Seeing Clearly Again: Using the Tear Lipidome To Detect Dry Eye After Laser Surgery
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In this interview, Dr. Tom Knapman, brand director, SCIEX, speaks to Lei Zhou, PhD, head of the Ocular Proteomics Platform at the world-renowned Singapore Eye Research Institute (SERI), about the development of a novel diagnostic test to guide precision medicine to prevent dry eye in patients after laser eye surgery.
Tom Knapman (TK): Laser eye surgery is becoming increasingly common. Although it can be prescribed to treat eye disease, it is becoming more ubiquitous as an elective surgical option to correct deficiencies in eyesight. So, what is dry eye and how is it related to laser eye surgery? And can it be treated?
Lei Zhou (LZ): Complications may arise with laser eye surgery – one of which is dry eye, which can occur after laser eye surgery. Dry eye is a condition that occurs if the person does not have enough tears or produce the right composition of tears to keep the eye lubricated and comfortable. Tears are necessary for maintaining the health of the front surface of the eye. When we blink, a thick film of tears coats the front of the eye, keeping it nourished and clear for good vision.
The tear film is made up of three layers: a lipid (oily) outer layer, a thick aqueous (watery) middle layer, and a mucin (mucus) inner layer. Each layer of the tear film serves a purpose. The lipid layer, produced by the meibomian glands, keeps the tear surface smooth and prevents tears from drying up too quickly. The aqueous layer contains fluid from the lacrimal glands to clean the eye and wash away particles that do not belong in the eye. The mucin layer works to stick tears to the eye, helping to spread the aqueous layer over the eye’s surface to keep it moist. Tears are normally produced continuously but if insufficient tears are generated or if something affects one or more layers of the tear film, dry eye can develop. Laser eye surgery causes damage to the corneal nerves, which affects the secretion of the tears and the normal conditions of the ocular surface. Post-surgical patients may suffer from dry eye symptoms.
Symptoms of dye eye include blurred vision, a scratchy or gritty feeling like something is in your eye, and strings of mucus in or around the eyes.1 Treatments include eyedrops of artificial tears to supplement the inadequate tear production, punctal plugs or surgery to block tear ducts and retain tears, warm eye compresses, and prescription medicines.1
TK: I hear that spotting dry eye development early may improve the therapeutic window. Can you tell me more about that?
LZ: To improve patient outcomes, biomarker-based clinical tests are being developed to detect early on any patients who may be developing dry eye, so that preventive measures can be taken. Currently, only 1–2 tests are available in the US and both focus on using only one biomarker. But dry eye is a multifactorial condition and can occur due to a variety of pathological mechanisms. Therefore, my collaborators and I are developing a test for dry eye based on multiple biomarkers. Applying a panel of biomarkers would better reflect the multiple potential ways in which dry eye can arise, be it due to damage to the ocular surface, lacrimal gland secretion deficiency, or ocular inflammation. Such a biomarker panel should also be highly accurate and could prove to be a better tool than the existing tests for guiding optimal treatment decisions and allowing early intervention.
TK: Is that something you and your colleagues have been working on?
LZ: To better understand the pathology and development of dry eye, as well as identify biomarkers of disease and potential treatment targets, we have been systematically investigating the components of tear fluid under a variety of conditions.2–10 This includes the analysis of tear proteomic, lipidomic and metabolomic profiles from patients before and after refractive eye surgery.11,12 Changes in profiles over time before and after surgery can be correlated with the development of dry eye, to identify key proteins, lipids and metabolites indicative of dry eye pathology. As the lipid layer of tear film has been shown to become thinner after (LASIK) surgery, our recent studies have focused on the lipidomics of patients before and after stromal ablation surgery, that is with LASIK, femtosecond laser (FS-LASIK), and Sub-Bowman’s keratomileusis (SBK) procedures.12
TK: How are your lipidomics studies performed?
LZ: Lipidomics studies require the use of highly specific, sensitive and precise (reproducible) analytical methods, especially when we may not know what particular lipids we are looking for. Tears contain a diverse range of lipid species, some at very high concentrations and some at trace amounts. Tear samples are also relatively small in volume, especially from patients with dry eye. This along with the complexities of qualitative and quantitative lipidomics analysis posed quite a challenge. To address this challenge, we – like many other researchers – use mass spectrometry, a highly sensitive and thus preferred approach in biomedical research to discern fine changes of lipid metabolism. MS has been used in tear lipidomics studies of ocular and non-ocular disorders, such as Meibomian gland dysfunction, dry eye syndrome, and multiple sclerosis.13–15
TK: MS research methods, especially for lipidomics analysis, can be very time-consuming. So, how do you optimize your analyses?
LZ: To fully evaluate and research the lipid profile of tear samples in a high-throughput fashion, we developed a high-resolution shotgun lipidomics method in conjunction with SCIEX using a technique called MS/MSALL with data-independent acquisition. This method has been specifically designed for global lipidomics analysis and is carried out on a quadrupole time-of-flight mass spectrometer like the TripleTOF 5600, which includes electrospray ionization (ESI) for the direct injection of the tear lipid sample into the instrument without any need for prior separation by liquid chromatography.12,16–18 The MS scans are run with both positive and negative polarities for complete lipidome coverage. The total run time for one MS/MSALL acquisition is rapid for us at less than two minutes, facilitating the high-throughput analysis of numerous samples.12 The acquired data is processed to identify and quantify each lipid detected, using LipidView software, which includes a searchable built-in library of glycerolipids, phospholipids, sphingolipids, sterol lipids and fatty acyls.12
TK: And was there a trade-off between speed and precision?
LZ: This MS/MSALL method proved to be highly reproducible, bias-free and required no further development once set up. Using this research method, we were able to detect around 300 lipid species present in tear samples from patients receiving stromal ablation surgery, as well as identify 76 new lipid species in human tears.
TK: What have your lipidomics studies revealed so far?
LZ: By characterizing the lipidomic profiles of tears before and after surgery, we found that patients who underwent SBK procedures had lipidomes at one year that were similar to their pre-operative profiles. Patients who had LASIK and FS-LASIK procedures showed greater variation in their lipidomes over the same time period. There were significant differences in certain lipid classes, such as phospholipids, over time, depending on the surgical procedure received. The lipids identified in correlation with the development of post-operative dry eye are not only potential biomarkers that could be used for the early detection of dry eye pathology but also to implicate mechanisms of disease that could be targeted early to provide maximal therapeutic efficacy.
TK: How could these potential and other biomarkers be used for early detection?
LZ: Using other protocols, MS could also be applied to detect and characterize other “omes” of tear fluid, such as proteomes and metabolomes. Our research team continues to detect, quantify and identify individual species using multi-omic analyses of tear fluid. Along with the ~300 lipids identified, my collaborators and I have banked almost 4,500 tear proteins and a few hundred small molecule metabolites in our database at the SERI.
TK: What is next for this research that you and your colleagues are working on?
LZ: Further details of our research into dry eye biomarkers are anticipated and we have filed for a patent regarding the diagnostic test we are developing for the early detection of dry eye after laser eye surgery. The test does not use MS as MS instruments are not standard equipment in clinical laboratories. The test is based on multiple biomarkers, it should pick up the development of dry eye early on, irrespective of its mechanism of disease. As the test works using tear fluid, samples should be quick and easy to collect. Its non-invasive nature also means it could be a convenient option for patients or caregivers to use repeatedly, at regular and frequent intervals, to monitor for the development of dry eye and profile post-operative recovery at home – thus helping people to see clearly again. Finally, as the content of tears are a reflection of the dynamic changes not only in the eye but of the entire body, tear multi-omic biomarkers possess great potential for discovery and development into companion diagnostic tools for the determination of disease pathogenesis, progression and treatment response in personalized and precision medicine.19
Dr. Lei Zhou was speaking to Dr. Tom Knapman, brand director, SCIEX.
References
1. Boyd K. What is dry eye? American Academy of Ophthalmology. Published September 15, 2021. Accessed March 2022. https://www.aao.org/eye-health/diseases/what-is-dry-eye
2. Zhou L, Beuerman RW, Chan CM, et al. Identification of tear fluid biomarkers in dry eye syndrome using ITRAQ quantitative proteomics. J Proteome Res. 2009;8(11):4889-4905. doi: 10.1021/pr900686s
3. Zhou L, Beuerman RW, Chew AP, et al. Quantitative analysis of n-linked glycoproteins in tear fluid of climatic droplet keratopathy by glycopeptide capture and ITRAQ. J Proteome Res. 2009;8(4):1992-2003. doi: 10.1021/pr800962q
4. Chen L, Zhou L, Chan ECY, Neo J, Beuerman RW. Characterization of the human tear metabolome by LC–MS/MS. J Proteome Res. 2011;10(10):4876-4882. doi: 10.1021/pr2004874
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6. Wong TT, Zhou L, Li J, et al. Proteomic profiling of inflammatory signaling molecules in the tears of patients on chronic glaucoma medication. Invest Ophthalmol Vis Sci. 2011, 52: 7385–91. doi: 10.1167/iovs.10-6532
7. Chng CL, Seah LL, Yang M, et al. Tear proteins calcium binding protein A4 (S100 A4) and prolactin induced protein (PIP) are potential biomarkers for thyroid eye disease. Sci Rep. 2018;8(1):16936. doi: 10.1038/s41598-018-35096-x
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10. OMICS International. Singapore Eye Research Institute (SERI). Global Medicine Experts. Dr Zhou Lei. Accessed April 2022. https://biography.omicsonline.org/singapore/singapore-eye-research-institute/dr-zhou-lei-222860.
11. Liu YC, Yam GHF, Lin MTY, et al. Comparison of tear proteomic and neuromediator profiles changes between small incision lenticule extraction (SMILE) and femtosecond laser-assisted in-situ keratomileusis (LASIK). J Adv Res. 2021;29:67-81. doi: 10.1016/j.jare.2020.11.001
12. Gao Y, Qi YY, Huang Y, Li XR, Zhou L, Zhao SZ. Lipidomics analysis of tears in patients receiving LASIK, FS-LASIK or SBK surgery. Front Med (Lausanne). 2021, 8: 731462. doi: 10.3389/fmed.2021.731462
13. Walter SD, Gronert K, McClellan AL, Levitt RC, Sarantopoulos KC, Galor A. ω-3 tear film lipids correlate with clinical measures of dry eye. Invest Ophthalmol Vis Sci. 2016, 57: 2472–8. doi: 10.1167/iovs.16-19131
14. Lam SM, Tong L, Reux B, et al. Lipidomic analysis of human tear fluid reveals structure-specific lipid alterations in dry eye syndrome. J Lipid Res. 2014, 55: 299–306. doi: 10.1194/jlr.P041780
15. Cicalini I, Rossi C, Pieragostino D, et al. Integrated lipidomics and metabolomics analysis of tears in multiple sclerosis: an insight into diagnostic potential of lacrimal fluid. IJMS. 2019, 20: 1265. doi: 10.3390/ijms20061265
16. Simons B, Kauhanen D, Sylvänne T, Tarasov T, Duchoslav E, Ekroos K. Shotgun lipidomics by sequential precursor ion fragmentation on a hybrid quadrupole time-of-flight mass spectrometer. Metabolites. 2012, 2: 195–213. doi: 10.3390/metabo2010195
17. Rockwell HE, Gao F, Chen EY, et al. Dynamic assessment of functional lipidomic analysis in human urine. Lipids. 2016, 51: 875–86. doi: 10.1007/s11745-016-4142-0
18. Gao F, McDaniel J, Chen EY, et al. Monoacylglycerol analysis using MS/MSALL quadruple time of flight mass spectrometry. Metabolites. 2016, 6: 25. doi: 10.3390/metabo6030025
19. Wang SS. In your eyes: What they reveal about your health. The Wall Street Journal. Published August 14, 2012. Accessed April 2022. https://www.wsj.com/articles/SB10000872396390444184704577587211317837868
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