Human meibum lipid conformation and thermodynamic changes with meibomian-gland dysfunction

Abstract

Purpose: Instability of the tear film with rapid tear break-up time is a common feature of aqueous-deficient and evaporative dry eye diseases, suggesting that there may be a shared structural abnormality of the tear film that is responsible for the instability. It may be that a change in the normal meibum lipid composition and conformation causes this abnormality. Principle component analyses of infrared spectra of human meibum indicate that human meibum collected from normal donors (Mn) is less ordered than meibum from donors with meibomian gland dysfunction (Md). In this study the conformation of Md was quantified to test this finding.

Methods: Changes in lipid conformation with temperature were measured by infrared spectroscopy. There were two phases to our study. In phase 1, the phase transitions of human samples, Mn and Md, were measured. In phase 2, the phase transitions of model lipid standards composed of different waxes and cholesterol esters were measured.

Results: The phase-transition temperature was significantly higher (4°C) for the Md compared with the Mn of age-matched donors with no history of dry-eye symptoms. Most (82%) of the phase-transition temperatures measured for Md were above the values for Mn. The small change in the transition temperature was amplified in the average lipid order (stiffness) at 33.4°C. The average lipid order at 33.4°C for Md was significantly higher (30%, P = 0.004) than for Mn. The strength of lipid-lipid interactions was 72% higher for Md than for Mn. The ability of one lipid to influence the melting of adjacent lipids is termed cooperativity. There were no significant differences between Mn and Md in phase-transition cooperativity, nor was there a difference between Mn and Md in the minimum order or maximum order that Mn and Md achieved at very low and very high temperatures, respectively. The model wax studies showed that the phase transition of complex mixtures of natural lipids was set by the level of unsaturation. A double bond decreased the phase-transition temperature by approximately 40°C. The addition of a second CH CH moiety decreased the phase-transition temperature by approximately 19°C. Unsaturated waxes were miscible with saturated waxes. When a saturated wax was mixed with an unsaturated one, the saturated wax disproportionately increased the phase transition of the mixture by approximately 30°C compared with the saturated wax alone. Cholesterol ester had little effect on the phase-transition temperature of the waxes. Model studies indicated that changes in the amount of lipid saturation, rather than the amount of cholesterol esters, could be a factor in the observed conformational changes.

Conclusions: Meibum lipid compositional changes with meibomian gland dysfunction reflect changes in hydrocarbon chain conformation and lipid-lipid interaction strength. Spectroscopic techniques are useful in studying the lipid-lipid interactions and conformation of lipid from individual patients. (ClinicalTrials.gov number, NCT00803452.).

Figures

Figure 1.

Multilamellar monolayers of ordered (solid, or unmelted) wax molecules that are arranged in an orthorhombic crystal structure in which the hydrocarbon chain conformation is all trans. The hydrocarbon chains of fluid waxes contain gauche rotomers.

Figure 2.

(a) A typical infrared spectrum in a 20-year-old man without dry eye symptoms. The infrared spectrum of meibum closely resembles the infrared spectrum of the wax oleyloleate (b) and the spectrum of cholesterylpalmitate (c). (d) The spectrum of lysozyme is scaled to the amide II band in (a) to show that even if all the intensity of the band marked amide II in (a) were from protein, there would be no significant overlap with the lipid CH stretching bands.

Figure 3.

(A) The relationship between donor age and phase-transition temperature for meibum lipid from donors with MGD. (B) The relationship between donor age and hydrocarbon chain order at lid temperature, 33.4°C, from meibum lipid from donors with MGD. Error bars are ± SE. Lines: linear fit and 95% confidence limits of data from meibum lipid donors without a history of dry eye symptoms, as reported in Reference . F, female; M, male; B, black; C, Caucasian; H, Hispanic.

Figure 4.

Linear relationship between lipid phase-transition temperature and order of meibomian lipid from donors with meibomian-gland dysfunction (●) and donors without a history of dry-eye symptoms (○).

Figure 5.

(A) Wax phase transition curves for OO oleyloleate, PO palmityloleate, PP palmitylpalmitate, SP sterylpalmitate. (●) Heating curve; (○) cooling curve. Lines are the fit of the data to equation 1 in Borchman et al. The lower the CH2 symmetric stretching frequency, the more ordered the wax hydrocarbon chains. (B) Melting curve for sterylpalmitate. The scale is enlarged from (A) so that hysteresis between the cooling (○) and heating (●) curves can be observed. (C) Equal molar mixture of sterylpalmitate and oleyloleate (●) heating and (○) cooling curve. Sterylpalmitate:oleyloleate:cholesterylpalmitate 2:2:1 (m:m:m) where m is moles. (▴) heating curve, (▵) cooling curve. (D) Enlargement of the scale in (C).

Figure 6.

Relationship between phase-transition temperature and hydrocarbon chain saturation. Samples measured in this study: OO oleyloleate, PO palmityloleate, PP palmitylpalmitate, SP sterylpalmitate, PPPO equimolar mixture of PP and PO, SPOO equimolar mixture of SP and OO. Data are reported in Ref. . Solid line: least-squares linear regression fit to all the data. Human meibum donor data are presented in Table 6. HL human lens lipid, ROS P bovine rod outer segment plasma membrane, SR F fast twitch rabbit muscle sarcoplasmic reticulum membrane, SR S slow twitch rabbit muscle sarcoplasmic reticulum membrane.

Figure 7.

Infrared spectra of the carbonyl region for (A) sterylpalmitate/oleyloleate, 1:1, m:m. Temperatures (°C) from top to bottom: 70.96, 59.47, 55.45, 52.14, 51.91, 51.37, 50.93, 50.57, 50.01, 49.41, 49.35, 48.78, 48.02, 42.74, 35.5, and 24.17. (B) Palmityloleate temperatures (°C) from top to bottom: 24.00, 20.25, 20.17, 20.14, 20.09, 20.02, 19.92, 19.85, 19.78 19.69, 19.63, 19.55 19.36, 19.16, 18.72, 18.47, 18.38, 18.35, 17.64, 16.93, 16.80, 13.59, and 12.83. (C) The carbonyl bandwidth at half height (▴) increased then decreased at the transition temperature measured by the carbonyl band center (○). All data are from heating curves. (D) Infrared spectra of the CO region: (top) sterylpalmitate/oleyloleate, 1:1, m:m, 70.96°C; sterylpalmitate/oleyloleate/cholesterylpalmitate, 2:2:1, m:m:m, 70.96°C; sterylpalmitate/oleyloleate/cholesterylpalmitate, 2:2:1, m:m:, 26.07°C; sterylpalmitate/oleyloleate, 1:1, m:m, 24.17°C; (bottom) cholesterylpalmitate 24°C.

Figure 8.

Average infrared spectra of human meibum showing (A) the carbonyl band region. Top: cholesteryl palmitate; middle: above the Tm at 55°C; bottom: below the Tm at 15°C. (B) The C—C rocking band region. Top: above the Tm at 55°C; middle: below the Tm at 15°C; bottom: cholesteryl palmitate.

Figure 9.

(A) Infrared spectra of the CH2 bending region for: (a) sterylpalmitate/oleyloleate, 1:1, m:m, 70.96°C; (b) sterylpalmitate/oleyloleate/cholesterylpalmitate, 2:2:1, m:m:m, 70.96°C; (c) sterylpalmitate/oleyloleate/cholesterylpalmitate, 2:2:1, m:m:, 26.07°C; and (d) sterylpalmitate/oleyloleate, 1:1, m:m, 24.17°C; (e) cholesterylpalmitate 24°C. Infrared spectra of the C—C rocking region for: (B) sterylpalmitate/oleyloleate, 1:1, m:m. Temperatures (°C) from top to bottom: 70.96, 59.47, 55.45, 52.14, 51.91, 51.37, 50.93, 50.57, 50.01, 49.41, 49.35, 48.78, 48.02, 42.74, 35.5, and 24.17. (C) Infrared spectra of the C—C rocking region: (a) sterylpalmitate/oleyloleate, 1:1, m:m, 70.96°C; (b) sterylpalmitate/oleyloleate/cholesterylpalmitate, 2:2:1, m:m:m, 70.96°C; (c) sterylpalmitate/oleyloleate/cholesterylpalmitate, 2:2:1, m:m:, 26.07°C; (d) sterylpalmitate/oleyloleate, 1:1, m:m, 24.17°C; and (e) cholesterylpalmitate 24°C. (D) C—C rocking bands of sterylpalmitate/oleyloleate, 1:1, m:m converge at the transition temperature.