Lipidomics of human Meibomian gland secretions: Chemistry, biophysics, and physiological role of Meibomian lipids

Abstract

Human Meibomian gland secretions (MGS) are a complex mixture of diverse lipids that are produced by Meibomian glands that are located in the upper and the lower eyelids. During blinking, MGS are excreted onto the ocular surface, spread and mix with aqueous tears that are produced by lachrymal glands, and form an outermost part of an ocular structure called "the tear film" (TF). The main physiological role of TF is to protect delicate ocular structures (such as cornea and conjunctiva) from desiccating. Lipids that are produced by Meibomian glands are believed to "seal" the aqueous portion of TF by creating a hydrophobic barrier and, thus, retard evaporation of water from the ocular surface, which enhances the protective properties of TF. As lipids of MGS are interacting with underlying aqueous sublayer of TF, the chemical composition of MGS is critical for maintaining the overall stability of TF. There is a consensus that a small, but important part of Meibomian lipids, namely polar, or amphiphilic lipids, is of especial importance as it forms an intermediate layer between the aqueous layer of TF and its upper (and much thicker) lipid layer formed mostly of very nonpolar lipids, such as wax esters and cholesteryl esters. The purpose of this review is to summarize the current knowledge on the lipidomics of human MGS, including the discussions of the most effective modern analytical techniques, chemical composition of MGS, biophysical properties of Meibomian lipid films, and their relevance for the physiology of TF. Previously published results obtained in numerous laboratories, as well as novel data generated in the author's laboratory, are discussed. It is concluded that despite a substantial progress in the area of Meibomian glands lipidomics, there are large areas of uncertainty that need to be addressed in future experiments.

Figure 1

Tear film and tear film lipid layer (reprinted from [43] with permission)

Figure 2

Measuring of tear film breakup time using fluorescein staining and a slit lamp. Panel A: uniform staining of the tear film after a blink. Panel B: irregular staining developed as the result of tear film deterioration caused by non-blinking

Figure 3

Visible light microscopy evaluation of meibomian glands (panel A) and manual expression of meibomian gland secretions from a lower eyelid of a volunteer using two cotton swabs. Panel A. Meibomian glands are marked with black arrows. Panel B. Expressed meibum is marked with a black arrow.

Figure 4

Infrared photographs of meibomian glands of a young healthy female volunteer (28 years old) (courtesy of Dr. R. Arita). The glands are visible as wavy white structures. Panel A. Upper eyelid. Panel B. Lower eyelid.

Figure 5

Histochemical staining of meibomian glands. An upper mouse meibomian gland is shown. The tissue lipids were stained with Oil Red O and counter-stained with hematoxylin. Notice accumulation of large amount of stained lipids (bright red) in the main (central) duct of the gland.

Figure 6

Publications on the topic of meibomian lipids since 1960 (data from PubMed; as of January, 2011).

Figure 7

Ultraviolet spectra of three oxidized derivatives of docosahexaenoic acid – 17-hydro(per)oxy-DHA (solid), non-conjugated dioxygenated 10,20-dihydroxy-DHA (long dashes), and a conjugated deoxygenated 10,17-dihydroxy-DHA (short dashes) in hexane-iso-propanol mixture (1:1, v/v)

Figure 8

Separation of chiral and positional isomers of monohydroxylated linoleic acid on a chiral column Chiralcel OD-H (reprinted from [116] with permission).

Figure 9

Direct GC-MS analysis of a test mixture of 10 underivatized wax esters (Panel A) and of a sample of human meibum collected from a healthy volunteer (Panel B). Elution of the analytes from a GC column was performed using a temperature gradient. Panel A. Ten WE were (in the order of elution): myristyl laurate (m/z 396), lauryl oleate (m/z 450), palmityl oleate (m/z 506), stearyl oleate (m/z 534), stearyl stearate (m/z 536), arachidyl oleate (m/z 562), arachidyl stearate (m/z 564), behenyl oleate (m/z 590), behenyl stearate (m/z 592), and behenyl behenate (m/z 648). Five chromatograms presented here depict (from top to bottom): 1) total ion chromatogram of the mixture; 2) elution profile of myristyl laurate; 3) elution profile of a common product ion m/z 283 (a proton adduct of oleic acid); the ion is generated due to spontaneous fragmentation of parent WE ions and allows all WE that contain oleic acid to be detected; 4) elution profile of a common product ion m/z 285 (a proton adduct of stearic acid); the ion is generated due to spontaneous fragmentation of parent WE ions and allows all WE that contain stearic acid to be detected; 5) elution profile of a unique for this mixture ion m/z 340 (a proton adduct of behenic acid) generated spontaneously from behenyl behenate. Panel B. Human meibum was analyzed similarly the test mixture of WE described above. From top to bottom, four chromatograms are shown: 1) total ion chromatogram of normal human meibum; 2) elution profile of ion m/z 283 (C_18:1-FA-based WE); 3) elution profile of ion m/z 271 (WE based on a C_17:0-FA); 4) elution profile of ion m/z 255 (WE based on a C_16:1-FA). The last three chromatograms show that the apparent abundance of C_16:1-FA-based WE is much lower than that of C_18:1- and C_17:0- FA-based WE.

Figure 10

HPLC-MS analysis of OAHFA (reprinted from [21] with permission). Panel A. Elution profile of free OAHFA as detected by RP HPLC-APCI MS in negative ion mode. Three most intense HPLC peaks are formed of three major OAHFA species present in human meibum (m/z 729, 757, and 785). Panel B. Elution profile of OAHFA m/z 757 Panel C. Averaged mass spectrum of OAHFA eluted between 6 and 16 min into the experiment. Panel D. Fragmentation mass spectrum of ion m/z 757 and its proposed structure Panel E. RP-HPLC profile of a synthetic OAHFA, (O-oleoyl)-16-hydroxypalmitic acid (m/z 535). Panel F. Zoom scan mass spectrum of authentic (O-oleoyl)-16-hydroxypalmitic acid Panel G. Fragmentation spectrum of (O-oleoyl)-16-hydroxypalmitic acid

Figure 11

Reverse phase chromatograms of an OAHFA (m/z 703) and a WE (m/z 673). Note that the compounds have the same number of carbons in their structures, but their retention times differ due to two additional oxygen atoms in OAHFA, which makes it much more hydrophilic that the corresponding WE.

Figure 12

Proposed orientation of an OAHFA at the air/water interface. Hydrophilic oxygen atoms are forming hydrogen and electrostatic bonds with water molecules and hydroniums, respectively. Electronegative oxygen atoms are labeled with their negative partial atomic charges (computed in a MM2/MMP2 molecular modeling experiment in MMX parametrization)

Figure 13

A positive ion mode ESI mass spectrum of a mixture of common plasticizers and plastic stabilizers extracted from an Eppendorf tube upon its exposure to a chloroform:methanol (1/1, v/v) solvent mixture. The signals of fatty acid amides are labeled according to the length of their carbon chains. The ions of both types, (M + H)⁺ and (M + Na)⁺, were detected. A very similar pattern was reported by Nichols et al [94], and was erroneously attributed to the lipids present in meibomian gland secretions.

Figure 14

Comparison of HPLC-UV and HPLC-MS approaches to phospholipid analysis. Panel A. An NP HPLC-UV method adopted from [63] was used to analyze a mixture of PE, PC, and SM. An aliquot of the mixture (800 ng of each lipid) was injected and the elution profile of the lipids was monitored at 220 nm. Note that there was no HPLC peaks of the phospholipids observed after the initial injection peak. A conclusion can be made that because of its insensitivity, the HPLC-UV approach is not suitable for analyzing complex lipid mixtures with small amounts of phospholipids, such as human meibum. Panel B. The same mixture of three lipids was analyzed by NP HPLC-ESI MS in positive ion mode. Note that only a 40 ng aliquot of each lipid was injected. Three lipids were clearly visible with a negligible background noise. Panel C. The same mixture of three lipids was analyzed by NP HPLC-ESI MS in positive ion mode. Note that this time only a 2 ng aliquot of each lipid was injected. Three lipids were still detectable albeit with a clearly declining signal-to-noise ratios.

Scheme 1