First, we tested the effects of increasing amounts of meibum loaded onto the air–liquid interface at a physiological temperature of 34°C. Unlike standard lipids (
Fig. 1B), meibum did not produce any kinks or bulges in the isotherms (which would have been indicative of classical collapsing) in
any of the tested conditions, regardless of the amount of meibum loaded. To the best of our knowledge, a rationale for choosing the amount of human meibum needed to form a condensed monolayer has never been provided before, primarily because of the insufficient information on the chemical structures, molecular masses, and relative ratios of lipids present in human meibum. At this moment, the exact calculations of the surface occupancies for a mixture of various compounds as complex as meibum are still not possible. However, our recent publications on the topic
13,33,34 allowed us to estimate the possible ranges of these parameters. Postulating that (1) an average molecular weight of a well-characterized meibomian lipid [MW
AV] is ∼700 (which is an average of 650 for wax esters and 750 for cholesteryl esters and (
O-acyl)-omega-hydroxy fatty acids
13,33,34 ), and (2) its apparent partial molecular area per molecule in a condensed monolayer (
A m) should be approximately 60 to 70 Å
2 (an average between ∼20 Å
2 for compounds such as free fatty acids and wax esters,
30,35 60 Å
2 for saturated triacylglycerols,
31,36 and 80 to 120 Å
2 for the bulkiest unsaturated triacylglycerols and cholesteryl esters
30,36 –38 ), one would need ∼1.4 × 10
16 molecules or ∼16 μg of meibum per 80-cm
2 area to form a condensed monolayer. Tragoulias et al.
16 used a very close number of molecules (1.72 × 10
16) in their calculations, but they worked with rabbit meibum, did not provide a rationale for this number, and did not specify whether they considered the rabbit meibum layers to be condensed. The largest amount of meibum used in our experiments presented in
Figure 2 was ∼21 μg, which was sufficient to form a condensed monolayer, even when the trough barriers were open, and guaranteed a multilayered structure at higher degrees of lateral compression of the layer. Of importance, under any tested conditions the meibum layer did not show any signs of the classic collapsing via fracturing or solubilization illustrated in
Figure 1B. At the same time, a slight drop in π
in on adding the increasing amounts of meibum was observed. The factors that lead to such an effect are not yet clear. However, several classes of compounds that cause measurable increases in the surface tension at the water-air interfaces are known and include carbohydrates, amino acids, and salts.
39 It is possible that meibum has a small, but important, pool of compounds in its composition that are capable of restructuring the surface layer, leading to the increase in its surface tension and the simultaneous decrease in the surface pressure. Note that the negative π
in is small, manifests itself only at very high meibum loads, and quickly disappears once the layers start to compress.