NMR spectra of meibum from 51 donors with MGD (Md) were compared with those obtained for meibum from 44 normal donors (Mn). Donor characteristics are provided in
Table 1.
1H-NMR spectra of human meibum provide a wealth of information about the amount and chemical shift δ of protons (
Fig. 1). The region below 1.39 ppm (CH
2 band) shows
1H resonances associated with CH
3 moieties. Resonances attributed to protons associated with carbon carbon double bonds appear between 5 and 6 ppm. Above 6 ppm, the resonances correspond to protons that are more deshielded due to the presence of nearby carbonyl groups. Detailed band assignments will appear in future reports.
The region between 4 and 4.8 ppm is of interest to this study because it contains
1H resonances associated with the ester groups of cholesteryl esters, glycerides, and waxes (
Fig. 1, inset). Waxes exhibit proton resonances between 3.9 and 4 ppm (
Fig. 1), corresponding to CH
2 protons (
Fig. 2). Glycerides display resonances between 4.1 and 4.3 ppm (
Fig. 1) also attributed to CH
2 protons of the glycerol backbone (
Fig 2). The broad band at 4.6 ppm (
Fig. 1) is attributed to the single hydrogen attached to carbon 3 of cholesterol that is adjacent to the acyl linkage of the cholesteryl ester (
Fig. 2). The region between 3.9 and 4.8 ppm was used to quantify the relative amount of cholesteryl esters, glycerides, and waxes.
1H-NMR spectra of model lipid compounds in known quantities were acquired and, as expected, the area of the resonances at 4.6 and 2.3 ppm (
Table 2) varied as the concentration of cholesteryl ester was changed (
Fig. 3). A calibration curve was constructed by plotting the ratio of integrated areas of the resonances at 4.6 ppm (due to cholesteryl ester) and 4.1 ppm (due to wax) versus the molar ratio of the model cholestoryl ester and wax (
Fig. 4A). The curve was linear with an intercept of 0, a slope of 0.459 mol/mol, and a correlation coefficient of 0.9995. A slope of 0.5 is expected since the band at 4.65 ppm arises from only one hydrogen per mole of cholesterol, whereas the band at 4.08 corresponds to two hydrogens per mole of wax. The difference between the expected value, 0.5, and the actual experimental value, 0.459, may be explained by the presence of impurities in the model compounds.
To evaluate all the possible spectral variations in the spectra collected for the model compounds (
Table 2), we used PCA.
44 –46 PCA has been used to analyze infrared spectra of human meibum.
32,33 PCA is a chemometric approach that enables the assessment of differences, even very subtle ones, in a set of spectra. Then, the differences are correlated to a “principal component” that is a variable, such as cholesteryl ester and wax content in our case. An eigenvector represents a constituent that changes its relative contribution from sample to sample. Using a press plot, we determined that only two eigenvectors were necessary to describe most of the variance in the NMR spectra of our model compounds. The actual values of cholesteryl ester to wax molar ratios were plotted versus those predicted using PCA analysis (
Fig. 4B). They were linearly correlated, with a slope of 0.89 and correlation coefficient of
r = 0.93. Like the curve in
Figure 4A and for the same reasons, the actual values of cholesteryl ester to wax molar ratios were slightly lower than the predicted values, thus leading to the lower than unity slope.
Because the ratio of the integrated areas (
Fig. 4A) provided a standard curve with a correlation coefficient closer to unity compared with that obtained in the PCA-derived standard curve (
Fig. 4B), we used the curve shown in
Figure 4A to measure the mole fraction of cholesteryl ester to wax (CE:Wax) in human meibum samples. The average value obtained for CE:Wax was significantly higher, about twofold (
P < 0.05), for the normal adolescent-to-adult group compared with the normal infant, child, and MGD groups (
Fig. 5,
Table 3). The median value for CE:Wax mole fraction was similar to the average values for Mn but slightly lower for Md (
Table 3). The CE:Wax molar ratios for “Mn child” were relatively tightly distributed compared with the molar ratios for “Mn adolescent to adult” and Md (
Fig. 6). The CE:Wax molar ratios for “Mn adolescent to adult” were distributed into three groups (
Fig. 6B) and those for Md were distributed into two groups. The major group for Md centered near 0.15 molar ratio encompassed approximately 60% of the samples (
Fig. 6C).
Triglyceride was quantified in a standard mixture with a detection limit of approximately 5 μmol (
Fig. 4C). The standard curve produced by the ratio of the areas due to triglycerides and wax resonances as a function of their molar ratio was linear,
r = 0.986. Even when the areas of the triglyceride resonances were not normalized to other resonances (
Fig. 4D), the triglyceride standard curve was also linear,
r = 0.988, suggesting that the magnetic field remains stable during the acquisition time. The two resonances between 4.2 and 4.3 ppm (
Fig. 1) were assigned to the four CH
2 protons associated with the glycerol backbone (
Fig. 2). The relative amounts of glycerides were not significantly different among the groups (
Table 3). The amount of glyceride in meibum was about two orders of magnitude above the detection limit of the instrument.
The intensity, width, and location of bands measured in our infrared and Raman spectroscopic studies of meibum are often sensitive to the conformation and the environment around the moieties associated with the band, thus complicating compositional analysis. Unlike vibrational and electronic spectroscopy, and if aggregation does not occur, the area of
1H-NMR resonances is proportional to the number of protons and is not affected by the surrounding environment. Therefore, quantitative studies based on
1H-NMR do not require standards for every different chain length and saturation. We acquired
1H-NMR spectra of eight different waxes spanning a wide range of chain lengths and saturation: steryl oleate, oleyl oleate, palmityl oleate, arachidyl oleate, steryl sterate, palmityl palmitate, myristyl laurate, and palmityl laurate. As expected, the ratios of the areas of the resonances at 3.98 and 0.88 ppm, assigned to the two protons of the first CH
2 group in the alkyl chain of the waxes (
Fig. 2) and to the six protons of the two terminal CH
3, respectively, were not dependent on saturation or chain length, and their averaged ratio was 0.322 with SD of 0.008. This is close to the expected ratio of 0.333 (two to six protons). Palmityl laurate contains the shortest alkyl chain length (12 carbons), whereas arachidyl oleate has the longest alkyl chain (20 carbons). The ratios were 0.328 and 0.327, the shortest and longest alkyl chain waxes, respectively.
Matrix-assisted laser desorption/ionization, time of flight mass spectrometry was used to detect free cholesterol in all our samples.
50 Free cholesterol was <5% of the cholesteryl esters, thus suggesting that cholesteryl esters were not hydrolyzed in our samples (data not shown).
Milligram quantities of model phospholipids were analyzed by 1H-NMR spectroscopy under the same conditions used for the meibum samples. No phospholipid was detected because glycero- and sphingophospholipids are not soluble in cyclohexane.