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. Author manuscript; available in PMC: 2018 Nov 1.
Published in final edited form as: J Cell Biochem. 2017 May 23;118(11):3920–3931. doi: 10.1002/jcb.26045

PHOSPHOLIPIDOMIC STUDIES IN HUMAN CORNEA FROM CLIMATIC DROPLET KERATOPATHY

M Fernanda Suarez 1, M Carmen Piqueras 2, Leandro Correa 3, Evangelina Esposito 3, M Fernanda Barros 3, Sanjoy K Bhattacharya 2, Julio A Urrets-Zavalia 3,#, Horacio M Serra 1,*,#
PMCID: PMC5603377  NIHMSID: NIHMS869865  PMID: 28401586

Abstract

Climatic droplet keratopathy (CDK) is an acquired degenerative disease predominantly affecting males over 40 years old. It results in progressive corneal opacities usually affecting both eyes. CDK is multifactorial and its etiology remains unknown. Our recent findings are consistent with CDK pathology being driven by environmental factors with oxidative stress playing an important role (for example, contributing to lipid peroxidation) rather than climate factors. The changes in corneal lipid composition affected by environmental factors remain understudied. The purpose of this study was to systematically investigate phospholipids profile [phosphatidylcholine (PC) and phosphatidylserine (PS)] in corneas from CDK patients using tandem mass spectrometry. Samples from CDK areas and from non-affected areas were obtained from patients diagnosed with CDK who underwent cataract surgery, were subjected to lipid extraction using a modified Bligh and Dyer method; protein concentrations were determined using the Bradford’s method. Lipids were identified and subjected to ratiometric quantification using TSQ Quantum Access Max triple quadrupole mass spectrometer, using appropriate class specific lipid standards. All phospholipid classes showed lower total amounts in affected areas compared to control areas from CDK’s corneas. Comparative profiles of two phospholipid classes (PC, PS) between CDK areas and control areas showed several common species between them. We also found a few unique lipids that were absent in CDK areas compared to controls and vice versa. Lower amount of phospholipids in CDK areas compared to control areas could be attributed to the lipid peroxidation in the affected corneal regions as a consequence of increased oxidative stress.

Keywords: CDK, Phospholipids, Phosphatidylcholine, Phosphatidylserine, Cornea, Tandem mass spectrometry

Climatic droplet keratopathy (CDK) is a bilateral degenerative corneal disease related to advanced age, lifestyle, and multiple environmental factors such as corneal micro traumas, low humidity and lack of adequate protection for exposure to ultraviolet radiation (UVR) (Suarez et al., 2015). CDK most commonly affects males over 40 years old, in the form of progressive opacities of the anterior layers of the cornea. This disease is rare in temperate latitudes, and it is commonly linked to overexposure to UVR, being endemic in certain rural communities around the world. Since the first description of the disease in 1898 (Baquis), CDK has been reported in different parts of the world following different criterions (presumed aetiology, geographic area, by eponym, clinical presentation, nature of corneal deposits and patient’s activities) and more recently in the Patagonia region of Argentina, as has been reviewed by Serra et al. (2015).

CDK slowly progresses to corneal opacity, through three grades of increasing severity. In the initial stages (grade 1), multiple tiny and tightly confluent translucent extracellular sub epithelial deposits, located in the Bowman layer close to the temporal and/or nasal limbus, giving the affected cornea a tarnished, hazy aspect. In grade 2, haziness spreads over the inferior 2/3rds of the cornea, affecting the central cornea. At this stage, visual acuity may be moderate to severely affected due to the visual axis involvement. Grade 3 is characterized by the presence of large golden sub-epithelial droplets of different sizes (some of them are 1 mm in diameter) grouped in clusters that grow and cover the cornea as the disease progresses. In advanced cases, areas of vascularized anterior stromal opacification or fibrosis may be observed. In general, corneal sensitivity and visual acuity are severely affected at this stage (Freedman, 1965; Urrets-Zavalia et al., 2007).

Another clinical finding observed in some of our patients in the Argentinian Patagonia is a mild to severe atrophy of the 1/2 inferior iris stroma, more frequently observed in grades 2 and 3 (Urrets-Zavalía et al., 2007). Despite the harsh environmental conditions in which individuals from this region live, dry eye was not common among CDK patients, or controls (Urrets-Zavalía et al., 2007).

We have investigated the biological features of matrix metalloproteinases (MMPs) and their inhibitors TIMPs in patients with CDK, as these molecules control the degradation of the corneal epithelium and stroma. Our studies showed enhanced MMP-2 and MMP-9 levels and a decreased expression of TIMP-1 in CDK patients’ tears (Holopainen et al., 2011). Immunohistochemistry showed that MMP-2 was expressed at the basement membrane zone in both control and affected corneas, but also marked the edges of the granular CDK deposits; MMP-9 expression was restrained to basal layers of the epithelium and was markedly induced in CDK corneas (Holopainen et al., 2012). We also investigated the effect of UVR in the production of MMPs and cytokines using an in vitro cellular model of immortalized human corneal epithelial cells (HCE). Exposure of HCE cells to UVR significantly increased MMP and pro-inflammatory cytokine secretion, suggesting an active participation of the corneal epithelium on CDK’s pathogenesis (Holopainen et al., 2012).

Many advances that could help to understand ethiopathogenic mechanisms involved in CDK have been made in the last years, recently reviewed by Serra et al (2015).

Lipids constitute a unique group of biomolecules that mediate a large number of functional and structural activities in the cell, tending to maintain homeostasis (Checa et al., 2015), and constitute about 5% of the weight of mammalian cells (Fahy et al., 2009). Cellular lipids are highly complex and dynamic (Yang and Han, 2016). There is evidence that phospholipids may play an important role in the regulation of several ocular homeostatic mechanisms due to their presence in aqueous humor and subsequent changes under injury conditions (Liliom et al., 1998). Lipids are also important components of the tear film. The lipid layer is highly organized, consisting of a monolayer of phospholipids at the water-air interface, which provides a hydrophobic interface on which non-polar lipids expand (Rantamäki et al., 2011). Defects in the lipid layer of the tear film results in increased evaporation, which perpetuates ocular surface inflammation and damage. Also, lipids have been involved in ocular surface pathologies such as dry eye, allergic keratoconjunctivitis, infections and glaucoma (Ham et al., 2004; Robciuc et al., 2014; Fujishima et al., 2013; Aribindi et al., 2013a; Edwards et al., 2014).

There are no previous reports on identification or determination of superficial corneal lipid composition of patients with CDK. Lipid peroxidation may occur at the epithelium of the cornea exposed to prolonged exposure to UVR without adequate protection, in certain individuals under certain circumstances, which could result in an oxidative stress. Our aim was to systematically study lipids in corneal epithelial cells from CDK corneas using tandem mass spectrometry, and identify a differential composition, if any, between healthy corneal epithelial areas and CDK affected areas.

MATHERIAL AND METHODS

Tissue samples procurement

The samples were procured according to the protocol approved by institutional review process and the tenets of the Declaration of Helsinki. Corneal epithelial cell specimens from the eyes of two CDK patients (a 62-year old and 63-year old males) (Figure 1) at the moment of a planned cataract surgery were obtained and kept at −70° C until they were further processed. One CDK affected area, and one control non-affected area, were collected by scraping these regions of the cornea using a crescent knife. No abnormalities of the iris were observed biomicroscopically, and the ocular tensions, as well as the ocular fundus were normal in both eyes patients.

Figure 1.

Figure 1

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Slit-lamp images of corneas in two CDK patients (A and C), and in vivo confocal microscopy (B and D). Grade 1 disease with peripheral nasal haziness (A) and grade 2 with diffuse haziness affecting also the central cornea (C), caused by tiny droplets of different sizes visualized with magnification and back-scattered slit-illumination. CDK grade 1 presents dot-like deposits at the level of Bowman’s layer and (B), and CDK grade 2 shows an increase in the density of the hyperreflective dot-like deposits in Bowman’s layer and superficial stroma (D).

Lipid extraction

Corneal tissue was subjected to lipid extraction using a modified Bligh and Dyer method (Iverson et al., 2001). The organic phase with extracted lipids was dried in a Speed-Vac (Model 7810014, Labconco, Kansas City, MO, USA). Samples were flushed with argon gas to prevent oxidation. All extractions and subsequent handling were made using glass vials; polyvinyl plastic was avoided completely to prevent contaminating impurities. Corresponding aqueous phase extracted proteins were subjected to determination of concentration using Bradford’s method (Bradford, 1976), and these concentrations were used to normalize lipids per amount of proteins.

Mass spectrometry

Dried lipid samples were re-suspended in LC-MS grade Acetonitrile: Isopropanol (1:1). Samples were infused with a flow rate of 5μl/min, using Tri Versa Nanomate (Advion Inc., Ithaca, NY, USA), a chip-based electrospray ionization machine controlled with Chipsoft8.3.3 version software.

A triple quadrupole electrospray mass spectrometer (TSQ Quantum Access Max; Thermo Fisher Scientific, Pittsburgh, PA, USA) was used for analysis of lipids in infusion mode using TSQ Tune software that is part of the Xcaliber 2.3 software package. Samples were analyzed for 2.00 minutes with a 0.500-second scan. Scans typically ranged from 200 to 1000 m/z. A peak width was set at 0.7 and collision gas pressure was set at 1 mTorr. Sheath gas (nitrogen) was set to 20 arbitrary units. Auxiliary gas (Argon) was set to 5 arbitrary units. Settings for analyses of different phospholipid classes were established based on previous studies (Enriquez-Algeciras and Bhattacharya, 2013). The 0.1% formic acid (FA) was used as an additive method for analyses of lipids in the positive ion mode only (that is, for analyses of PC but not for PS). Control and CDK corneal epithelial cells were utilized for each of the two different class of phospholipid analyzed. Class specific lipids were quantified using class specific quantitative lipid standards:1,2-ditridecanoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (Avanti Polar Lipids, Inc; Alabaster, AL, USA). Approximately 5 scans each with and without internal standard (usually in the range of 0.1–10pmol) were performed for each sample. Ratiometric quantification was achieved using the MZmine 2.9 program. Lipid concentration was normalized to protein amount determined from the corresponding aqueous phase as described above.

Representative spectra for each sample were carefully and manually inspected by two independent observers from 5 spectra collected for each sample with and without the internal standard (total 10 spectra) and then used for further analyses. Spectra were converted to netCDF files from Thermo RAW files using the Xcalibur 2.3 software suite, subsequently imported into MZmine 2.9 (Edwards et al., 2014). Identification was carried out against a custom database created from the Lipid Maps Database (LMDB).

Identified lipids were subjected to analysis for determination of common and unique species using an Excel macro (Aribindi et al., 2013b). We defined unique when a given lipid species was found in only one group (control areas or CDK affected areas). Common species are those that have been found in samples from both control and CDK affected areas of both patients. All unique lipid experimental readings (the amount of lipid species pM/μg protein) were found to be significantly different from 0.0 by one sample t-test (p≤0.05).

Statistical analysis

Student’s t-test was used for comparison between lipid concentrations in control areas vs CDK affected areas. A p-value ≤0.05 was considered significant.

RESULTS

We obtained lipid profiles for two classes of phospholipids: phosphatidylcholine (PC) and phosphatidylserine (PS), using established parameters (Enriquez-Algeciras and Bhattacharya, 2013). A representative PC and PS spectrum for control corneal samples without and with ratiometric standards (Fig. 2 A, 3A and Fig. 2 B, 3B, respectively) are shown. Using Excel Macros all data from two phospholipids were analyzed to determine the presence of common and unique lipid species in control areas and CDK affected areas.

Figure 2.

Figure 2

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Representative electro spray ionization tandem mass spectrometric analysis of PC lipid class extracted from CDK human corneal epithelium control area, in positive ion mode. (A) A parent ion scan (PIS) scanning of m/z =184.0 for PC class. (B) Representative PIS as presented above with internal standard addition (arrow; m/z= 650.3, 10pmol) to perform ratiometric quantification of all identified lipids in PC class.

Figure 3.

Figure 3

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Representative electro spray ionization tandem mass spectrometric analysis of PS lipid class extracted from CDK human corneal epithelium control area, in neutral loss scan (NLS). (A) NLS scanning of m/z =87.1 for PS class. (B) Representative NLS as presented above with internal standard addition (arrow; m/z= 810.63, 10pmol) to perform ratiometric quantification of all identified lipids in PS class.

All phospholipids classes showed higher total amounts in control areas compared to CDK affected areas. In grade 1, total phospholipids amount was 7.6 times higher in control than in CDK areas. In grade 2, phospholipids concentration was 35 times higher in control areas than in CDK affected areas. PC and PS concentrations were higher in the control than in CDK area in grade 1 (4.7 times and 10 times, respectively) as well as in grade 2 (5.7 times and 36 times, respectively). The total amount of all two classes of phospholipids normalized to total amount of proteins in the corresponding aqueous phase extractions are presented in table 1.

Table 1.

Total average protein normalized phosphatidylcholine and phosphatidylserine in the CDK corneal specimens.

Patient / CDK grade Phosphatidylcholines (pmol/μg protein) Phosphatidylserines (pmol/μg protein) Total phospholipids amount (pmol/μg protein)
Control CDK Control CDK Control CDK
Grade 1 11792.35 2510.78 29936.93 2981.93 41729.28 5492.71
Grade 2 2583.76 450.44 489172.16 13572.31 491755.92 14022.75
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We found 35 unique PC species in the control areas and 38 species in CDK affected areas from grade 1 patient’s sample. No unique PC species were found in samples from grade 2 (table 2, figure 4). For common lipids, we found 47 common PC species in control and CDK affected areas in grade 1 and 85 common PC species for grade 2 (table 3, figure 4).

Table 2.

Unique phospholipid species identified in control and CDK affected areas.

m/z* Amount, pmol per Species/μg protein LIPIDMAPS ID
Phosphatydilcholines
Grade 1
              Control
PC(6:2(2E,4E)/6:2(2E,4E)) 449.5343475 35.10504116 LMGP01011235
PC(6:0/6:0) 461.611969 60.97552036 LMGP01011229
PC(8:2(2E,4E)/8:2(2E,4E)) 508.7760621 336.7085264 LMGP01011254
PC(O-16:0/2:0) 532.708313 30.49454999 LMGP01020046
PC(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/0:0) 576.223999 27.63661101 LMGP01050056
PC(12:0/14:1(9Z)) 653.0681152 188.8764232 LMGP01011316
PC(18:0/11:1(10E)) 697.8502197 123.003872 LMGP01010735
PC(12:0/18:4(6Z,9Z,12Z,15Z)) 705.4578247 16.83355497 LMGP01011326
PC(13:0/18:4(6Z,9Z,12Z,15Z)) 719.3928223 261.6877919 LMGP01011351
PC(14:0/18:4(6Z,9Z,12Z,15Z)) 734.7036133 33.99468733 LMGP01010499
PC(14:0/18:3(9Z,12Z,15Z)) 736.7713623 139.3388865 LMGP01010497
PC(O-16:0/17:2(9Z,12Z)) 739.5371704 10.7416212 LMGP01020184
PC(10:0/22:0) 743.0431061 95.55680672 LMGP01010397
PC(14:0/20:5(5Z,8Z,11Z,14Z,17Z)) 760.842041 14.79965082 LMGP01010508
PC(14:0/20:4(5Z,8Z,11Z,14Z)) 761.8012695 11.29438532 LMGP01010506
PC(16:0/18:3(6Z,9Z,12Z)) 764.8153687 16.84177156 LMGP01010598
PC(13:0/22:2(13Z,16Z)) 780.9053548 141.4017152 LMGP01011360
PC(18:0/18:2(10Z,12Z)) 793.9078979 4.870731935 LMGP01010764
PC(15:1(9Z)/22:4(7Z,10Z,13Z,16Z)) 802.3403931 0.292454956 LMGP01011460
PC(16:0/22:6(4E,7E,10E,13E,16E,19E)) 814.689621 35.41917971 LMGP01010650
PC(16:0/22:4(7Z,10Z,13Z,16Z)) 818.5667572 35.02464907 LMGP01010642
PC(18:0/20:2(11Z,14Z)) 822.8417358 3.113532506 LMGP01010788
PC(17:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) 829.2927246 19.62032538 LMGP01010720
PC(18:0/22:3(10Z,13Z,16Z)) 848.0913086 10.55761574 LMGP01010812
PC(20:5(5Z,8Z,11Z,14Z,17Z)/22:5(7Z,10Z,13Z,16Z,19Z)) 861.9257813 12.02594653 LMGP01011058
PC(20:3(8Z,11Z,14Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) 864.4815369 96.29052353 LMGP01011896
PC(20:1(11Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) 869.3223114 4.978646959 LMGP01011834
PC(23:0/18:0) 869.6445313 9.01741973 LMGP01011130
PC(20:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) 870.8692932 18.18734822 LMGP01011028
PC(20:0/22:5(7Z,10Z,13Z,16Z,19Z)) 871.7922363 63.0557569 LMGP01011027
PC(20:1(11Z)/22:2(13Z,16Z)) 877.6144409 0.34022481 LMGP01011832
PC(22:4(7Z,10Z,13Z,16Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) 890.7302246 10.41447043 LMGP01012096
PC(22:1(11Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) 897.191803 58.34030765 LMGP01012034
PC(22:1(11Z)/22:2(13Z,16Z)) 905.4731445 11.1785486 LMGP01012032
PC(20:0/24:1(15Z)) 908.4393921 55.37025535 LMGP01011032
              CDK
PC(P-15:0/0:0) 475.3136597 2.258060045 LMGP01070003
PC(0:0/14:0) 475.7080078 8.760073598 LMGP01050073
PC(17:2(9Z,12Z)/0:0) 512.0413666 139.3999566 LMGP01050127
PC(20:3(8Z,11Z,14Z)/0:0) 555.0397949 9.151008296 LMGP01050133
PC(10:0/10:0) 574.9521484 58.01008602 LMGP01010380
PC(16:0/9:0(COOH)) 674.8492432 43.25367692 LMGP20010007
PC(12:0/17:2(9Z,12Z)) 695.5105082 59.84938759 LMGP01011322
PC(12:0/18:3(6Z,9Z,12Z)) 709.3492432 29.40629858 LMGP01011324
PC(13:0/18:3(6Z,9Z,12Z)) 722.7244721 23.22549354 LMGP01011349
PC(14:0/18:2(11Z,14Z)) 738.9689941 9.61773106 LMGP01010494
PC(14:0/18:1(11Z)) 740.4227498 10.14359376 LMGP01010490
PC(13:0/20:3(8Z,11Z,14Z)) 751.0203247 6.071596573 LMGP01011355
PC(15:0/18:1(11Z)) 754.4980469 16.34204123 LMGP01010541
PC(10:0/23:0) 757.4887085 7.0248453 LMGP01010399
PC(16:0/18:2(10E,12Z)) 765.9030151 19.37539039 LMGP01010585
PC(16:0/18:1(9Z)) 767.6375427 5.401143878 LMGP01010005
PC(18:2(9Z,12E)/17:2(9Z,11E)) 777.0445557 8.175188459 LMGP01010931
PC(14:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) 787.3533936 8.379876858 LMGP01010512
PC(16:0/20:5(5Z,8Z,11Z,14Z,17Z)) 789.5361938 5.682940604 LMGP01010633
PC(18:0/18:1(11Z)) 797.3813477 1.802717735 LMGP01010750
PC(16:1(9Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) 812.9232788 5.572865494 LMGP01010696
PC(17:2(9Z,12Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) 823.7950745 5.964470179 LMGP01011580
PC(18:3(6Z,9Z,12Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) 836.2522125 8.435849515 LMGP01011670
PC(18:2(9Z,12Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) 838.1468811 2.276002421 LMGP01010947
PC(18:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) 842.451416 5.65016685 LMGP01010821
PC(18:0/22:5(4Z,7Z,10Z,13Z,16Z)) 844.7405396 0.22199696 LMGP01010816
PC(16:0/24:1(15Z)) 852.4516602 0.506300409 LMGP01010659
PC(19:1(9Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) 854.8773652 7.943575465 LMGP01011786
PC(19:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) 857.3092651 0.602203978 LMGP01011755
PC(19:1(9Z)/22:4(7Z,10Z,13Z,16Z)) 858.6088867 0.439391289 LMGP01011785
PC(16:0/26:2(5Z,9Z)) 878.7354736 7.857161887 LMGP01010665
PC(16:0/26:0) 882.1089478 3.17402956 LMGP01010663
PC(22:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) 899.3740845 6.28295672 LMGP01012003
PC(22:1(11Z)/22:4(7Z,10Z,13Z,16Z)) 900.31604 4.064865082 LMGP01012033
PC(22:0/22:4(7Z,10Z,13Z,16Z)) 902.128006 12.31269729 LMGP01012002
PC(22:1(13E)/22:1(13E)) 906.6358643 8.243314415 LMGP01011108
PC(18:0/26:0) 910.7911377 23.81364481 LMGP01010828
PC(20:0/26:0) 938.448761 5.159413759 LMGP01011034
Phosphatydilserines
Grade 1
              Control
PS(15:0/0:0) 486.2679443 349.4908061 LMGP03050031
PS(16:1(9Z)/0:0) 490.930191 1351.894279 LMGP03050010
PS(18:0/0:0) 530.1671143 456.3458717 LMGP03050006
PS(20:0/0:0) 554.648584 684.5651013 LMGP03050012
PS(22:0/0:0) 585.0875854 179.8953782 LMGP03050025
PS(14:1(9Z)/14:1(9Z)) 670.7094727 32.97216305 LMGP03010919
PS(12:0/22:2(13Z,16Z)) 756.0275879 97.04654863 LMGP03010066
PS(13:0/22:2(13Z,16Z)) 769.5744629 191.1471607 LMGP03010090
PS(18:0/18:1(9Z)) 792.4706421 41.71102472 LMGP03010025
PS(17:0/22:2(13Z,16Z)) 826.5164795 394.1171434 LMGP03010246
PS(17:0/22:1(11Z)) 826.6235962 173.221204 LMGP03010245
PS(19:0/22:2(13Z,16Z)) 853.6619873 434.259899 LMGP03010477
PS(22:0/22:2(13Z,16Z)) 894.7367554 5.440477649 LMGP03010723
              CDK
PS(22:1(11Z)/0:0) 580.0860748 33.72772797 LMGP03050023
PS(12:0/16:1(9Z)) 673.6097412 6.176727075 LMGP03010049
PS(12:0/20:2(11Z,14Z)) 728.3601685 57.60975185 LMGP03010060
PS(13:0/22:4(7Z,10Z,13Z,16Z)) 766.5620728 2.502793177 LMGP03010091
PS(18:1(9Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) 829.5198975 4.677840797 LMGP03010045
PS(19:0/21:0) 852.2431335 50.02072802 LMGP03010474
PS(20:0/22:2(13Z,16Z)) 868.54953 7.575445338 LMGP03010526
PS(21:0/22:0) 890.7318624 57.13378798 LMGP03010698
Grade 2
              Control
PS(12:0/0:0) 439.9199524 20160.19245 LMGP03050008
PS(16:1(9Z)/0:0) 492.0351257 16760.19337 LMGP03050010
PS(19:1(9Z)/0:0) 535.6446533 38229.62984 LMGP03050019
PS(22:4(7Z,10Z,13Z,16Z)/0:0) 575.9509277 5370.664303 LMGP03050014
PS(22:0/0:0) 584.8668213 399.0301165 LMGP03050025
PS(12:0/13:0) 639.6738281 1485.057448 LMGP03010001
PS(14:0/12:0) 655.169632 2134.979121 LMGP03010931
PS(12:0/15:1(9Z)) 658.8825073 1073.721191 LMGP03010048
PS(14:1(9Z)/14:1(9Z)) 672.0128784 5326.561203 LMGP03010919
PS(14:0/14:0) 681.993866 1454.833471 LMGP03010028
PS(13:0/18:2(9Z,12Z)) 713.6038208 1105.076469 LMGP03010078
PS(12:0/20:2(11Z,14Z)) 727.508667 1256.958181 LMGP03010060
PS(12:0/22:2(13Z,16Z)) 754.8304749 664.3245694 LMGP03010066
PS(13:0/22:4(7Z,10Z,13Z,16Z)) 767.3230438 4198.550223 LMGP03010091
PS(17:0/21:0) 824.0448914 1161.380728 LMGP03010243
PS(17:0/22:1(11Z)) 827.3017273 109.6109416 LMGP03010245
PS(19:0/22:2(13Z,16Z)) 853.5385742 42064.46901 LMGP03010477
PS(20:0/22:2(13Z,16Z)) 867.8551636 711.0927161 LMGP03010526
PS(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) 877.0893402 1496.288525 LMGP01030017
              CDK
PS(18:2(9Z,12Z)/0:0) 517.1746216 44.51834435 LMGP03050011
PS(18:0/0:0) 529.024292 152.087076 LMGP03050006
PS(12:0/16:1(9Z)) 674.1259766 2.737743873 LMGP03010049
PS(13:0/18:3(6Z,9Z,12Z)) 711.3513184 24.13983471 LMGP03010079
PS(16:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) 805.9547729 13.41827269 LMGP03010043
PS(18:0/20:4(5Z,8Z,11Z,14Z)) 815.3720093 125.3012959 LMGP03010039
PS(17:0/22:2(13Z,16Z)) 826.2357788 277.8600108 LMGP03010246
PS(19:0/22:0) 863.70578 25.48655768 LMGP03010475
PS(22:4(7Z,10Z,13Z,16Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) 887.5043945 106.6639534 LMGP01030018
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Figure 4.

Figure 4

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Venn diagram showing common and unique lipid species found in control areas and CDK affected areas, for CDK grade 1 (superior panel) and CDK grade 2 (inferior panel), for PC and PS. Diagram intersections’ show common lipids between control and affected areas.

Table 3.

Common phospholipid species identified in both, control and CDK affected areas.

m/z* Amount, pmol per Species/μg protein LIPIDMAPS ID
Phosphatydilcholines
Grade 1
PC(2:0/0:0) 294.646521 119.0274412 LMGP01050043
PC(0:0/3:1(2E)) 312.3085632 0.955340492 LMGP01050088
PC(0:0/4:0) 328.5174103 171.4947089 LMGP01050089
PC(2:0/2:0) 345.2589264 25.29684283 LMGP01010992
PC(6:0/0:0) 360.7000733 85.34025255 LMGP01050062
PC(3:0/3:0) 373.3088633 158.4236004 LMGP01011215
PC(8:0/0:0) 390.3349152 28.19330422 LMGP01050066
PC(O-10:1(9E)/0:0) 399.1379204 50.14397867 LMGP01060027
PC(10:0/0:0) 414.0388184 168.5467928 LMGP01050005
PC(5:0/5:0) 430.6288147 301.5102334 LMGP01011225
PC(12:0/0:0) 442.1364975 41.97399351 LMGP01050009
PC(O-11:1(10E)/2:0) 458.1137187 142.0015605 LMGP01020146
PC(14:1(9Z)/0:0) 468.9347534 134.0907124 LMGP01050014
PC(15:1(9Z)/0:0) 482.7285462 316.4143978 LMGP01050125
PC(16:1(9Z)/0:0) 498.2089463 308.5061021 LMGP01050022
PC(18:4(9E,11E,13E,15E)/0:0) 520.5874023 1156.005517 LMGP01050040
PC(18:3(6Z,9Z,12Z)/0:0) 525.5578613 188.3306353 LMGP01050128
PC(19:3(10Z,13Z,16Z)/0:0) 537.7588501 1.693566016 LMGP01050003
PC(16:1(9Z)/2:0) 542.2512207 69.99420119 LMGP01010693
PC(20:5(5Z,8Z,11Z,14Z,17Z)/0:0) 549.6884155 496.3744142 LMGP01050050
PC(20:2(11Z,14Z)/0:0) 555.9118652 328.6174258 LMGP01050132
PC(6:2(3E,5E)/14:2(11E,13E)) 563.8403321 95.79849495 LMGP01011236
PC(18:1(9E)/2:0) 569.2772339 861.9458746 LMGP01010878
PC(22:4(7Z,10Z,13Z,16Z)/0:0) 579.3897247 264.4194871 LMGP01050124
PC(22:2(13Z,16Z)/0:0) 582.6686401 34.07005381 LMGP01050135
PC(18:1(9Z)/4:0) 598.3166504 305.8745425 LMGP01010916
PC(24:0/0:0) 611.7825394 475.51746 LMGP01050057
PC(12:0/12:0) 624.421875 311.3969386 LMGP01010429
PC(12:0/13:0) 643.6590983 463.2832235 LMGP01010001
PC(12:0/15:1(9Z)) 664.0793991 841.602332 LMGP01011318
PC(10:0/18:2(9Z,12Z)) 677.8133087 689.7408956 LMGP01010393
PC(10:0/18:1(9Z)) 684.8070069 220.5491298 LMGP01010392
PC(10:0/18:0) 686.6455383 54.59223528 LMGP01010390
PC(12:0/18:1(9Z)) 712.737854 17.62268556 LMGP01010440
PC(10:0/21:0) 728.0479736 2.847456952 LMGP01010396
PC(12:0/20:5(5Z,8Z,11Z,14Z,17Z)) 732.6423645 98.46496859 LMGP01011333
PC(13:0/20:5(5Z,8Z,11Z,14Z,17Z)) 747.0627747 22.27042735 LMGP01011357
PC(13:0/20:4(5Z,8Z,11Z,14Z)) 749.2914124 41.31325122 LMGP01011356
PC(15:1(9Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) 799.3400269 16.9228596 LMGP01011461
PC(17:0/20:4(5Z,8Z,11Z,14Z)) 804.3778992 54.65130021 LMGP01010003
PC(17:1(9Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) 826.1033936 17.78568203 LMGP01011550
PC(16:0/23:5(8E,11E,14E,17E,20E)) 830.2391357 8.993563427 LMGP01010656
PC(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) 886.4762269 41.85462361 LMGP01011119
PC(23:1(9Z)/23:1(9Z)) 927.0930237 134.2862697 LMGP01011141
PC(24:1(15Z)/24:1(15Z)) 952.9333598 203.7302272 LMGP01011163
PC(22:0/26:0) 965.081604 37.28316211 LMGP01011107
PC(34:0/16:0) 984.7036705 51.08762534 LMGP01011218
Grade 2
PC(2:0/0:0) 300.7475204 3.981478114 LMGP01050043
PC(0:0/3:1(2E)) 316.1439412 25.57478466 LMGP01050088
PC(0:0/4:0) 330.2403361 2.286290765 LMGP01050089
PC(2:0/2:0) 343.6709595 22.23310934 LMGP01010992
PC(6:0/0:0) 358.9837341 95.66402265 LMGP01050062
PC(3:0/3:0) 370.5003433 195.937209 LMGP01011215
PC(8:0/0:0) 391.1658936 0.226786815 LMGP01050066
PC(O-10:1(9E)/0:0) 397.5191345 3.512329217 LMGP01060027
PC(4:0/4:0) 406.9981689 15.65223013 LMGP01011222
PC(10:0/0:0) 416.5296326 196.4323143 LMGP01050005
PC(5:0/5:0) 429.5228831 23.91039676 LMGP01011225
PC(12:0/0:0) 440.451149 8.562903876 LMGP01050009
PC(6:2(2E,4E)/6:2(2E,4E)) 451.0426025 1.262407207 LMGP01011235
PC(O-11:1(10E)/2:0) 459.2329407 2.376642324 LMGP01020146
PC(14:1(9Z)/0:0) 470.2901306 0.666841011 LMGP01050014
PC(15:1(9Z)/0:0) 484.2517853 117.2943671 LMGP01050125
PC(16:1(9Z)/0:0) 497.7138443 0.415454367 LMGP01050022
PC(O-14:0/2:0) 504.4213715 0.191977505 LMGP01020019
PC(O-17:0/0:0) 505.3683929 135.6667898 LMGP01060013
PC(8:2(2E,4E)/8:2(2E,4E)) 509.2782364 9.147315957 LMGP01011254
PC(17:2(9Z,12Z)/0:0) 514.7207642 145.624321 LMGP01050127
PC(17:1(9Z)/0:0) 516.0500488 173.0182473 LMGP01050126
PC(18:3(6Z,9Z,12Z)/0:0) 525.7462158 210.0639731 LMGP01050128
PC(0:0/18:1(9Z)) 529.6395264 44.11870543 LMGP01050082
PC(O-16:0/2:0) 531.8207397 0.202057957 LMGP01020046
PC(19:3(10Z,13Z,16Z)/0:0) 538.7858124 1.225915616 LMGP01050003
PC(16:1(9Z)/2:0) 542.2966309 2.055870035 LMGP01010693
PC(20:5(5Z,8Z,11Z,14Z,17Z)/0:0) 549.8468933 1.663656827 LMGP01050050
PC(6:2(3E,5E)/14:2(11E,13E)) 562.7392883 129.1237968 LMGP01011236
PC(18:1(9E)/2:0) 570.3476563 3.703695673 LMGP01010878
PC(22:4(7Z,10Z,13Z,16Z)/0:0) 580.1956787 21.28910443 LMGP01050124
PC(16:0/5:1(4E)) 586.2541809 9.473000368 LMGP01010673
PC(18:1(9Z)/4:0) 601.3341064 0.548999836 LMGP01010916
PC(24:0/0:0) 610.3458252 48.39189195 LMGP01050057
PC(12:0/12:0) 620.5292664 69.65722502 LMGP01010429
PC(12:0/13:0) 638.8419088 83.83930245 LMGP01010001
PC(12:0/14:1(9Z)) 645.7624207 0.657894935 LMGP01011316
PC(12:0/15:1(9Z)) 664.4971771 3.292237794 LMGP01011318
PC(10:0/18:2(9Z,12Z)) 677.1966705 128.2524298 LMGP01010393
PC(10:0/18:0) 685.7401123 96.26273872 LMGP01010390
PC(12:0/17:2(9Z,12Z)) 691.0081787 2.483937833 LMGP01011322
PC(18:0/11:1(10E)) 699.3982849 1.484629256 LMGP01010735
PC(12:0/18:4(6Z,9Z,12Z,15Z)) 701.6916199 0.222956068 LMGP01011326
PC(10:0/20:0) 713.9262695 1.332221436 LMGP01010395
PC(13:0/18:4(6Z,9Z,12Z,15Z)) 718.4020233 2.416812887 LMGP01011351
PC(14:0/18:4(6Z,9Z,12Z,15Z)) 734.6452637 50.92343205 LMGP01010499
PC(14:0/18:3(9Z,12Z,15Z)) 735.803833 57.74795118 LMGP01010497
PC(14:0/18:2(11Z,14Z)) 738.2484741 1.165634299 LMGP01010494
PC(14:0/18:1(11Z)) 741.1770325 0.763261748 LMGP01010490
PC(13:0/20:5(5Z,8Z,11Z,14Z,17Z)) 744.3664246 1.517228029 LMGP01011357
PC(15:0/18:1(11Z)) 754.477356 0.833595162 LMGP01010541
PC(12:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) 757.8933716 0.830715782 LMGP01010447
PC(14:0/20:4(5Z,8Z,11Z,14Z)) 762.9011078 31.29192158 LMGP01010506
PC(16:0/18:2(10E,12Z)) 765.7141113 16.23479689 LMGP01010585
PC(10:0/24:0) 770.1452637 2.13800887 LMGP01010400
PC(15:0/20:3(8Z,11Z,14Z)) 778.9407043 1.169170041 LMGP01011423
PC(16:0/20:4(5Z,8Z,11Z,14Z)) 789.7157288 37.82410851 LMGP01010007
PC(15:1(9Z)/22:4(7Z,10Z,13Z,16Z)) 801.9890137 1.363450938 LMGP01011460
PC(17:0/20:4(5Z,8Z,11Z,14Z)) 804.8378601 0.448589654 LMGP01010003
PC(16:0/22:6(4E,7E,10E,13E,16E,19E)) 814.1509705 1.124265111 LMGP01010650
PC(16:0/22:4(7Z,10Z,13Z,16Z)) 818.4989014 1.446115056 LMGP01010642
PC(18:0/20:3(5Z,11Z,14Z)) 821.5475159 1.178012109 LMGP01010795
PC(17:1(9Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) 826.6335449 43.84880809 LMGP01011550
PC(18:4(6Z,9Z,12Z,15Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) 835.464447 1.54416391 LMGP01011730
PC(18:3(6Z,9Z,12Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) 836.2970886 1.422869593 LMGP01011670
PC(18:2(9Z,12Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) 838.7856903 2.5963024 LMGP01010947
PC(18:0/22:4(7Z,10Z,13Z,16Z)) 847.0201721 42.56806052 LMGP01010813
PC(16:0/24:1(15Z)) 852.4560547 0.756871684 LMGP01010659
PC(20:2(11Z,14Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) 867.4215088 1.056135248 LMGP01011865
PC(20:1(11Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) 868.4871216 1.518624943 LMGP01011834
PC(20:0/22:4(7Z,10Z,13Z,16Z)) 873.7175903 1.454865821 LMGP01011804
PC(18:0/24:1(15Z)) 880.6356201 1.500554735 LMGP01010826
PC(16:0/26:0) 882.6246948 0.947456718 LMGP01010663
PC(21:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) 884.5966187 0.994330628 LMGP01010004
PC(21:0/22:4(7Z,10Z,13Z,16Z)) 889.1891479 0.273602214 LMGP01011979
PC(22:2(13Z,16Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) 894.450531 1.460927121 LMGP01012065
PC(22:1(11Z)/22:2(13Z,16Z)) 905.4901733 1.428776634 LMGP01012032
PC(22:1(13E)/22:1(13E)) 906.8718414 2.833697169 LMGP01011108
PC(18:0/26:0) 910.887207 0.778367357 LMGP01010828
PC(23:1(9Z)/23:1(9Z)) 927.9228668 2.87855425 LMGP01011141
PC(22:0/24:1(15Z)) 936.1557312 0.531377092 LMGP01011105
PC(20:0/26:0) 938.6951904 0.372342145 LMGP01011034
PC(24:1(15Z)/24:1(15Z)) 950.0507507 121.4692795 LMGP01011163
PC(22:0/26:0) 965.9285583 1.449679534 LMGP01011107
PC(34:0/16:0) 984.3062998 7.685921442 LMGP01011218
Phosphatydilserines
Grade 1
PS(12:0/0:0) 440.4143036 2793.388271 LMGP03050008
PS(6:0/6:0) 457.8782501 772.5812763 LMGP03010020
PS(14:0/0:0) 470.6537933 2681.892134 LMGP03050009
PS(15:1(9Z)/0:0) 480.2579575 2310.389423 LMGP03050015
PS(16:0/0:0) 496.9708455 365.5803313 LMGP03050002
PS(18:1(9Z)/0:0) 522.1900024 1940.676303 LMGP03050001
PS(19:1(9Z)/0:0) 535.2797623 850.3865587 LMGP03050019
PS(20:4(5Z,8Z,11Z,14Z)/0:0) 545.4216919 111.6436172 LMGP03050007
PS(10:0/10:0) 565.9069214 1667.852742 LMGP03010022
PS(12:0/12:0) 622.0140991 175.9625895 LMGP03010027
PS(12:0/13:0) 638.8652649 268.9540205 LMGP03010001
PS(12:0/14:1(9Z)) 653.2467041 153.7468114 LMGP03010046
PS(14:0/12:0) 654.826416 285.2899071 LMGP03010931
PS(12:0/15:0) 663.7553711 79.64711642 LMGP03010047
PS(14:0/14:0) 675.4645996 45.88818134 LMGP03010028
PS(12:0/17:2(9Z,12Z)) 686.1657104 131.8011401 LMGP03010052
PS(12:0/17:1(9Z)) 686.7450867 376.4542771 LMGP03010051
PS(12:0/17:0) 693.3683065 150.2897921 LMGP03010050
PS(12:0/18:2(9Z,12Z)) 699.5421143 140.4609379 LMGP03010053
PS(13:0/18:2(9Z,12Z)) 714.3778687 25.41099612 LMGP03010078
PS(17:0/14:1(9Z)) 718.2476654 122.289291 LMGP03010006
PS(12:0/19:0) 726.2710266 42.10891676 LMGP03010057
PS(16:0/16:0) 735.1644775 569.4678808 LMGP03010029
PS(12:0/21:0) 748.2671509 1740.017591 LMGP03010064
PS(16:0/18:1(11Z)) 761.6001587 22.96151068 LMGP03010007
PS(13:0/22:1(11Z)) 770.7945557 2.285220437 LMGP03010089
PS(13:0/22:0) 775.6061401 94.64329165 LMGP03010088
PS(18:2(9Z,12Z)/18:2(9Z,12Z)) 781.9361877 112.3843186 LMGP03010023
PS(17:0/20:4(5Z,8Z,11Z,14Z)) 795.7001953 998.0748387 LMGP03010003
PS(16:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) 804.4544678 88.11515529 LMGP03010043
PS(18:0/20:4(5Z,8Z,11Z,14Z)) 806.6505737 97.02972519 LMGP03010039
PS(18:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) 836.5066528 322.1845042 LMGP03010040
PS(18:0/22:1(11Z)) 845.3986003 1022.9785 LMGP03010320
PS(19:0/22:0) 865.8910522 257.3924868 LMGP03010475
PS(20:0/22:1(11Z)) 870.6346842 168.337861 LMGP03010525
PS(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) 883.2276306 84.36108425 LMGP01030017
PS(22:4(7Z,10Z,13Z,16Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) 888.2210693 100.7549591 LMGP01030018
PS(22:0/22:1(11Z)) 902.2740682 739.7834825 LMGP03010722
PS(22:0/22:0) 908.0455933 99.37037004 LMGP03010943
PS(8:0/8:0) 1535.316406 3393.87959 LMGP03010021
Grade 2
PS(6:0/6:0) 456.8153992 30698.24075 LMGP03010020
PS(14:0/0:0) 465.2920074 203.8807057 LMGP03050009
PS(15:1(9Z)/0:0) 484.0389252 1108.446661 LMGP03050015
PS(16:0/0:0) 494.9042969 52469.15679 LMGP03050002
PS(17:2(9Z,12Z)/0:0) 505.7750702 25458.10288 LMGP03050016
PS(18:1(9Z)/0:0) 522.9552917 27456.93029 LMGP03050001
PS(20:4(5Z,8Z,11Z,14Z)/0:0) 544.1977437 2318.998246 LMGP03050007
PS(20:0/0:0) 553.0429688 526.7623955 LMGP03050012
PS(10:0/10:0) 569.5486145 9698.171378 LMGP03010022
PS(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/0:0) 572.4798279 8951.025196 LMGP03050013
PS(22:1(11Z)/0:0) 582.1560364 16767.76244 LMGP03050023
PS(12:0/12:0) 624.1285095 4540.48997 LMGP03010027
PS(12:0/14:1(9Z)) 650.6720479 19941.5584 LMGP03010046
PS(12:0/15:0) 669.9039307 514.3605395 LMGP03010047
PS(12:0/17:0) 697.3886719 5536.324964 LMGP03010050
PS(12:0/18:2(9Z,12Z)) 700.0890503 472.5421882 LMGP03010053
PS(17:0/14:1(9Z)) 721.6215363 26279.15042 LMGP03010006
PS(16:0/16:0) 737.6786804 4369.155673 LMGP03010029
PS(13:0/20:2(11Z,14Z)) 741.8208008 1566.624098 LMGP03010084
PS(13:0/20:1(11Z)) 742.5445557 985.938965 LMGP03010083
PS(12:0/21:0) 751.5957794 18965.41756 LMGP03010064
PS(16:0/18:1(11Z)) 764.1244914 4240.293661 LMGP03010007
PS(13:0/22:0) 775.519104 119.9065759 LMGP03010088
PS(18:2(9Z,12Z)/18:2(9Z,12Z)) 787.125 665.3150824 LMGP03010023
PS(18:0/18:1(9Z)) 791.3975525 12003.57914 LMGP03010025
PS(17:0/20:4(5Z,8Z,11Z,14Z)) 797.8404948 3217.740952 LMGP03010003
PS(16:0/22:1(11Z)) 820.0691732 3110.351725 LMGP03010199
PS(18:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) 839.9198608 1050.759611 LMGP03010040
PS(18:0/22:1(11Z)) 845.3218384 2133.385921 LMGP03010320
PS(19:0/21:0) 851.4665375 29861.94664 LMGP03010474
PS(19:0/22:1(11Z)) 854.967041 1085.657158 LMGP03010476
PS(20:0/22:1(11Z)) 870.8126221 3280.083414 LMGP03010525
PS(21:0/22:0) 889.1227112 1632.461271 LMGP03010698
PS(22:0/22:2(13Z,16Z)) 896.3721924 1235.249654 LMGP03010723
PS(22:0/22:1(11Z)) 901.10554 2192.570608 LMGP03010722
PS(8:0/8:0) 1023.023682 19258.31146 LMGP03010021
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In case of PS, 13 unique species were found in control areas and 8 in CDK affected areas from grade 1. For grade 2, 19 PS and 9 PS species were found in control and CDK areas, respectively (table 2, figure 4). Forty PS and 36 species were common between control and affected areas from grade 1 and grade 2, respectively (table 3, figure 4).

DISCUSION

CDK is a rare corneal degenerative disease characterized by progressive opacity because of accumulation of translucent material in the Bowman layer of the cornea within the interpalpebral fringe (Gray et al., 1992; Urrets-Zavalía et al., 2006, 2007). We have previously shown that CDK occurred in individuals who have lived their entire life under certain unfavorable environmental conditions such as high exposure to UVR, chronic micro erosions of the cornea, low levels of ascorbic acid (AA) in their diets and serum, and lack of protection with sunglasses or hats (Suarez et al., 2015). For those reasons we have previously proposed that this corneal disease should be call environmental proteinaceous corneal degenerative disease instead of climatic droplet keratopathy (Suarez et al, 2015).

The precise chemical nature of globules accumulated under the corneal epithelium remains uncertain despite many efforts have been made to unravel its composition (Tabbara, 1986; Kaji et al., 2007; Menegay et al., 2008; Kaji et al., 2010). It has been suggested that corneal deposits are derived from plasma proteins that, after reaching an inflamed cornea from the limbal vessels, are degraded by excessive exposure to UVR (Gray et al., 1992). Although no mechanistic link between UVR exposure and CDK has been established, Holopainen et al. have addressed this issue analyzing tears and corneal specimens obtained from CDK affected eyes. They found that CDK samples have higher levels of MMP-2, MMP-9, and pro inflammatory cytokines than unaffected individuals (Holopainen et al., 2012). They hypothesized that these proteins are produced by the corneal epithelium, and showed that UVR-B is capable of inducing pro-inflammatory response in these cells with concomitant increase in MMP-2 and MMP-9 levels. This is then likely to increase the apoptotic/ necrotic response of the corneal epithelial cells, harm the integrity of the basement membrane, and, eventually, lead to visible changes in the corneal architecture.

Recently, we performed a study in which two groups of guinea pigs were exposed during 30 months to daily doses of UVR-B similar to the one received by CDK patients living in Patagonia Argentina, fed with AA sufficient or AA partially deficient diets, respectively. Superficial corneal debridement wounds were made in one eye using a rotating burr (Algerbrush II, Ambler Surgical, Exton, PA, USA) at the nasal or temporal corneal limbus (once a week, alternating each region) previous topical anesthetic applied on the ocular surface (proparacaine ophthalmic solution, Alcon laboratories Argentina, Buenos Aires, Argentina). We found that guinea pig corneal epithelial cell levels of MMP-9 were increased, and that ALDH3A1 activity (most abundant soluble protein in the cornea, Estey et al., 2007) was increased during the first 18 months and then it decayed. The lipid peroxidation marker malondialdehyde (MDA) (Grotto, 2009) concentration followed the same pattern than ALDH3A1 activity. Moreover, we found that animals fed with deficient AA diet showed more pronounced abnormalities in the cornea and in the crystalline lens (unpublished data). All these findings are compatible with a state of chronic oxidative stress.

It is well known that combination of near continual exposure of the cornea and ocular surface to UVR and molecular oxygen can provoke oxidative stress and tissue damage. Oxidative stress can be defined as an imbalance between reactive oxygen species (ROS) and antioxidants (Cejka and Cejkova, 2015). Among the antioxidants we can mention enzymatic and non-enzymatic molecules. Some of the enzymatic antioxidants are constituted by aldehyde dehydrogenases (ALDHs), catalase, and superoxide dismutase (Chance et al., 1979; Fridovich, 1995; Harrison and Arosio, 1996; Chen et al., 2013), whereas AA, reduced glutathione (GSH), α-tocopherol and NAD(P)H are among the non-enzymatic antioxidants (Dickinson and Forman, 2002; Machlin and Bendich, 1987).

Oxidative stress can cause peroxidation and further damage of lipids, nucleic acids, bases, and proteins. The eye is one of the major targets of the ROS and reactive nitrogen species (RNS) attack due to exposition to several environmental factors like high pressure of oxygen, light, UVR, ionizing radiation, chemical pollutants and pathogenic microbes, which are able to shift the redox status of a cell towards oxidizing conditions. There is increasing evidence indicating that persistent oxidative stress contributes to the development of many ocular diseases such as certain types of keratitis, pterygium, and pinguecula, among others (Kruk et al., 2015).

Our hypothesis for CDK genesis is that individuals with prolonged corneal exposure to multiple unfavorable environmental conditions (e.g., excessive UVR-B exposure, lack of vegetation/shade, dry/windy climate, particle bombardment, AA partial nutritional deficiency, lack of eye protection, genetic factors, etc.) would develop inflammatory processes and oxidative stress leading to progressive degradation and accumulation of proteinaceous material in Bowman’s layer and, in advances cases of the disease, in the superficial stroma and deep epithelium (Urrets-Zavalia et al., 2012; Holopainen et al., 2012; Serra et al., 2015).

In the present study, we have determined phospholipids (PC and PS) present in control and CDK affected areas from patients’ corneas using triple quadrupole mass spectrometry, in parent-ion and neutral loss scan modes with parameters previously established in the lipid field and widely used for ocular tissue in the study of some ophthalmological diseases (Han et al., 2012; Bhattacharya, 2013; Wang et al., 2016). Phospholipids are the basic building blocks of cell membranes, arranged as bilayer membranes. Membrane phospholipids create a hydrophobic environment for transmembrane protein function and communication. Some membrane lipids are substrates for production of lipid second messengers, which are metabolized by enzymatic activity from phospholipid precursors (Fahy et al., 2011; Aribindi et al., 2013b; Edwards et al., 2014; Li et al., 2015).

Corneal lipid content was studied in humans and animals many years ago (Feldman, 1967; Broekhuyse, 1968; Bazan and Bazan, 1984). Bazan et al. (1984) established that corneal rabbit epithelium has larger phospholipid content and more saturated than in the rest of the cornea. Conversely, corneal endothelium has a higher unsaturation level, according with water permeability of this layer (three times higher than in epithelium). Herein, we report data that clearly shows decreased levels of total phospholipids, PC and PS in the affected CDK epithelium area (in grade 1 as well as grade 2) compared to the non-affected areas. We also describe phospholipids uniquely enriched in control areas, and others only present in CDK affected areas (table 2). At the same time, corneal epithelial cells from CDK grade 1 and grade 2 patients’ eyes present a common PC and PS composition (table 3).

It is known that UVR can induce lipid peroxidation in many cell types including human corneal epithelial cells. UVR leads to the production of ROS that initiate lipid peroxidation by attacking the polyunsaturated fatty acids chains in cell membrane phospholipids, and may cause the accumulation of reactive aldehydes. Unlike ROS, aldehydes are generally long-lived compounds that can diffuse through the cell and react with biological targets distant from the site of origin (Estey et al., 2007). Lipid peroxidation has been involved in several disorders such as atherogenesis, neurodegenerative diseases, cataractogenesis, and retinopathy (Awasthi et al., 1996; Witting et al., 1999; Butterfield et al., 2001; Kumar et al., 2001; Totan et al., 2001). Lipid peroxidation as a consequence of oxidative stress may have been taking place in corneas from CDK patients. This could account for the reduction in phospholipid concentrations that was observed.

In summary, we have studied for the first time phospholipid composition in corneal epithelial cells from CDK patients using shotgun lipidomics (direct infusion in triple quadrupole mass spectrometry), and we have found new evidences in favor of our hypothesis about the etiopathogenesis of CDK. The lower total amount of phospholipids observed in affected areas compared to control areas, and a differential composition between healthy corneal epithelial areas and CDK affected areas, could be signs of an active oxidative stress process occurring in CDK corneas.

Acknowledgments

We thank Yennifer Guerra for assistance with bioinformatic analyses.

This work was partly supported by a grant FONCYT from Argentina, and an unrestricted grant to University of Miami from Research to Prevent Blindness (RPB), NIH grants EY016112 and EY14801, Department of Defense grant W81XWH-15-1-0079.

Footnotes

CONFLICT OF INTERESTS

The authors declare that there is no conflict of interests regarding the publication of this paper.

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