Changes in human meibum lipid composition with age using nuclear magnetic resonance spectroscopy

Abstract

Purpose: Human tear film stability decreases with increasing age. In this study, the changes in meibum composition were measured in search of markers of tear film instability.

Methods: (1)H NMR nuclear magnetic resonance (NMR) spectra of 43 normal donors aged 1 to 88 years were acquired.

Results: Compared with meibum from adolescents and adults, meibum from infants and children contains less CH(3) and C═C groups and an increased aldehyde-to-lipid hydroperoxide ratio.

Conclusions: It is reasonable that tear film stability is higher in infants than in adults. Their meibum contains less CH(3) and C═C groups and higher levels of protein, and as a result, the lipid is more ordered because of the tighter and stronger lipid-lipid interactions. For water to evaporate, it must first pass through the tight lipid-lipid barrier. For tears to break up, lipid-lipid interactions must be broken. It is reasonable that because the lipid-lipid interactions are stronger in infants' and children's tears compared with those of adolescents and adults, the tear film in the younger groups is more stable and provides a better barrier to evaporation than does the tear film of adults. Lipid saturation could be the critical feature in meibum that stabilizes tears in infants.

Figure 1.

An average ¹H NMR spectrum of human meibum from donors with meibomian gland dysfunction. The ¹H NMR spectra of human meibum may be divided into five regions: the CH₂ and CH₃ region from 0 to 1.4 ppm, the CH and deshielded CH₂ region from 1.4 to 3.9 ppm, the ester region from 3.9 to 4.7, the C═C region from 4.7 to 7 ppm, and the oxidative products region above 7 ppm.

Figure 2.

The relative areas of bands that changed between the infant group and child-adult groups. Area total is the total area for all the proton resonances. Resonance assignments are given in Table 2. Sample data are provided in Table 1.

Figure 3.

¹H NMR spectrum of meibum from a 4-year-old Caucasian girl. The region contains protons assigned to CH₃ moieties. The proton moiety associated with the resonance is underscored.

Figure 4.

(A) A standard curve was prepared from the ¹H NMR spectra of mixtures of palmityl palmitate and oleyl oleate, using the area ratio of the resonances at 3.98 and 5.31 ppm to determine hydrocarbon chain saturation. The standard curve was linear (r = 0.999), with a slope of 2.06 ± 0.02. (B) Some of the ¹H NMR spectra of mixtures of palmityl palmitate and oleyl oleate used to make the standard curve in (A). Proton resonances associated with the ester protons next to the ester bond of wax appear at 3.98 ppm. Proton resonances associated with HC═CH bonds are located at 5.31. The lowest intensity in the 5.31-ppm resonance region is due to palmityl palmitate with no double bonds. The highest intensity in the 5.31-ppm resonance is due to oleyl oleate, with two double bonds per molecule. Between these spectra at 5.31 ppm are spectra of palmityl palmitate/oleyl oleate at 0.25 and 0.6 (wt:wt).

Figure 5.

The relative areas of bands that change between the child group and adolescent-adult groups. Area total is the total area for all the resonances. Resonance assignments are given in Table 2. Sample data are provided in Table 1.

Figure 6.

Two proton resonances related to aldehydes appear at 9.7 and 9.65 ppm. Two proton resonances related to hydroperoxides appear at 7.9 and 8.5 ppm. The areas of the two aldehyde resonances were divided by the areas of the two hydroperoxide resonances in the plot at the top. Sample data are provided in Table 1.