CLINICAL INVESTIGATION AND MEIBUM COLLECTION

This study was approved by the Institutional Review Board of the Yonsei University College of Medicine (IRB No. 4-2017-0708) and followed the tenets of the Declaration of Helsinki. Written informed consent was obtained from all the subjects. A total of 94 dry eye patients and 11 normal subjects were enrolled in this study at Severance Hospital. Dry eye was diagnosed according to the following Tear Film and Ocular Surface Society Dry Eye Workshop II diagnostic criteria: 1) ocular surface disease index (OSDI) score ≥13 and 2) tear film break-up time (TBUT) <10 seconds or Oxford score ≥1. Exclusion criteria were: patients aged <20 years and those with a history of ocular diseases such as ocular infection, ocular allergy, and autoimmune disease; ocular injury; patients using a punctal plug or topical ocular medications other than nonpreserved artificial tears; and contact lens wearers.

All subjects underwent a detailed ophthalmological examination sequentially as follows: (1) Tear lipid interference and noninvasive TBUT tests were performed using a DR-1α interferometer (Kowa). ^{, ,} The subjects were asked to blink twice naturally and then to keep both eyes open as long as possible. (2) Lipid layer thickness (LLT) measurements and MG imaging were conducted using the LipiView II interferometer (Johnson & Johnson Vison Inc.). (3) Ocular surface staining was graded from 0 to 5 according to the Oxford staining score based on the patterns of fluorescein staining notes in slit-lamp biomicroscopy. (4) Schirmer’s test I was performed by placing a Schirmer strip in the mid-lateral portion of the lower fornix for 5 minute without topical anesthesia. (5) The OSDI questionnaire, a validated 12-item questionnaire, was completed by the subjects. (6) Meibomian gland dysfunction grading was performed as previously described. ^, The degree of lid margin abnormalities, MG expressibility, and meibum quality were checked, and the grade of MGD was assessed using all these clinical parameters and the ocular symptom scores. There was at least a 10-minute interval between each test. All the clinical examinations were performed by one ophthalmologist (I.J) and data were obtained from one eye, which was randomly chosen using randomization tables, regardless of degree of ocular signs or symptoms.

After the clinical examinations, the meibum of the subjects was collected by squeezing the eyelids and the expressed meibum was transferred to a tube. The samples were immediately stored at –80°C until further analysis.

LIPID EXTRACTION

All the lipid standard solutions were prepared by dissolving the lipid standards in chloroform/methanol (1:1, v/v) and stored at –20°C. These standard solutions were diluted to the 100 ng/μL concentration for lipid extraction from biological samples. To effectively extract anionic lipids, a two-step extraction method that can divide ionic interactions between acidic lipids and proteins was used. First, 990 μL of chloroform/methanol (1:2, v/v) containing 10 μL of the 1 μg/mL of lipid standard mixture as the internal standard was added to the samples. Samples were gently vortexed for 30 seconds and sonicated for 1 minute 3 times for 15 minutes. After centrifugation (13,800 x g, 2 minutes at 4°C), 950 μL of the supernatant was transferred to a new tube. Next, for extracting acidic lipids, the remaining pellets were resuspended in 750 μL chloroform/methanol/37% HCl (40:80:1, v/v/v) and incubated for 15 minutes at room temperature with vortexing for 30 seconds every 5 minutes. Then, 250 μL of cold chloroform and 450 μL of cold 0.1 N HCl were added into the solution, followed by vortexing for 1 minute and centrifugation (6500 x g, 2 minutes at 4°C). The lower organic phase was collected and transferred to a prior extract. Subsequently, the samples were divided into two equal aliquots and dried under vacuum in a SpeedVac vacuum concentrator. Solvent A consisted of an acetonitrile–methanol–water mixture (19:19:2) with 20 mM ammonium formate and 0.1% (v/v) formic acid, and solvent B consisted of 2-propanol with 20 mM ammonium formate and 0.1% (v/v) formic acid. One of the dried samples was added to 100 μL of solvent A/solvent B (2:1, v/v) for neutral and positive lipid analysis, the other sample was added to 100 μL methanol followed by the addition of TMSD to undergo methylation for analyzing anionic lipids.

TRIMETHYLSILYDIAZOMETHANE METHYLATION

A solution of 2 M TMSD in hexane was added in equal volumes to the prior lipid extracts dissolved in methanol. After gently vortexing for 30 seconds, the TMSD methylation reaction was performed at 37°C for 15 minutes. The reaction was quenched by adding 10% volume of acetic acid. The samples were then subjected to LC-MS analysis.

GLOBAL LIPID ANALYSIS OF TEAR SAMPLES USING ULTRA-PERFORMANCE LIQUID CHROMATOGRAPHY-MASS SPECTRONOMY

Ultra-performance liquid chromatography and mass spectrometry conditions were previously optimized. UPLC analysis was performed using an ACQUITY UPLC instrument (Waters) with a Hypersil GOLD column (2.1 × 100 mm ID; 1.9 μm, Thermo Fisher Scientific). The temperatures of the autosampler and column oven were set at 10°C and 40°C, respectively. The flow rate was 0.3 mL/minute and the injection volume was 8 μL for each run. The lipid elution gradient was performed as follows: 0 to 5 minutes, B 5%; 5 to 15 minutes, B 5 to 30%; 15 to 22 minutes, B 30 to 95%; 22 to 27 minutes, B 95%; 27 to 28 minutes, B 95 to 5%; 28 to 33 minutes, B 5%. The total run time was 33 minute for each analysis.

Lipid analysis of meibum was performed using a hybrid linear ion trap mass spectrometer (QTRAP 5500, AB Sciex) equipped with a Turbo V ion source, together with the Analyst 1.5.1 software package. Ultra-pure nitrogen gas was used as the collision gas. The operating conditions were as follows: capillary voltage = 5500 V, nebulizer gas pressure = 30 to 50 psi, heater gas pressure = 30 to 50 psi, collision gas setting = high, and source temperature = 400 to 500°C. All lipids were analyzed in the MRM mode using computed transitions for each lipid class.

DATA PROCESSING OF INDIVIDUAL DATA OBTAINED BY MULTIPLE REACTION MONITORING

The Analyst 1.5.1 software package (AB Sciex) was used to acquire LC-MS data. Retention time (RT) of the internal standard value was referenced from LIPID MAPS Lipidomics Gateway ( https://www.lipidmaps.org/ ) and all the targeted lipid peaks were assigned by comparing RT with the internal standard values. The peak area of each assigned lipid compound was determined from replicated experimental raw data using the Skyline software package (MacCoss Lab) based on the same Q1 and Q3 transition databases that were analyzed for lipids by LC-MS. The concentrations of lipids are presented as relative expression level or as mol% of total lipids or lipid category.

MEASUREMENT OF SURFACE PROPERTIES OF MEIBUM

Each sample was dissolved in chloroform and 60 µL of the 1 mg/mL sample was added to the saline solution between the barriers of the Langmuir-Blodgett trough (KN2002, KSV NIMA). After allowing 15 minutes for chloroform to evaporate from the sample, surface pressure–area isotherms were measured with a barrier speed of 140 mm/minute and 10 cycles were performed. Surface pressure is defined by the following equation:

Where π is the surface pressure, γ ₀ is the surface tension of saline solution, and γ is the surface tension of lipids. The isotherm reversibility was calculated by using the following formula:

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