Letters Controversies Regarding the Role of Polar Lipids in Human and Animal Tear Film Lipid Layer Letter to the Editor ecause an abundance of data, often controversial, on the presence (or absence) and the role(s) of polar (or, more precisely, amphiphilic) lipids (PLs) in the human and animal tear film lipid layer (TFLL) has been published in recent years, a review on the topic is highly relevant for the clinical and scientific community working on the tear film. This is the important goal set by Pucker and Haworth (Ocul Surf 2015; 13[1]:2642), who made an exhaustive effort to aggregate and comment on the diverse range of published data and hypotheses. However, in their review, Section III. B, “Significance of Polar Tear Lipids,” contains some controversies and misinterpretations that need to be commented on and corrected in order to present to the readership of The Ocular Surface the most precise view with regard to the current understanding of the tear film structure and function. A major question in the field of TFLL is whether meibomian lipids alone can be considered to represent the overall properties of TFLL or whether the eventual admixtures of PLs (primarily phospholipids [PhL] and sphingolipids [SL]) from the aqueous tear can have a significant impact on TFLL structure and properties. It is clear that meibomian lipids represent the major constituent of TFLL. The main PLs of meibum are the (O-Acyl)-u-hydroxy fatty acids (OAHFA) that represent 3%-5% or 30,000-40,000 ppm of meibomian lipids.1,2 As confirmed by virtually all
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recent lipidomics studies, the amount of PhL and SL in meibum is negligible and can be due to trace amounts picked up as an artifact during the sample collection. As Butovich has noted,2 “Generally, almost any two (or more) types of lipids can mix to form a more or less homogeneous mixture, but only when one of the components is present in sufficiently small amounts compared to the other (PL are). Some lipids are quite miscible, while the others have limited mutual miscibility. When a threshold of miscibility has been reached, the lipid mixtures start to segregate, forming separate phases enriched with one of the components. Phase diagrams of these binary, ternary, etc. types of mixtures are used to illustrate what state the mixture is in at a particular ratio of its components.” Based on a range of published data on the amount of PhL and SL in aqueous tears3-5 and even exaggeratedly assuming that all PhL and SL found in aqueous tears get associated with TFLL (which, of course, will never be the case), Butovich1,2 concludes that the maximum possible amount of PhL and SL that can be reached in TFLL will be 1200 ppm, i.e., still an extremely low amount compared to the 30,000-40,000 ppm of OAHFA present in meibomian lipids. As further as rationalized by Butovich et al,6 “considering the results published by Georgiev et al (Colloids Surf B Biointerfaces 2010; 78: 317e27), who demonstrated that to have a significant effect on the biophysical properties of MLF, phospholipids have to be present in the amounts 0.2 molar parts (i.e., 200,000 ppm) or more, the impact of the tiny amounts of phospholipids
observed in our earlier experiments (Butovich et al: Lipids 2007; 42:765e76) and in a recent paper (Dean and Glasgow: Invest Ophthalmol Vis Sci 2012; 53:1773e82) on the properties of meibomian lipid films is expected to be minor.” Thus, as already recognized in the literature, the study of Georgiev et al7 does not try to identify the minimal amount of PLs necessary for TFLL to function but makes an effort to clarify what is the minimum amount of “exogenous” PhL and/or SL that will prevent homogeneous mixing with meibomian lipids and will seriously perturb the structure of the meibomian films at the air/water surface. The threshold of 0.2 molar parts indicates that meibomian films are quite robust and can sustain a significant amount of external “contamination” without their structure being altered. This can be due to good miscibility of OAHFA with the exogenous PLs or other more intricate mechanism (e.g., solubilization of PLs in the non-polar stratum of the meibomian films2). The statement of Pucker and Haworth on page 28 of their review is completely different. Citing Butovich’s paper,1 they state: “Butovich once estimated based on past literature that, from a structural standpoint, at least 5% of the tears needed to be polar lipids to form an interphase.” Continuing, citing our 2010 paper,7 the authors state: “In sharp contrast, Georgiev et al demonstrated with a Langmuir trough using egg sphingomyelin (SM), dipalmitoylphosphatidylcholine (DPPC), and bovine meibum that the minimum polar lipid concentration needed to simulate an interaction between the two layers was about 20% (0.2 molar).”
Contrary to the statements of Pucker and Haworth, the study of
THE OCULAR SURFACE / JULY 2015, VOL. 13 NO. 3 / www.theocularsurface.com
LETTERS / Georgiev; Pucker and Haworth Georgiev et al7 examines among others a completely different point: What is the maximum amount of polar lipids exogenous to meibum that can be added to meibomian lipid films and still have homogeneous mixing between the two occur? Thus, as stated by Butovich et al,6 the ultimate question is whether the amount of PhL and SL that can be brought by aqueous tears to the TFLL is large enough. If the estimate of 1200 ppm (vs 30,000-40,000 ppm of OAHFA) is correct, then the impact of PhL and SL on meibum can be neglected and meibum can be considered as the determinative source of lipids for TFLL. However, later data8,9 suggest that the amount of PLs in aqueous tear is far higher than previously thought and the amphiphilic part of TFLL consists of 2.5% OAHFAs, 6.0% phospholipids, and 1.8% sphingolipids. Here, careful analysis has to be performed on the reasons behind the discrepancies between older and more recent measurements. If the recent values stand the test of time, then the partition coefficients have to be evaluated of the PLs between the aqueous tear and the TFLL. Concerning the intricacies posed by the presence of lipidbinding proteins, e.g., tear lipocalin and surfactant proteins A/B/C and D,4,10,11 and amphiphilic molecules (competing for the air/tear surface) in tears, this is a challenging task that goes beyond the scope of this letter. Also, as recently demonstrated,12-14 surface rheology studies can be far more sensitive than the commonly used Langmuir film tensiometry measurements, and these are yet to be performed concerning the interactions of meibum and polar lipids. Further, it is important to consider the notion for a minimum, “threshold,” concentration of PLs needed in TFLL for its proper function, assuming that “at least 5% of the tears needed to be polar lipids to form an interphase.”1 The estimate is used in the referenced study of Butovich1 to outline once more that OAHFA should be the most probable amphiphilic compound of TFLL as the then (pre-2009)
estimate was that PhL and SL are in trace amount in tears and therefore cannot compete with OAHFA for the TFLL/aqueous tear interface. As already explained in detail, the question of the minimal amount of polar lipids necessary to be present in TFLL is not addressed at all in our 2010 study7 or studies by other groups. This aspect is indeed very hard to address because it is not possible to obtain the NPL lipids of TFLL alone, free of PL admixtures, and to see how this “purely NPL fraction” will behave by itself. As mentioned above, meibum contains 3%-5% of its own “endogenous” amphiphilic lipids, OAHFA,1,2,6,8.9 and, as confirmed by numerous biophysical studies of mutually independent groups (e.g., Millar’s, Fuller’s, and ours), those are enough to allow meibomian lipids alone to form stable films at the air/water surface, although, as mentioned by Pucker and Haworth, “OAHFA alone fail to meet or barely meet Butovich’s 5% threshold.” Actually, it is not at all clear that the aqueous interface should be entirely covered by PLs in order for stable TFLL to be formed. In the detailed study of Petrov et al,15 a set of state-of-the-art biophysical techniques (Langmuir monolayers, glancing-incidence x-ray diffraction, Brewster angle microscopy) were used to characterize the surface properties and structure of a composite synthetic replica of TFLL: “70.2% (w/w) oleic acid ethyl ester (OEA) and 21.1% cholesteryl stearate (CS) as the hydrophobic part, and 4.5% phosphatidylcholine (PC), 1.9% phosphatidylethanolamine (PE), 0.9% sphingomyelin (SM), 0.7% phosphatidylserine (PS) 0.5% phosphatidylinositol (PI) and 0.2% lysophosphatidylcholine (LPC) as a mimic to the polar part, all with 16 carbon atoms saturated aliphatic chains except PI (soybean) and SM (egg yolk).” The synthetic replica proved capable of forming non-collapsible, rough films of multilayer thickness similar to the layers formed by bovine meibum. The study confirmed that even at high,
>30 mN/m, surface pressures, the aqueous interface got covered by a mixture of PLs and a significant amount of non-polar lipids. Thus, some presence of non-polar lipids mixed with the PLs at the TFLL/water interface cannot be excluded. Of course, if the recently reported8-11 excess of PLs and lipid-binding proteins in aqueous tears receives further confirmation, then the covering of the aqueous/TFLL interface with a layer composed entirely of PLs protruding in the aqueous tear and interlinked with lipid-binding proteins looks highly probable. Still, the current level of knowledge does not allow definitive interpretation, and great care is necessary in the analysis of the accumulated data.
Georgi As. Georgiev, PhD Associate Professor Department of Biochemistry Faculty of Biology St. Kliment Ohridski University of Sofia Sofia, Bulgaria E-mail address: [email protected]fia.bg
REFERENCES 1. Butovich IA. The meibomian puzzle: combining pieces together. Prog Retin Eye Res 2009;28:483-98 2. Butovich IA. Tear film lipids. Exp Eye Res 2013;117:4-27 3. Campbell D, Griffiths G, Tighe BJ. Tear analysis and lens-tear interactions: part II. Ocular lipids-nature and fate of meibomian gland phospholipids. Cornea 2011;30: 325-32 4. Dean AW, Glasgow BJ. Mass spectrometric identification of phospholipids in human tears and tear lipocalin. Invest Ophthalmol Vis Sci 2012;53:1773-82 5. Saville JT, Zhao Z, Willcox MD, et al. Detection and quantification of tear phospholipids and cholesterol in contact lens deposits: the effect of contact lens material and lens care solution. Invest Ophthalmol Vis Sci 2010;51: 2843-51 6. Butovich IA, Lu H, McMahon A, et al. Toward an animal model of the human tear film: biochemical comparison of the mouse, canine, rabbit, and human meibomian lipidomes. Invest Ophthalmol Vis Sci 2012;53: 6881-96
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LETTERS / Georgiev; Pucker and Haworth 7. Georgiev GA, Kutsarova E, Jordanova A, et al. Interactions of Meibomian gland secretion with polar lipids in Langmuir monolayers. Colloids Surf B Biointerfaces 2010;78:317-27 8. Lam SM, Tong L, Duan X, et al. Extensive characterization of human tear fluid collected using different techniques unravels the presence of novel lipid amphiphiles. J Lipid Res 2014;55:289-98 9. Brown SH, Kunnen CM, Duchoslav E, et al. A comparison of patient matched meibum and tear lipidomes. Invest Ophthalmol Vis Sci 2013;54:7417-24
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10. Bräuer L, Kindler C, Jäger K, et al. Detection of surfactant proteins A and D in human tear fluid and the human lacrimal system. Invest Ophthalmol Vis Sci 2007;48:3945-53 11. Bräuer L, Paulsen FP. Tear film and ocular surface surfactants. J Epithel Biol Pharmacol 2008;1:62-7 12. Georgiev GA, Yokoi N, Ivanova S, et al. Surface relaxations as a tool to distinguish the dynamic interfacial properties of films formed by normal and diseased meibomian lipids. Soft Matter 2014;10: 5579-88
13. Bhamla MS, Giacomin CE, Balemans C, et al. Influence of interfacial rheology on drainage from curved surfaces. Soft Matter 2014;10:6917-25 14. Svitova TF, Lin MC. Lens-care-solutioninduced alterations in dynamic interfacial properties of human tear-lipid films. Contact Lens Anterior Eye 2014;37: 368-76 15. Petrov PG, Thompson JM, Abdul Rahman IB, et al. Two-dimensional order in mammalian pre-ocular tear film. Exp Eye Res 2007;84:1140-6
THE OCULAR SURFACE / JULY 2015, VOL. 13 NO. 3 / www.theocularsurface.com
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© 2015 Elsevier Inc. All rights reserved.
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recent lipidomics studies, the amount of PhL and SL in meibum is negligible and can be due to trace amounts picked up as an artifact during the sample collection. As Butovich has noted,2 “Generally, almost any two (or more) types of lipids can mix to form a more or less homogeneous mixture, but only when one of the components is present in sufficiently small amounts compared to the other (PL are). Some lipids are quite miscible, while the others have limited mutual miscibility. When a threshold of miscibility has been reached, the lipid mixtures start to segregate, forming separate phases enriched with one of the components. Phase diagrams of these binary, ternary, etc. types of mixtures are used to illustrate what state the mixture is in at a particular ratio of its components.” Based on a range of published data on the amount of PhL and SL in aqueous tears3-5 and even exaggeratedly assuming that all PhL and SL found in aqueous tears get associated with TFLL (which, of course, will never be the case), Butovich1,2 concludes that the maximum possible amount of PhL and SL that can be reached in TFLL will be 1200 ppm, i.e., still an extremely low amount compared to the 30,000-40,000 ppm of OAHFA present in meibomian lipids. As further as rationalized by Butovich et al,6 “considering the results published by Georgiev et al (Colloids Surf B Biointerfaces 2010; 78: 317e27), who demonstrated that to have a significant effect on the biophysical properties of MLF, phospholipids have to be present in the amounts 0.2 molar parts (i.e., 200,000 ppm) or more, the impact of the tiny amounts of phospholipids
observed in our earlier experiments (Butovich et al: Lipids 2007; 42:765e76) and in a recent paper (Dean and Glasgow: Invest Ophthalmol Vis Sci 2012; 53:1773e82) on the properties of meibomian lipid films is expected to be minor.” Thus, as already recognized in the literature, the study of Georgiev et al7 does not try to identify the minimal amount of PLs necessary for TFLL to function but makes an effort to clarify what is the minimum amount of “exogenous” PhL and/or SL that will prevent homogeneous mixing with meibomian lipids and will seriously perturb the structure of the meibomian films at the air/water surface. The threshold of 0.2 molar parts indicates that meibomian films are quite robust and can sustain a significant amount of external “contamination” without their structure being altered. This can be due to good miscibility of OAHFA with the exogenous PLs or other more intricate mechanism (e.g., solubilization of PLs in the non-polar stratum of the meibomian films2). The statement of Pucker and Haworth on page 28 of their review is completely different. Citing Butovich’s paper,1 they state: “Butovich once estimated based on past literature that, from a structural standpoint, at least 5% of the tears needed to be polar lipids to form an interphase.” Continuing, citing our 2010 paper,7 the authors state: “In sharp contrast, Georgiev et al demonstrated with a Langmuir trough using egg sphingomyelin (SM), dipalmitoylphosphatidylcholine (DPPC), and bovine meibum that the minimum polar lipid concentration needed to simulate an interaction between the two layers was about 20% (0.2 molar).”
Contrary to the statements of Pucker and Haworth, the study of
THE OCULAR SURFACE / JULY 2015, VOL. 13 NO. 3 / www.theocularsurface.com
LETTERS / Georgiev; Pucker and Haworth Georgiev et al7 examines among others a completely different point: What is the maximum amount of polar lipids exogenous to meibum that can be added to meibomian lipid films and still have homogeneous mixing between the two occur? Thus, as stated by Butovich et al,6 the ultimate question is whether the amount of PhL and SL that can be brought by aqueous tears to the TFLL is large enough. If the estimate of 1200 ppm (vs 30,000-40,000 ppm of OAHFA) is correct, then the impact of PhL and SL on meibum can be neglected and meibum can be considered as the determinative source of lipids for TFLL. However, later data8,9 suggest that the amount of PLs in aqueous tear is far higher than previously thought and the amphiphilic part of TFLL consists of 2.5% OAHFAs, 6.0% phospholipids, and 1.8% sphingolipids. Here, careful analysis has to be performed on the reasons behind the discrepancies between older and more recent measurements. If the recent values stand the test of time, then the partition coefficients have to be evaluated of the PLs between the aqueous tear and the TFLL. Concerning the intricacies posed by the presence of lipidbinding proteins, e.g., tear lipocalin and surfactant proteins A/B/C and D,4,10,11 and amphiphilic molecules (competing for the air/tear surface) in tears, this is a challenging task that goes beyond the scope of this letter. Also, as recently demonstrated,12-14 surface rheology studies can be far more sensitive than the commonly used Langmuir film tensiometry measurements, and these are yet to be performed concerning the interactions of meibum and polar lipids. Further, it is important to consider the notion for a minimum, “threshold,” concentration of PLs needed in TFLL for its proper function, assuming that “at least 5% of the tears needed to be polar lipids to form an interphase.”1 The estimate is used in the referenced study of Butovich1 to outline once more that OAHFA should be the most probable amphiphilic compound of TFLL as the then (pre-2009)
estimate was that PhL and SL are in trace amount in tears and therefore cannot compete with OAHFA for the TFLL/aqueous tear interface. As already explained in detail, the question of the minimal amount of polar lipids necessary to be present in TFLL is not addressed at all in our 2010 study7 or studies by other groups. This aspect is indeed very hard to address because it is not possible to obtain the NPL lipids of TFLL alone, free of PL admixtures, and to see how this “purely NPL fraction” will behave by itself. As mentioned above, meibum contains 3%-5% of its own “endogenous” amphiphilic lipids, OAHFA,1,2,6,8.9 and, as confirmed by numerous biophysical studies of mutually independent groups (e.g., Millar’s, Fuller’s, and ours), those are enough to allow meibomian lipids alone to form stable films at the air/water surface, although, as mentioned by Pucker and Haworth, “OAHFA alone fail to meet or barely meet Butovich’s 5% threshold.” Actually, it is not at all clear that the aqueous interface should be entirely covered by PLs in order for stable TFLL to be formed. In the detailed study of Petrov et al,15 a set of state-of-the-art biophysical techniques (Langmuir monolayers, glancing-incidence x-ray diffraction, Brewster angle microscopy) were used to characterize the surface properties and structure of a composite synthetic replica of TFLL: “70.2% (w/w) oleic acid ethyl ester (OEA) and 21.1% cholesteryl stearate (CS) as the hydrophobic part, and 4.5% phosphatidylcholine (PC), 1.9% phosphatidylethanolamine (PE), 0.9% sphingomyelin (SM), 0.7% phosphatidylserine (PS) 0.5% phosphatidylinositol (PI) and 0.2% lysophosphatidylcholine (LPC) as a mimic to the polar part, all with 16 carbon atoms saturated aliphatic chains except PI (soybean) and SM (egg yolk).” The synthetic replica proved capable of forming non-collapsible, rough films of multilayer thickness similar to the layers formed by bovine meibum. The study confirmed that even at high,
>30 mN/m, surface pressures, the aqueous interface got covered by a mixture of PLs and a significant amount of non-polar lipids. Thus, some presence of non-polar lipids mixed with the PLs at the TFLL/water interface cannot be excluded. Of course, if the recently reported8-11 excess of PLs and lipid-binding proteins in aqueous tears receives further confirmation, then the covering of the aqueous/TFLL interface with a layer composed entirely of PLs protruding in the aqueous tear and interlinked with lipid-binding proteins looks highly probable. Still, the current level of knowledge does not allow definitive interpretation, and great care is necessary in the analysis of the accumulated data.
Georgi As. Georgiev, PhD Associate Professor Department of Biochemistry Faculty of Biology St. Kliment Ohridski University of Sofia Sofia, Bulgaria E-mail address: [email protected]fia.bg
REFERENCES 1. Butovich IA. The meibomian puzzle: combining pieces together. Prog Retin Eye Res 2009;28:483-98 2. Butovich IA. Tear film lipids. Exp Eye Res 2013;117:4-27 3. Campbell D, Griffiths G, Tighe BJ. Tear analysis and lens-tear interactions: part II. Ocular lipids-nature and fate of meibomian gland phospholipids. Cornea 2011;30: 325-32 4. Dean AW, Glasgow BJ. Mass spectrometric identification of phospholipids in human tears and tear lipocalin. Invest Ophthalmol Vis Sci 2012;53:1773-82 5. Saville JT, Zhao Z, Willcox MD, et al. Detection and quantification of tear phospholipids and cholesterol in contact lens deposits: the effect of contact lens material and lens care solution. Invest Ophthalmol Vis Sci 2010;51: 2843-51 6. Butovich IA, Lu H, McMahon A, et al. Toward an animal model of the human tear film: biochemical comparison of the mouse, canine, rabbit, and human meibomian lipidomes. Invest Ophthalmol Vis Sci 2012;53: 6881-96
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LETTERS / Georgiev; Pucker and Haworth 7. Georgiev GA, Kutsarova E, Jordanova A, et al. Interactions of Meibomian gland secretion with polar lipids in Langmuir monolayers. Colloids Surf B Biointerfaces 2010;78:317-27 8. Lam SM, Tong L, Duan X, et al. Extensive characterization of human tear fluid collected using different techniques unravels the presence of novel lipid amphiphiles. J Lipid Res 2014;55:289-98 9. Brown SH, Kunnen CM, Duchoslav E, et al. A comparison of patient matched meibum and tear lipidomes. Invest Ophthalmol Vis Sci 2013;54:7417-24
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10. Bräuer L, Kindler C, Jäger K, et al. Detection of surfactant proteins A and D in human tear fluid and the human lacrimal system. Invest Ophthalmol Vis Sci 2007;48:3945-53 11. Bräuer L, Paulsen FP. Tear film and ocular surface surfactants. J Epithel Biol Pharmacol 2008;1:62-7 12. Georgiev GA, Yokoi N, Ivanova S, et al. Surface relaxations as a tool to distinguish the dynamic interfacial properties of films formed by normal and diseased meibomian lipids. Soft Matter 2014;10: 5579-88
13. Bhamla MS, Giacomin CE, Balemans C, et al. Influence of interfacial rheology on drainage from curved surfaces. Soft Matter 2014;10:6917-25 14. Svitova TF, Lin MC. Lens-care-solutioninduced alterations in dynamic interfacial properties of human tear-lipid films. Contact Lens Anterior Eye 2014;37: 368-76 15. Petrov PG, Thompson JM, Abdul Rahman IB, et al. Two-dimensional order in mammalian pre-ocular tear film. Exp Eye Res 2007;84:1140-6
THE OCULAR SURFACE / JULY 2015, VOL. 13 NO. 3 / www.theocularsurface.com
