The purpose of this study was to investigate in more detail the evaporation-retarding effect of WEs, because they are a major lipid component in TFLL. In meibum, the most abundant WEs are based on oleic (∼75%), or palmitoleic acid (∼8%) and 24:0, 25:0, 26:0 and 27:0 branched fatty alcohols.
3,20,21 For this study, the WEs were selected as close as possible to those found in meibum. We endeavored to reveal more details about the evaporation-retarding effect and the physical behavior of these somewhat hydrophobic molecules at the air–water interface, using mainly Langmuir film techniques.
It must be mentioned that studying WEs using the Langmuir film techniques is challenging, because their hydrophobic characteristics lead to aggregation at the air–water interface. Therefore, the concentration and volume of the liquid in which WEs are applied to the surface affect the liftoff area of the isotherms. Second, the profiles of the WE isotherms are not as repeatable as with phospholipids due to the low compression stability of the layers. Therefore, the results must be interpreted cautiously.
To begin with, we measured the melting points of WEs because, surprisingly, this data could not be found from the literature. Based on the melting points, the WEs were grouped into three categories: those that have their melting points much lower than the physiological temperature, those that have their melting points close the physiological temperature, and those that have extremely high melting points (
Table).
We measured the evaporation rates at the near-physiological temperature of 35°C and found out that four WEs were evaporation retardant (
Fig. 1). BP, BLN, and BLNN, in addition to BO discovered in our previous study
13 decreased evaporation rates by 20% to 40%. A common factor for these lipids was that they melted within 2°C of the physiological temperature. By visual inspection alone we noticed clear differences in the spreading of the lipids. The saturated long-chain WEs spread poorly, forming raft-like aggregates at the air–water interface, whereas shorter-chain and unsaturated WEs spread more uniformly.
The observations above suggested that the differences in the evaporation-retarding effects of WEs were caused by certain peculiar physical properties of WEs, when they are very close to their solid–liquid phase transition. More simply, if the WEs are in their solid state, a large area of the interface is not covered by lipid and the lipid rafts float like icebergs on the sea. Similarly, when WEs are in a very fluid-like state, the extensive wobbling motion of lipids allows for a large free-volume to accumulate within the lipid membrane, and this allows the water molecules to travel through the WE membrane.
22 At close to their melting point, WEs form a structure, tentatively reminiscent of percolated or soft monolayers,
23 which retards evaporation. This is distinctive behavior, as the evaporation-retarding effect is not related to the melting point for the more typical retardants such as BAl (melting point of 71°C ± 1°C).
24 In these layers the evaporation-retarding effect is achieved by very tight packing of the acyl chains, with exceedingly low free volume within the lipid layer.
As the evaporation-retarding effect was obviously related to the melting point, we also measured the evaporation rates through BO, BP, BLN, and BLNN layers at 30°C and 41°C. The purpose was to discover whether these lipids still retarded evaporation when they were more clearly in solid state at 30°C or in liquid state at 41°C. At 30°C BLN and BLNN, having the lowest melting points of the four WEs, decreased evaporation by ∼50%, whereas BP and BO retarded evaporation by only 5% to 10%. At 41°C, the decrease in evaporation was only 2% to 4%, when all the WEs were in liquid state. In summary, the data suggest that the melting point of the lipid has to be at or just above the interfacial temperature for the retardation of evaporation to occur.
To further test this theory of melting point dependency, we measured the evaporation-retarding effect of AO also at 30°C and 41°C. AO was selected because it melts at 33°C ± 1°C and, therefore, should retard evaporation at 30°C, based on our theory. As expected, this indeed was the case. AO decreased the evaporation rate by 16% ± 1%, but it did not retard evaporation at 41°C as expected. The evaporation-retarding effect was noticeably smaller than those of BLN and BLNN and only ∼5% larger than that of BP.
At 35°C, the differing spreading behavior of WE species was observed using BAM (
Fig. 2). Long-chain saturated LL formed thick three-dimensional aggregates at the interface, whereas short-chain LO spread more uniformly, showing only few small aggregates. Based on the BAM images alone, we cannot assume that the black areas are covered with a lipid layer; however, the higher surface pressure produced by LO suggests that it is more uniformly spread than LL. The similar aggregate formation and surface pressures of BO, BLNN, and BLN accompanied with the similar evaporation-retarding effect imply that these WE layers were in the same phase. BP also spread uniformly when applied to the interface but also formed larger areas of layered/aggregated lipid layer. The somewhat stagnant appearance of BP also reminded us of a phase that is condensed or gel-like compared to the more liquid BO, BLN, and BLNN layers. As a reference,
Figure 2 (upper panel) shows a uniformly spread BAl layer. The gray shading, presented more clearly in the high-exposure image on the right-hand side, indicates that the layer is in a condensed phase. The effective evaporation-retarding effect of BAl is caused by the small cross-sectional area of the molecule (small polar head group and only one hydrophobic alkyl chain), which enables close packing of the molecules.
Based on the BAM images, it seems likely that the evaporation-retarding effect for these four WEs is the result of the condensed-like phase existing at the proximity of the liquid-condensed phase transition, as hypothesized above. BP seems somewhat more condensed than the BO, BLN, and BLNN layers; hence, the more efficient evaporation-retarding effect of the BP layer. The WEs possibly organize at the air–water interface so that the carboxyl group is in the aqueous phase and the alcohol and acyl chains point toward the air in a V-shaped manner.
25 However, aggregation or stacking of layered structures is also expected, as 500 nmol of WE spread uniformly results here as a mean area of ∼9 Å
2/molecule, whereas the minimum molecular area for a fatty alcohol is ∼20 Å
2 . Therefore, the approximated thickness of these layers is <100 nm, depending on the minimum mean molecular area occupied by the WEs. This is the same order of magnitude as the in vivo thickness of TFLL.
26
The isotherms indicate that the selected WE layers share common properties (
Fig. 3). The transfer of the liftoff area is simply explained by increased aggregation, that is, excess WEs are pushed out of the interface to form a new overlaying layer, either as an organized structure or as bulky three-dimensional aggregates. Consecutive compression cycles cause the WE layer to aggregate more, and because of the hydrophobic nature, it is energetically more favorable for the “excess” molecules not to return to the interface on relaxation. This poor respreading shows as large hysteresis observed in the isotherms. The decrease in compressibility instead results in steeper isotherm profiles when compared to the first cycles. Finally, the isotherms also suggest that WEs at the air–water interface take a somewhat organized lateral structure since the layers are tolerant to high surface pressures without collapsing.
As expected, the liquid state LO and AO were the most compressible of the WEs tested. They also showed less hysteresis, especially at higher surface pressures. The kinks at ∼8 mN/m for AO and ∼23 mN/m for LO also suggest phase transition, but the phase transitional behavior of these WE layers is beyond the scope of this study, as the evaporation-retarding effect existed already at lower surface pressures (<5 mN/m) than where the potential phase transitions took place. In contrast, solid state BL at the other extreme has a spike-like isotherm profile. The profile is easily explained by visual inspection of the lipid layer, or moreover a lipid raft, during compression. By the first compression, BL behaved somewhat similarly to the other WE layers, but by relaxation, no spreading took place due to the solid state of the lipid. BL stayed floating as a vast raft in the middle of the trough. Therefore, during consecutive compression–relaxation cycles the changes in surface pressure took place during very minimal movement of the Langmuir balance barriers due to the close-to-zero compressibility of the raft. The surfactant properties of evaporation-retardant WEs located well between the solid state BL and liquid state AO, as expected based on the melting point of these four evaporation-retardant lipids. However, based on the isotherms it is challenging to draw any further conclusions about the structural properties of the lipid layers that induce the evaporation-retardant nature of the WE layer. It is clear that these four retardant layers have very similar surfactant properties and, therefore, most likely very similar structures. Overall, the Langmuir film experiments unveiled the poor compression–expansion behavior of the WE layers.
The dependency between the physical state of the WE layer at given temperatures and the ability to retard evaporation may partially explain the aging-related changes in the meibum composition and therefore in the stability of the tear film.
27,28 One may speculate that because of the changes in WE structures, such as changed degree of saturation and branching of the carbon chains, the order and melting temperature of the TFLL decrease and lead to a reduced ability to retard evaporation. Therefore it is possible that the infant TFLL retards evaporation more effectively than an adult TFLL, which would also explain the lower blinking frequency of the infants.
In summary, we have shown here that only certain WEs retard evaporation at physiological temperatures. The ability to retard evaporation is dependent on the physical properties of the WEs at a given temperature. The evaporation-retarding effect may be explained by formation of a specific condensed-like phase of the WE layer, which exists at the proximity of the solid–liquid phase transition. Under conditions of consecutive compression–relaxation cycles, the buildup of multilayered or aggregated WE structures continues until a pressure-tolerant WE layer is formed. Despite the high surface pressure tolerance, WEs are missing the main hallmarks of a stable lipid layer. They organize very slowly into a layer form, prefer aggregation over uniform spreading, are incompressible, and finally, create lipid layers that are unstable in a dynamic environment. Therefore, they need to be accompanied by more efficient surfactants such as polar phospholipids. As emphasized in our previous studies, phospholipids, such as phosphatidylcholine and sphingomyelin, are prerequisites for the spreading and controlled function of TFLL-like lipid layers.
4,29–31 If such an evaporation-retardant TFLL-like lipid layer composed of nonpolar and polar lipids exists, WEs most likely play a major role in that structure.