydrophobic chain−air interface is pH dependent. The latter interface is the dominant contributor to the interfacial tension. The minimum interfacial tension was obtained when the pH approached the isoelectric point, reaching 41 mN m−1 at pH 4.05. As the subphase pH was increased, the interfacial tension increased until pH 6, at which the interfacial tension was approximately 47.50 mN m−1. These results were used to calculate the interfacial tensions of the SMH+ (40.51 mN m−1) and SMOH− (47.27 mN m−1) forms. The interfacial tension values at pH levels below 2 were calculated using eq 17 and are presented in Figure 3. Remarkably, the experimental measurements were in good agreement with the calculated values presented in the figure as a solid line. The predicted molecular areas were verified using eq 12, and the interfacial tensions were verified using eq 19. The estimated values (solid line) are very close to the experimental results represented by points. The results confirm the existence of four sphingomyelin forms, regardless of whether the sphingomyelin is present as a monolayer or a bilayer. The P− end of the sphingomyelin headgroup is anchored at the air/water interface, while the hydrocarbon chains are oriented toward the air. The resulting hydrophobic effect drives the methyl and methylene groups around the N+ charge toward the hydrocarbon region. In order to reduce hydrocarbon−water contact, the polar head groups are closely packed. This restricts the freedom of the lipid chains and results in exertion of a lateral pressure on their surroundings. The area per molecule is entirely determined by the area of the headgroup.
Figure 3. Interfacial tension of sphingomyelin monolayer at air/water interface vs pH of subphase.
only slightly from pH 2 to 6. A further decrease in the H+ concentration resulted in an abrupt change in the plot, with the interfacial tension increasing continuously to pH 8−10, then remaining relatively constant at higher OH− concentrations. In the Langmuir approach,22 the monolayer surface tension is composed of an air−hydrophobic layer component and a polar layer−aqueous subphase component. The interfacial tension for the hydrophobic chain−hydrophobic chain interface in 13334
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and Transition Phenomena in the Film. Proc. R. Soc. London, Ser. A 1932, 138, 430−450. (9) Brzozowska, I.; Figaszewski, Z. A. The Influence of pH on Phosphatidylcholine Monolayer at the Air/Aqueous Solution Interface. Colloids Surf., B 2003, 27, 303−309. (10) Tessier, C.; Quinn, P.; Koumanov, K.; Trugnan, G.; Rainteau, D.; Wolf, C. Modulation of the Phase Heterogeneity of Aminoglycerophospholipid Mixtures by Sphingomyelin and Monovalent Cations: Maintenance of the Lamellar Arrangement in the Biological Membranes. Eur. Biophys. J. 2004, 33, 513−521. (11) Tatulian, S. A. Ionization and Ion Binding. In Phospholipid Handbook, Cevc, G., Ed.; Marcel Dekker: New York, 1993. (12) Rydall, J. R.; Macdonald, P. M. Investigation of Anion Binding to Neutral Lipid Membranes Using 2H NMR. Biochemistry 1992, 31, 1092−1099. (13) Petrache, H. I.; Zemb, T.; Belloni, L.; Parsegian, V. A. Salt Screening and Specific Ion Adsorption Determine Neutral-Lipid Membrane Interactions. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 7982− 7987. (14) Vaknin, D.; Kelley, M. S.; Ocko, B. M. Sphingomyelin at the AirWater Interface. J. Chem. Phys. 2001, 115, 7697−7704. (15) Petelska, A. D.; Naumowicz, M; Figaszewski, Z. A. The Equilibrium of Phosphatidylcholine-Sphingomyelin in a Monolayer at the Air/Water Interface. Pol. J. Chem. 2008, 82, 2323−2330. (16) Petelska, A. D.; Figaszewski, Z. A. pH Effect of the Sphingomyelin Membrane Interfacial Tension. J. Membr. Biol. 2009, 230, 11−19. (17) Petelska, A. D.; Figaszewski, Z. A. The Equilibria of Phosphatidylethanolamine-Cholesterol and Phosphatidylcholine− Phosphatidylethanolamine in Monolayers at the Air/Water Interface. J. Macromol. Sci. A 2009, 46, 607−614. (18) Petelska, A. D.; Figaszewski, Z. A. The Equilibria of Phosphatidylcholine−Fatty Acid and Phosphatidylcholine−Amine in Monolayers at the Air/Water Interface. Colloids Surf., B 2011, 82, 340−344. (19) Petelska, A. D.; Naumowicz, M; Figaszewski, Z. A. Interfacial Tension of the Lipid Membrane Formed From Lipid-Fatty Acid and Lipid-Amine Systems. Bioelectrochemistry 2007, 70, 28−32. (20) Shaikh, S. R.; Dumaual, A. C.; Jenski, L. J.; Stillwell, W. Lipid Phase Separation in Phospholipid Bilayers and Monolayers Modeling the Plasma Membrane. Biochim. Biophys. Acta 2001, 1512, 317−328. (21) Engineering Handbook; Wydawnictwa Naukowo-Techniczne: Warsaw, 1974. (22) Langmuir, I. Oil Lenses on Water and the Nature of Monomolecular Expanded Films. J. Chem. Phys. 1933, 1, 756−776. (23) Jähning, F. Lipid Exchange Between Membranes. Biophys. J. 1984, 46, 687−694. (24) Nagle, J. Theory of Lipid Monolayer and Bilayer Phase Transitions: Effect of Headgroup Interactions. J. Membr. Biol. 1976, 27, 233−250.
The surface charge is pH-dependent and easily modified by free protons. The pH of the subphase therefore affects the molecular packing of the sphingomyelin headgroup and subsequently the area occupied by chains at the air− hydrocarbon interface. When comparing Figures 2 and 3, it is clear that the interfacial tension at the air−water interface is not as strongly dependent on pH as it is in the case of air− phospholipid chains.
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CONCLUSIONS
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AUTHOR INFORMATION
The proposed model is based on the additivity of the interfacial tension and molecular area of the various sphingomyelin forms. The relative contribution of each sphingomyelin form depends on the pH of the subphase. The interfacial tension and molecular area were calculated for the SMH+ and SMOH− forms of sphingomyelin. These were 2.05 × 105 m2 mol−1 (34 Å2 molec.−1) and 40.51 mN m−1 for SMH+ and 2.29 × 105 m2 mol−1 (38 Å2 molec.−1) and 47.27 mN m−1 for SMOH−. The area of the hydrophilic head groups in sphingomyelin determines the surface concentration and consequently the interfacial tension at the air/hydrophobic chain interface. The difference between the experimentally obtained monolayer interfacial tension and the bilayer interfacial tension is equal to the interfacial tension of the hydrophobic layer−air interface. The model agreed well with the experimental values. The mathematically derived and experimentally confirmed results presented here are of great importance for the interpretation of surface phenomena occurring in lipid monolayer. These results can lead to a better understanding of the physical properties of biological membranes. The simple and very interesting methods proposed in this paper and in earlier studies15−19 may be used with success to determine the lipid−lipid and lipid−ion equilibria in the lipid monolayer.
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Karp, G. Cell and Molecular Biology: Concepts and Experiments, Wiley and Sons: New York, 2004. (2) Koval, M.; Pagano, R. E. Intracellular Transport and Metabolism of Sphingomyelin. Biochim. Biophys. Acta 1991, 1082, 113−125. (3) Thudicum, J. L. W. in A Treatise on the Chemical Constitution of Brain; Bailliere, Tindall and Cox: London, 1884. (4) Merrill, A. H., Jr.; Schmelz, E.-M.; Dillehay, D. L.; Spiegel, S.; Shayman, J. A.; Schroeder, J. J.; Riley, R. T.; Voss, K. A.; Wang, E. Sphingolipids-The Enigmatic Lipid Class: Biochemistry, Physiology, and Pathophysiology. Toxicol. Appl. Pharmacol. 1997, 142, 208−215. (5) Nyberg, L.; Duan, R. D.; Nilsson, A. Sphingomyelin - a Dietary Component with Structural and Biological Function. Prog. Colloid Polym. Sci. 1998, 108, 119−128. (6) Talbott, C. M.; Vorobyov, I.; Borchman, D.; Taylor, K. G.; DuPre, D. B.; Yappert, M. C. Conformational Studies of Sphingolipids by NMR Spectroscopy. II. Sphingomyelin. Biochim. Biophys. Acta 2000, 1467, 326−337. (7) Ramstedt, B.; Slotte, J. P. Membrane Properties of Sphingomyelins. FEBS Lett. 2002, 531, 33−37. (8) Schulman, J. H.; Hughes, A. H. On the Surface Potentials of Unimolecular Films. Part IV. The Effect of the Underlying Solution 13335
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