Longitudinal changes in tear fluid lipidome brought about by eyelid-warming treatment in a cohort of meibomian gland dysfunction

Meibomian gland dysfunction (MGD) is a leading cause of evaporative dry eye and ocular discomfort characterized by an unstable tear film principally attributed to afflicted delivery of lipids to the ocular surface. Herein, we elucidated longitudinal tear lipid alterations associated with disease alleviation and symptom improvement in a cohort of MGD patients undergoing eyelid-warming treatment for 12 weeks. Remarkably, eyelid-warming resulted in stark reductions in lysophospholipids (P < 0.001 for lyso-plasmalogen phosphatidylethanolamine, lysophosphatidylcholine, and lysophosphatidylinositol), as well as numerous PUFA-containing diacylglyceride species in tears, accompanied by significant increases in several PUFA-containing phospholipids. These changes in tear lipidomes suggest that eyelid-warming leads to diminished activity of tear phospholipases that preferentially target PUFA-containing phospholipids. In addition, treatment led to appreciable increases (P < 0.001) in O-acyl-ω-hydroxy-FAs (OAHFAs), which are lipid amphiphiles critical to the maintenance of tear film stability. Longitudinal changes in the tear lipids aforementioned also significantly (P < 0.05) correlated with reduced rate of ocular evaporation and improvement in ocular symptoms. The foregoing data thus indicate that excess ocular surface phospholipase activity detrimental to tear film stability could be alleviated by eyelid warming alone without application of steroids and identify tear OAHFAs as suitable markers to monitor treatment response in MGD. Meibomian gland dysfunction (MGD) is a leading cause of evaporative dry eye and ocular discomfort characterized by an unstable tear film principally attributed to afflicted delivery of lipids to the ocular surface. Herein, we elucidated longitudinal tear lipid alterations associated with disease alleviation and symptom improvement in a cohort of MGD patients undergoing eyelid-warming treatment for 12 weeks. Remarkably, eyelid-warming resulted in stark reductions in lysophospholipids (P < 0.001 for lyso-plasmalogen phosphatidylethanolamine, lysophosphatidylcholine, and lysophosphatidylinositol), as well as numerous PUFA-containing diacylglyceride species in tears, accompanied by significant increases in several PUFA-containing phospholipids. These changes in tear lipidomes suggest that eyelid-warming leads to diminished activity of tear phospholipases that preferentially target PUFA-containing phospholipids. In addition, treatment led to appreciable increases (P < 0.001) in O-acyl-ω-hydroxy-FAs (OAHFAs), which are lipid amphiphiles critical to the maintenance of tear film stability. Longitudinal changes in the tear lipids aforementioned also significantly (P < 0.05) correlated with reduced rate of ocular evaporation and improvement in ocular symptoms. The foregoing data thus indicate that excess ocular surface phospholipase activity detrimental to tear film stability could be alleviated by eyelid warming alone without application of steroids and identify tear OAHFAs as suitable markers to monitor treatment response in MGD. Dry eye syndrome (DES) is a prevalent ophthalmic condition adversely affecting up to 80% of the population over the age of 80, with potential debilitating effects on specific segments of the population such as contact lens wearers, people who have undergone refractive surgeries, postmenopausal women, and patients suffering from a variety of autoimmune disorders (1Aquavella J.V. Dry eyes: are new ideas drying up?.Br. J. Ophthalmol. 2013; 97: 801-802Crossref PubMed Scopus (2) Google Scholar). DES is a multifactorial disease of the tears and ocular surface caused by a deficiency in tear production or excessive evaporation (2The definition and classification of dry eye disease: report of the Definition and Classification Subcommittee of the International Dry Eye WorkShop.Ocul Surf. 2007; 5: 75-92Crossref PubMed Google Scholar). Regardless of the initiating causes, chronic dryness and the resultant tear film hyperosmolarity leads to inflammation that jeopardizes the structural and functional integrity of the lacrimal gland, meibomian gland, and corneal and conjunctival epithelial tissues. The gradual destruction of these tissues that are major contributors of various tear film components further disturbs tear film homeostasis and results in a vicious cycle of inflammatory events that represents the major pathological mechanism in DES (3Baudouin C. The pathology of dry eye.Surv. Ophthalmol. 2001; 45: S211-S220Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). Meibomian gland dysfunction (MGD) is a major cause of dry eye and ocular discomfort (3Baudouin C. The pathology of dry eye.Surv. Ophthalmol. 2001; 45: S211-S220Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). MGD is a diffuse condition of the eyelids characterized by progressive obstruction of meibomian gland terminal ducts due to ductal hyperkeratinization or inspissation of secretion (4Foulks G.N. The correlation between the tear film lipid layer and dry eye disease.Surv. Ophthalmol. 2007; 52: 369-374Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). In MGD, pathological alterations in the compositions of the meibomian gland secretions, also known as the meibum, the predominant source of lipids for the human tear film, result in the thickening of the meibum, subsequently leading to the blockage of the glandular ducts. The occlusion may also be attributed to excessive colonization by bacterial commensals as well as exfoliated skin materials and crusts as a result of hyperkeratinization of the glandular ducts. These aberrations cumulatively result in a hyposecretion of lipids into the tear reservoir at the lid margins (5Blackie C.A. Korb D.R. Knop E. Bedi R. Knop N. Holland E.J. Nonobvious obstructive meibomian gland dysfunction.Cornea. 2010; 29: 1333-1345Crossref PubMed Scopus (101) Google Scholar). The currently accepted view of MGD states that the disease is fundamentally a result of (1) a deficiency in meibum and/or (2) abnormal lipids to constitute the tear film lipid layer, either of which will substantially compromise the overall structural integrity of the tear film, leading to reduced tear film stability, loss of lubrication, and damage to the corneal epithelium due to excessive evaporation and the resultant desiccation. In addition, reactive lipid species may elicit inflammation at the ocular surface. For instance, elevated ocular phospholipase A2 (PLA2) activity has been shown to exacerbate ocular inflammation in chronic blepharitis and experimental murine dry eye model (6Wang J. Kolko M. Phospholipases A2 in ocular homeostasis and diseases.Biochimie. 2010; 92: 611-619Crossref PubMed Scopus (15) Google Scholar, 7Wei Y. Epstein S.P. Fukuoka S. Birmingham N.P. Li X.M. Asbell P.A. sPLA2-IIa amplifies ocular surface inflammation in the experimental dry eye (DE) BALB/c mouse model.Invest. Ophthalmol. Vis. Sci. 2011; 52: 4780-4788Crossref PubMed Scopus (21) Google Scholar). As the disease progresses, these pathophysiological alterations eventually lead to the emergence of disease symptoms including ocular discomfort and afflicted visual quality (8Driver P.J. Lemp M.A. Meibomian gland dysfunction.Surv. Ophthalmol. 1996; 40: 343-367Abstract Full Text PDF PubMed Scopus (246) Google Scholar). Warm compresses currently represent the most frequently prescribed treatment for patients with MGD or glandular obstruction leading to DES symptoms. The fundamental therapeutic aims of heat therapy are (1) to heat the meibomian gland secretions, thus facilitating their secretion into the tear film; (2) to reduce glandular obstruction; and (3) to increase vascular flow to the tissues surrounding the meibomian glands (9Blackie C.A. Solomon J.D. Greiner J.V. Holmes M. Korb D.R. Inner eyelid surface temperature as a function of warm compress methodology.Optom. Vis. Sci. 2008; 85: 675-683Crossref PubMed Scopus (84) Google Scholar). Eyelid warming is also often used with additional treatment methodologies such as oral (systemic) and topical antibiotics, manual expression of glands, artificial tears, and steroid ointments (5Blackie C.A. Korb D.R. Knop E. Bedi R. Knop N. Holland E.J. Nonobvious obstructive meibomian gland dysfunction.Cornea. 2010; 29: 1333-1345Crossref PubMed Scopus (101) Google Scholar). In the disease state, compositional changes in meibum could lead to elevated melt temperature and enhanced viscosity. Indeed, it has been previously demonstrated that the average lipid order (i.e., stiffness) of meibum for MGD patients is significantly increased compared with that of controls (10Borchman D. Foulks G.N. Yappert M.C. Bell J. Wells E. Neravetla S. Greenstone V. Human meibum lipid conformation and thermodynamic changes with meibomian-gland dysfunction.Invest. Ophthalmol. Vis. Sci. 2011; 52: 3805-3817Crossref PubMed Scopus (115) Google Scholar). Apart from compromised meibum secretion, the altered composition of the meibum would also lead to considerable changes in viscoelasticity, thus adversely affecting the spreading of the lipid layers after a blink. Warming the eyelid therefore represents one of the earliest therapies for treating MGD, as it is expected that raising the eyelid temperature would lead to conformational changes in the lipid hydrocarbon chains in the meibum, thus increasing the disorder in the packing of these lipids and enhancing the delivery and secretion of meibum out of the glandular ducts. Indeed, numerous studies have demonstrated the temperature-induced elevation in hydrocarbon chain disorder in meibomian lipids (11Leiske D.L. Leiske C.I. Leiske D.R. Toney M.F. Senchyna M. Ketelson H.A. Meadows D.L. Fuller G.G. Temperature-induced transitions in the structure and interfacial rheology of human meibum.Biophys. J. 2012; 102 ([Erratum. 2012. Biophys. J. 102: 719.]): 369-376Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 12Borchman D. Foulks G.N. Yappert M.C. Ho D.V. Temperature-induced conformational changes in human tear lipids hydrocarbon chains.Biopolymers. 2007; 87: 124-133Crossref PubMed Scopus (66) Google Scholar). While the biophysical changes in meibum lipids upon warming are well documented, the precise molecular changes in tear lipid composition, if any, that might accompany routine eyelid warming eventually leading to DES symptom alleviation have remained largely unknown. Thus, the primary aim of the current study is to elucidate longitudinal changes in the molecular compositions of tear lipids with eyelid warming over an extended period of routine treatment. A molecular signature associated with disease alleviation and symptom improvement will confer novel insights pertaining to disease pathogenesis and reveal potential markers to monitor disease progression. In addition, longitudinal changes in tear lipid profiles were correlated with improvement in clinical indicators of DES including ocular discomfort and ocular evaporation rate. As the current study focuses on MGD, it would undoubtedly yield a more discerning picture of the contribution by meibomian gland function to the multifactorial syndrome of dry eye per se. This study involves patients (n = 32) from a 3-month longitudinal study evaluating the effects of eyelid warming in a cohort of MGD patients. Written informed consent was obtained from all participants in the current study. The clinical procedure was specifically approved by the Singhealth Centralised Institutional Review Board (CIRB Ref No.: 2011/197/A) and registered at the ClinicalTrials.gov database (NCT01448369). We adhered to the tenets of the Declaration of Helsinki for all human research conducted in this study. Withdrawal from the study followed the usual good clinical practice in clinical trials. For withdrawn subjects, no data were obtained after the date of the withdrawal. Withdrawn subjects were not replaced. Patients at the Singapore National Eye Centre dry eye clinic who met the eligibility criteria (supplementary Table I) were invited for screening. Participants were then enrolled with written informed consent obtained by the clinical trial coordinator. Eligible patients (supplementary Table II) were randomly assigned into three respective treatment arms each utilizing a different treatment modality (supplementary Fig. I): traditional method of warm compresses using a hot towel (n = 10); Blephasteam (n = 10); and EyeGiene (n = 12). Blephasteam (Spectrum Théa, France) is an eyelid-warming device available in Europe that can be conveniently used at home (supplementary Fig. IIA). The goggles provide standardized heat of ∼38°C to liquefy lipids and humidify the chambers with mineral water to ensure optimal moisture levels. EyeGiene (Eyedetec Medical Inc.) is a self-contained, convenient warm compress system for the eyes (supplementary Fig. IIB). The system is composed of a reusable eye mask and one-time use warmers that are inserted into the eye mask during usage. The warming units are activated by squeezing just prior to usage and deliver 40°C heat for up to 5 min within 30–60 s. The production of heat is based on a sustained thermochemical reaction. Screening visit was performed at the regular dry eye clinic, and baseline examination was subsequently carried out. Follow-up visits were conducted after 12 weeks of treatment. A window period of ±3 days was permitted for this visit. Tear fluid samples were collected from the right eye of each participant at baseline visit (week 0) and at the end of the treatment period (week 12) using Schirmer's strips as described previously (13Lam S.M. Tong L. Duan X. Petznick A. Wenk M.R. Shui G. Extensive characterization of human tear fluid collected using different techniques unravels the presence of novel lipid amphiphiles.J. Lipid Res. 2014; 55: 289-298Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Tear samples collected were frozen immediately and kept at −80°C until further analyses. Patients in each treatment arm carried out routine treatment using the assigned eyelid-warming modality for 10 min each time and for two times a day. All patients were allowed to continue their regular management of MGD in the form of lid scrub with Blephagel. The frequency of use of these measures was monitored in a daily diary, and other types of MGD treatment such as omega-3 tablets, antibiotics or steroid ointments, and the manual expression of meibomian glands were prohibited. A visual analog scale (VAS) was applied to evaluate DES symptoms as previously described (supplementary Figs. III, IV) (14Schaumberg D.A. Gulati A. Mathers W.D. Clinch T. Lemp M.A. Nelson J.D. Foulks G.N. Dana R. Development and validation of a short global dry eye symptom index.Ocul. Surf. 2007; 5: 50-57Crossref PubMed Scopus (133) Google Scholar). The outcome was taken as the change in the global score at week 12 from that at week 0, which was calculated from the discomfort frequency and severity as previously described (14Schaumberg D.A. Gulati A. Mathers W.D. Clinch T. Lemp M.A. Nelson J.D. Foulks G.N. Dana R. Development and validation of a short global dry eye symptom index.Ocul. Surf. 2007; 5: 50-57Crossref PubMed Scopus (133) Google Scholar). Other outcome measures include differences in the VAS of visual outcomes (i.e., blurred vision and light sensitivity) (supplementary Figs. III, IV), tear breakup time (TBuT), Schirmer's I test (Schir I), and corneal fluorescent staining. Details on the clinical procedures have been reported previously (15Lam S.M. Tong L. Yong S.S. Li B. Chaurasia S.S. Shui G. Wenk M.R. Meibum lipid composition in Asians with dry eye disease.PLoS ONE. 2011; 6: e24339Crossref PubMed Scopus (136) Google Scholar).The severity of MGD was also graded in this study. Microscopic signs of MGD including the presence of misdirected lashes, fragility of lashes, scurf formation, irregularity of meibomian gland orifices, loss of meibomian gland expressibility, formation of plaques, and the number of blocked meibomian gland orifices (i.e., plugs) were recorded. The Yamaguchi grading scheme was used to identify microscopic signs of MGD (16Tong L. Chaurasia S.S. Mehta J.S. Beuerman R.W. Screening for meibomian gland disease: its relation to dry eye subtypes and symptoms in a tertiary referral clinic in Singapore.Invest. Ophthalmol. Vis. Sci. 2010; 51: 3449-3454Crossref PubMed Scopus (81) Google Scholar) and essentially indicates the position of the Marx's line relative to the meibomian gland orifices, which has been previously shown to correlate strongly with meibomian gland function (17Yamaguchi M. Kutsuna M. Uno T. Zheng X. Kodama T. Ohashi Y. Marx line: fluorescein staining line on the inner lid as indicator of meibomian gland function.Am. J. Ophthalmol. 2006; 141: 669-675Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Ocular evaporation rate was measured based on infrared thermography in a clinical room setting as reported previously (18Petznick A. Tan J.H. Boo S.K. Lee S.Y. Acharya U.R. Tong L. Repeatability of a new method for measuring tear evaporation rates.Optom. Vis. Sci. 2013; 90: 366-371Crossref PubMed Scopus (25) Google Scholar). Lipids were extracted from the Schirmer's strips using a modified version of the Bligh and Dyer's method as optimized previously (13Lam S.M. Tong L. Duan X. Petznick A. Wenk M.R. Shui G. Extensive characterization of human tear fluid collected using different techniques unravels the presence of novel lipid amphiphiles.J. Lipid Res. 2014; 55: 289-298Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Polar lipids and neutral lipids were analyzed using an Agilent HPLC 1200 system coupled with ABSciex QTRAP 4000 and ABSciex 3200, respectively. Lipidomic analyses were chiefly based on the principle of HPLC/multiple-reaction-monitoring of individual lipid species. The detailed lipidomic platform optimized for human tear fluid analyses have been previously described in details elsewhere (13Lam S.M. Tong L. Duan X. Petznick A. Wenk M.R. Shui G. Extensive characterization of human tear fluid collected using different techniques unravels the presence of novel lipid amphiphiles.J. Lipid Res. 2014; 55: 289-298Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 19Lam S.M. Tong L. Reux B. Lear M.J. Wenk M.R. Shui G. Rapid and sensitive profiling of tear wax ester species using high performance liquid chromatography coupled with tandem mass spectrometry.J. Chromatogr. A. 2013; 1308: 166-171Crossref PubMed Scopus (21) Google Scholar, 20Lam S.M. Tong L. Reux B. Duan X. Petznick A. Yong S.S. Khee C.B.S. Lear M.J. Wenk M.R. Shui G. Lipidomic analysis of human tear fluid reveals structure-specific lipid alterations in dry eye syndrome.J. Lipid Res. 2014; 55: 299-306Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). One-way ANOVA with post hoc Tukey was first performed to compare the differences in the changes of clinical indices before and after treatment among the three groups (i.e., week 12 − week 0). Following this, paired-t comparisons were performed on the tear lipid profiles of a combined group of patients from the three individual treatment arms, obtained at week 0 and week 12 of the study. This would reveal lipid alterations under an extended period of routine eyelid warming. False discovery rate was controlled for based on q values calculated using R 3.0.1 (supplementary Table III). Correlation analyses between the changes in individual lipid species/classes with changes in clinical signs following 12-week treatment was performed using Spearman's correlation. Ellipse demarcates 95% confidence region of correlating parameters and lipid species/classes. No appreciable difference was observed in the clinical outcomes among the three treatment arms at the end of the 12-week period (supplementary Table IV), except for a marginal difference (P = 0.06) in the improvement of ocular discomfort using EyeGiene over Blephasteam (supplementary Table IV). The evaluation was based on changes in clinical parameters before and after routine eyelid warming using the respective modality, for a total duration of 12 weeks. The foregoing results thus imply that participants in all three treatment arms essentially received a comparable degree of lid warming throughout the course of treatment considered in terms of clinical outcomes. In other words, while patients in each treatment arm utilized different treatment modalities, the actual treatment received was, in principle, similar among the three groups (i.e., eyelid warming). Therefore, we then grouped together patients from the three treatment arms to evaluate the longitudinal effects of eyelid warming per se on tear lipid profiles over the 12-week treatment period. Eyelid warming for 12 weeks resulted in appreciable alleviation of symptoms of ocular discomfort (P < 0.01) (Table 1, supplementary Table V). The number of plugged orifices were significantly reduced (P < 0.01), and there was a noticeable improvement in TBuT (P < 0.10), which is in agreement with an earlier study demonstrating that the application of heat to the inner surface of the eyelids on a routine basis leading to steady increases in both TBuT and the number of meibomian glands yielding liquid secretion over a 12-week treatment period (21Korb D.R. Blackie C.A. Restoration of meibomian gland functionality with novel thermodynamic treatment device-a case report.Cornea. 2010; 29: 930-933Crossref PubMed Scopus (48) Google Scholar). On the other hand, no significant changes were observed in Schir I after treatment, which was not surprising because the current MGD cohort did not have discernible lacrimal dysfunction to begin with, even at week 0 (i.e., mean Schir I > 5.5 mm) (supplementary Table II). On another note, eyelid-warming treatment resulted in a reduction in ocular evaporation rate with marginal significance (P < 0.10).TABLE 1Changes in ocular symptoms and signs after routine eyelid-warming treatment for 12 weeksWeek 0Week 12PSymptomsOcular discomfort35.7 ± 3.930.7 ± 3.9cP < 0.01.Light sensitivity23.1 ± 5.318.7 ± 4.9a0.05 < P < 0.10.Blurred vision25.9 ± 5.225.5 ± 5.6NSSummed global score84.7 ± 10.774.9 ± 10.7a0.05 < P < 0.10.SignsTBuT2.5 ± 0.34.0 ± 0.8a0.05 < P < 0.10.Schir I9.8 ± 1.59.6 ± 1.4NSTotal corneal staining3.5 ± 0.64.1 ± 0.7NSTotal Yamaguchi's score5.1 ± 0.66.5 ± 0.4cP < 0.01.Blocked glands18.5 ± 2.013.9 ± 2.2cP < 0.001.Ocular evaporation rate80.6 ± 3.271.6 ± 3.6a0.05 < P < 0.10.Values were presented as means ± SEs. A total of 32 MGD patients successfully completed the entire course of treatment. NS, nonsignificant.b P < 0.05.a 0.05 < P < 0.10.c P < 0.01.d P < 0.001. Open table in a new tab Values were presented as means ± SEs. A total of 32 MGD patients successfully completed the entire course of treatment. NS, nonsignificant. b P < 0.05. Routine eyelid warming did not result in a discernible increase in the absolute amount of total lipids in the tear fluid (Fig. 1A), which is rather surprising considering the reductions in the number of plugged meibomian glands (see previous discussion) following heat treatment. This could imply that the relief of meibomian plugs resulted in a restoration of normal lipid turnover, instead of an enhanced amount of lipids at the eyelid margin. In fact, these MGD patients did not have an absolute deficiency in total lipids to begin with because their mean molar concentration of total lipids in tears before treatment (∼0.58 µmol ml−1) (Fig. 1A) was comparable to that of a healthy cohort asymptomatic for DES (∼0.50 µmol ml−1) as we have previously reported (20Lam S.M. Tong L. Reux B. Duan X. Petznick A. Yong S.S. Khee C.B.S. Lear M.J. Wenk M.R. Shui G. Lipidomic analysis of human tear fluid reveals structure-specific lipid alterations in dry eye syndrome.J. Lipid Res. 2014; 55: 299-306Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar).This observation is in good agreement with our previous report that the quality of meibomian lipids, instead of the quantity, might be the actual cause of MGD-associated signs and symptoms of ocular discomfort (20Lam S.M. Tong L. Reux B. Duan X. Petznick A. Yong S.S. Khee C.B.S. Lear M.J. Wenk M.R. Shui G. Lipidomic analysis of human tear fluid reveals structure-specific lipid alterations in dry eye syndrome.J. Lipid Res. 2014; 55: 299-306Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Indeed, stark changes in the compositions of tear lipids were observed after routine eyelid warming for 12 weeks, as elaborated subsequently. The most striking changes after lid warming were observed in lysophospholipids (Figs. 1B, 2). Molar fractions of major lysophospholipid classes, including lyso-plasmalogen phosphatidylethanolamines (LpPEs), lysophosphatidylcholines (LPCs), and lysophosphatidylinositols (LPIs), were reduced by almost half (P < 0.001) after treatment, while total lysophosphatidylethanolamine (LPE) also displayed a significant decrease (P < 0.05) (Figs. 1B, 2). Remarkably, several individual species of LpPE, LPE, LPI, and LPC were also significantly reduced after treatment (Fig. 3A–D), consistent with the overall trends observed for the respective lipid classes. Interestingly, the drastic reduction in total LpPE was paralleled by concomitant increases in total PE (Fig. 1C) and several plasmalogen PE species that possessed a high degree of unsaturation (containing six or more double bonds) (Fig. 4A). In addition, numerous diacyl PE, diacyl PC, and diacyl PI species were also significantly increased after the 12-week treatment period (Fig. 4), which corresponded well with the observed reductions in their respective lysophospholipid counterparts. Moreover, similar to plasmalogen PE, various unsaturated PC, PS, and PI species containing two or more double bonds seem preferentially increased after eyelid warming. In addition, while total DAG (Fig. 1D) did not change significantly after the treatment period, levels of individual DAG species containing PUFAs in their structures, namely DAG 16:1/22:5 (P < 0.05), DAG 18:0/20:4 (P < 0.01), and DAG 18:0/22:6 (P < 0.05), were almost halved following eyelid-warming treatment (supplementary Table VI). In addition, DAG 16:0/22:5 and DAG 16:0/22:6 were reduced by ∼30% following treatment with marginal significance (P < 0.10) (supplementary Table VI). The reductions in the various lysophospholipids and PUFA-containing DAG, together with the concomitant increases in PUFA-containing phospholipid species, indicated attenuation of PLA2 and PLC activities, respectively, following eyelid-warming treatment. In particular, these phospholipases seem to preferentially target PUFA-containing phospholipid species over their more saturated counterparts.Fig. 3Changes in individual lysophospholipid species with eyelid-warming treatment. Heat maps illustrate molar fractions of individual species of LpPE (A), LPE (B), LPI (C), and LPC (D) at week 0 and week 12 of the study. # 0.05 < P < 0.10, * P < 0.05, ** P < 0.01, *** P < 0.001.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4Changes in individual phospholipid species with eyelid-warming treatment. Bar plots illustrate the molar fractions of individual species of PE (A), PC (B), PS (C), and PI (D) in the tear samples of MGD patients (n = 32) at week 0 and week 12 of the study. Values were plotted as mean ± SEs. # 0.05 < P < 0.10, * P < 0.05, ** P < 0.01, *** P < 0.001.View Large Image Figure ViewerDownload Hi-res image Download (PPT) On another note, molar fractions of major tear film lipid amphiphiles, namely CS (P < 0.10) and OAHFA (P < 0.001), were noticeably increased following heat treatment (Fig. 5A, B). Also, the increases in total OAHFA and numerous OAHFA species, including OAHFA 18:1/26:1, OAHFA 18:1/30:2, and OAHFA 18:1/34:1, were significantly correlated (P < 0.05) with the reductions in ocular discomfort (Fig. 5C), suggesting that the alleviation of ocular discomfort with treatment was associated with enhanced levels of OAHFA in the tear fluid. The increase in total OAHFA, as well as several other OAHFA species including OAHFA 18:1/25:0, OAHFA 18:1/28:1, OAHFA 16:1/32:1, OAHFA 18:1/30:1, OAHFA 18:1/31:0, OAHFA 18:2/32:1, OAHFA 18:1/32:2, and OAHFA 18:1/34:2, was significantly (P < 0.05) correlated with the improvement in ocular evaporation rate following 12-week treatment (Fig. 5D, supplementary Table VII). On the other hand, the reduction in total LpPE, as well as several other LpPE species including LpPE 16:0p, LpPE 18:1p, LpPE 18:0p, and LpPE 20:1p, was significantly correlated (P < 0.05) with the improvement in ocular evaporation rate (Fig. 5E, supplementary Table VII). Similar trends were observed between changes in molar fractions of OAHFA and LpPE with improvement in corneal evaporation rate and scleral (conjunctival) evaporation rates (supplementary Table VII). These observations implied that while OAHFA might have a stabilizing effect on tear film structural integrity, elevated levels of LpPE might compromise tear film stability, leading to an increase in evaporation rate. This is the very first study to investigate longitudinal changes in the tear lipid profiles of MGD patients over a 12-week treatment period. The longitudinal design allows detection of changes in tear lipid profiles corresponding to improvement in ocular symptoms in the same group of individuals over the treatment period, while minimizing confounding factors such as hormonal, genetic, and interind