Differential scanning calorimetric evaluation of human meibomian gland secretions and model lipid mixtures: transition temperatures and cooperativity of melting

Abstract

Meibomian gland secretions (or meibum) are produced by holocrine meibomian glands and are secreted in melted form onto the ocular surface of humans and animals to form a protective tear film lipid layer (TFLL). Its protective effect strongly depends on the composition and, hence, thermotropic behavior of meibum. The goal of our study was to quantitatively evaluate the melting characteristics of human meibum and model lipid mixtures using differential scanning microcalorimetry. Standard calorimetric parameters, e.g. changes in calorimetric enthalpy, transition temperatures Tm, cooperativity of melting, etc. were assessed. We found that thermotropic behavior of meibum resembled that of relatively simple mixtures of unsaturated wax esters, but showed a lower change in calorimetric enthalpy, which can be indicative of a looser packing of lipids in meibum compared with pure standards and their simple mixtures. The cooperativity of melting of meibomian lipids was comparable to that of an equimolar mixture of four oleic-acid based wax esters. We demonstrated that the phase transitions in meibum start at about 10-15°C and end at 35-36°C, with Tm being about 30°C. The highly asymmetrical shape of the thermotropic peak of meibum is important for the physiology and biophysics of TFLL.

Figure 1

A typical baseline-corrected DSC thermogram of a wax ester stearyl oleate and thermodynamic parameters computed from the data.

Figure 2

Validation of the experimental procedures. Panel A. A correlation between the literature data on melting temperatures of standard wax esters and T_m values determined in experiments with dry-loaded samples. Panel B. A correlation between the T_m values of standard wax esters determined using the “dry-loading” procedure and in experiments with chloroformic solutions of the analytes (“wet loading”). Panel C. Raw, baseline-corrected thermograms of palmityl oleate obtained in four repetitive experiments are shown. Note an incremental, but unilateral, shift in T_m values toward lower temperatures with each repetitive cycle. Panel D. Four repetitive raw, baseline-corrected thermograms of stearyl oleate are shown. The shift in T_m was much less noticeable than in experiments with palmityl oleate.

Figure 3

Thermograms of a four-component lipid mixture. The mixture composition was as follows: arachidyl oleate:behenyl oleate:stearyl stearate:behenyl stearate = 1:1:0.5:0.5 (mol). The first melting/cooling cycle (labeled as 1-st scan) is considered to be a preparatory cycle during which the lipids mix, distribute, and assume their final shape inside the microcalorimeter’s cell. Note the bimodal nature of the thermogram. The ΔH_cal of the peaks were comparable.

Figure 4

A sample thermogram of a binary mixture of palmityl laurate and behenyl stearate. Solid line: experimental data; broken line: fitted curve. The curve-fitting procedure was performed using the ThermoCal/Origin software. Note the bimodal nature of the thermotropic transitions of the mixture. The ΔH_cal of the peaks were close to each other. The thermograms were baseline-corrected.

Figure 5

Effects of mixing of four unsaturated wax esters on their phase transition behavior. Panel A. Raw thermograms of four unsaturated wax esters tested individually and as a four-component equimolar mixture. Tested lipids: palmityl oleate (PO), stearyl oleate (SO), arachidyl oleate (AO), and behenyl oleate (BO). Solid lines: individual wax esters; broken line: the mixture of four waxes. The thermograms were baseline-corrected. Panel B. Deconvolution of a thermogram of the four-lipid mixture shown in Panel A. Note that at least four individually melting domains with comparable ΔH_cal values are needed to adequately describe the experimental thermogram. For this particular mixture of four lipids, the following individual T_m were calculated: 16.2, 19.9, 22.8, and 25.0 °C. The value of T_1/2 for the entire thermotropic peak was calculated to be about 8.4 min, T_m was 24.9 °C, while ΔH_cal was 16 kCal/mole. Solid line: experimental data; broken line: deconvoluted peaks; dotted line: the sum of individual melting peaks.

Figure 6

Effects of mixing of four saturated wax esters on their phase transition behavior. Tested sample – an equimolar mixture of palmityl laurate, palmityl palmitate, stearyl stearate, and behenyl stearate. Note an approximately equal sizes of Peak I and Peak II. Solid line: experimental data; broken line: deconvoluted peaks I and II; dotted line: the sum of individual melting peaks a-d.

Figure 7

Effects of mixing of four unsaturated wax esters on ΔH_cal. Lipids were mixed in the equimolar ratio (0.5 μmol each). The experimental ΔH_cal of the mixture (the left-most bar) was about 80% of the arithmetic sum of its individual components (the right-most bar).

Figure 8

Thermotropic behavior of human meibum. Panel A. Five repetitive raw thermograms of a sample of human male meibum. Approximately 0.4 mg of meibum was loaded. Panel B. Repetitive raw thermograms of a sample of human meibum after baseline correction. Panel C. A deconvoluted thermogram of a male meibum sample. At least three T_m were needed to adequately model the thermotropic behavior of human meibum. Cooperativity of melting of these three peaks, expressed as n, rose from 10 to 40 to 100 (from left to right). Note a striking resemblance of the meibum thermogram and a thermogram of a four-component mixture of unsaturated wax esters depicted in Figure 5, Panel B. Panel D. Comparison of two human samples. Both samples were loaded into the microcalorimeter in the amount of 0.4 mg (male, solid line; female, broken line). Note almost identical values of T_m (about 30 °C), T_1/2 (10.0 ± 0.5 °C), and the completion temperature of transition (35.5 ± 0.5 °C) determined for both the samples.