a total of 81 participants , including 52 patients with des ( 21 males and 31 females ; mean age , 52.37  10.18 years ; range , 32 to 75 years ) and 29 age- and sex - matched healthy individuals as controls ( 17 males and 12 females ; mean age , 50.69  10.37 years ; range , 24 to 68 years ) , were enrolled in this study .
des was diagnosed by a physician based on the results of a patient questionnaire and clinical examinations according to the international dry eye workshop report . in the 52 patients with des not associated with sjgren 's syndrome , coexisting lid margin disease , or altered tear distribution and clearance , des severity was categorized into 1 of 4 levels , according to the delphi panel classification ( des grade 1 , n = 10 ; grade 2 , n = 10 ; grade 3 , n = 22 ; grade 4 , n = 10 ) .
then , each individual underwent a complete examination of the ocular surface in the following order : tear break - up time ( tbut ) , corneal staining with f luorescein , conjunctival staining with lissamine green , and schirmer 's test without anesthesia .
corneal staining was evaluated using the national eye institute method , and conjunctival staining was evaluated using the oxford scheme .
the exclusion criteria included an age less than 18 years , previous usage of ophthalmic medications except artificial tears , a history of surgical intervention , chemical injury , a corneal pathology such as a corneal infection , complaints of ocular pain or discomfort due to any recent history of ocular disease besides des , use of contact lenses in the previous 6 months , connective tissue disease ( other than rheumatoid arthritis ) , autoimmune disease , diabetes mellitus , and parkinson 's disease .
all control individuals had no abnormalities in their ocular surface examinations , i.e. , a schirmer test i value greater than 10 mm/5 min , a tbut value greater than 10 seconds , no complaint of ocular discomfort , and no usage of ocular medications .
the research followed the tenets of the declaration of helsinki and informed consent was obtained from the participants after explanation of the nature and possible consequences of the study .
the research was approved by an institutional review board ( no . c2012136 - 831 ) .
samples of pure , undiluted tear fluid were collected from all 81 participants in 50-l glass capillaries placed with one end at the lateral canthus in contact with the tear film of the right eye .
each tear sample was placed in a 1.5-ml eppendorf tube and immediately stored at -70 until further examination . to keep tear stimulation at a low level , contact with the conjunctival epithelium
no additional solution that could induce irritation and stimulate tear fluids was used during tear collection . from each participant ,
approximately 10 l of unstimulated tears were collected by a single physician ( sww ) .
the total tear protein concentration was evaluated with a nano - drop spectrophotometer ( nd-1000 ; nano - drop technologies , wilmington , de , usa ) .
as the concentrations of ang and lf in an individual 's tear fluids used for elisa may vary , we normalized the concentration of each protein to the total tear protein concentration for both groups .
we used 3 l of tear sample from each normal individual and des patient , and diluted this aliquot to a volume of 1 ml with buffer .
tear protein profiling of all participants was performed with an immunodot blot assay following the manufacturer 's instructions .
tear proteins such as basic fibroblast growth factor ( bfgf ) , vascular endothelial growth factor , transforming growth factor ( tgf)-2 , tumor necrosis factor- , interleukin ( il)-10 , il-17 , angiostatin , stromal cell - derived factor-1 , fas ligand , and ang were investigated with a human cytokine antibody array kit ( raybiotech , atlanta , ga , usa ) .
collected tear samples were centrifuged to eliminate any cell debris and were applied to a membrane .
the tear samples were applied to each membrane for 2 hours at room temperature , after which the non - specific areas of the membranes were blocked for 30 minutes .
all membranes were washed three times for 5 minutes with wash buffer 1 , and then washed twice for 5 minutes with wash buffer 2 .
tear proteins were detected by diluted biotin - conjugated antibodies added to each membrane for 2 hours of incubation .
the membranes were visualized using a chemidoc xrs ( bio - rad laboratories , berkeley , ca , usa ) .
the density of each blot was measured with a personal molecular imager fx ( bio - rad laboratories ) supported by imaging analysis software ( quantity one , bio - rad laboratories ) .
each tear sample was diluted to a total volume of 300 l with buffer . to confirm the results of the immunodot blot assay ,
the amounts of ang and lf in tears from normal controls and des patients were compared quantitatively by elisa using a human lf detection kit ( bethyl laboratories , montgomery , tx , usa ) and a human ang elisa detection kit ( abcam , cambridge , england ) following the manufacturers ' instructions . in brief , standards or samples
were added to 96-well plates and incubated for 1 hour at 4. the plates were washed four times , and a biotin antibody for ang or lf detection was added to each well .
the plates were covered , incubated for 1 hour at room temperature , and then washed four times . a streptavidin solution for ang or an horseradish peroxidase solution
the plates were covered and incubated at room temperature for 45 minutes with ang or 30 minutes with lf .
after the washing step was repeated , the 3,3',5,5'-tetramethylbenzidine liquid substrate solution was added to each well .
the plates were incubated for 30 minutes at room temperature in the dark , and the stop solution was added to each well .
the optical density was measured at 450 nm on a microplate photometer spectramax 340pc using softmax pro 5.4.1 ( mds inc . ,
16.0 ( spss inc . , chicago , il , usa ) . for all tests , the significance level was set at p < 0.05 .
based on the normality of the data distribution , comparative analyses among individual samples of proteins were performed using the mann - whitney u - test , student 's t - test , and the chi - square test .
correlations between the protein concentrations and dry eye severity were assessed using spearman 's correlation test .
a total of 81 participants , including 52 patients with des ( 21 males and 31 females ; mean age , 52.37  10.18 years ; range , 32 to 75 years ) and 29 age- and sex - matched healthy individuals as controls ( 17 males and 12 females ; mean age , 50.69  10.37 years ; range , 24 to 68 years ) , were enrolled in this study .
des was diagnosed by a physician based on the results of a patient questionnaire and clinical examinations according to the international dry eye workshop report . in the 52 patients with des not associated with sjgren 's syndrome , coexisting lid margin disease , or altered tear distribution and clearance , des severity was categorized into 1 of 4 levels , according to the delphi panel classification ( des grade 1 , n = 10 ; grade 2 , n = 10 ; grade 3 , n = 22 ; grade 4 , n = 10 ) .
then , each individual underwent a complete examination of the ocular surface in the following order : tear break - up time ( tbut ) , corneal staining with f luorescein , conjunctival staining with lissamine green , and schirmer 's test without anesthesia .
corneal staining was evaluated using the national eye institute method , and conjunctival staining was evaluated using the oxford scheme .
the exclusion criteria included an age less than 18 years , previous usage of ophthalmic medications except artificial tears , a history of surgical intervention , chemical injury , a corneal pathology such as a corneal infection , complaints of ocular pain or discomfort due to any recent history of ocular disease besides des , use of contact lenses in the previous 6 months , connective tissue disease ( other than rheumatoid arthritis ) , autoimmune disease , diabetes mellitus , and parkinson 's disease .
all control individuals had no abnormalities in their ocular surface examinations , i.e. , a schirmer test i value greater than 10 mm/5 min , a tbut value greater than 10 seconds , no complaint of ocular discomfort , and no usage of ocular medications .
the research followed the tenets of the declaration of helsinki and informed consent was obtained from the participants after explanation of the nature and possible consequences of the study .
the research was approved by an institutional review board ( no . c2012136 - 831 ) .
samples of pure , undiluted tear fluid were collected from all 81 participants in 50-l glass capillaries placed with one end at the lateral canthus in contact with the tear film of the right eye .
each tear sample was placed in a 1.5-ml eppendorf tube and immediately stored at -70 until further examination . to keep tear stimulation at a low level , contact with the conjunctival epithelium
no additional solution that could induce irritation and stimulate tear fluids was used during tear collection . from each participant ,
approximately 10 l of unstimulated tears were collected by a single physician ( sww ) .
the total tear protein concentration was evaluated with a nano - drop spectrophotometer ( nd-1000 ; nano - drop technologies , wilmington , de , usa ) .
as the concentrations of ang and lf in an individual 's tear fluids used for elisa may vary , we normalized the concentration of each protein to the total tear protein concentration for both groups .
we used 3 l of tear sample from each normal individual and des patient , and diluted this aliquot to a volume of 1 ml with buffer .
tear protein profiling of all participants was performed with an immunodot blot assay following the manufacturer 's instructions .
tear proteins such as basic fibroblast growth factor ( bfgf ) , vascular endothelial growth factor , transforming growth factor ( tgf)-2 , tumor necrosis factor- , interleukin ( il)-10 , il-17 , angiostatin , stromal cell - derived factor-1 , fas ligand , and ang were investigated with a human cytokine antibody array kit ( raybiotech , atlanta , ga , usa ) .
collected tear samples were centrifuged to eliminate any cell debris and were applied to a membrane .
the tear samples were applied to each membrane for 2 hours at room temperature , after which the non - specific areas of the membranes were blocked for 30 minutes .
all membranes were washed three times for 5 minutes with wash buffer 1 , and then washed twice for 5 minutes with wash buffer 2 .
tear proteins were detected by diluted biotin - conjugated antibodies added to each membrane for 2 hours of incubation .
the membranes were visualized using a chemidoc xrs ( bio - rad laboratories , berkeley , ca , usa ) .
the density of each blot was measured with a personal molecular imager fx ( bio - rad laboratories ) supported by imaging analysis software ( quantity one , bio - rad laboratories ) .
was diluted to a total volume of 300 l with buffer . to confirm the results of the immunodot blot assay ,
the amounts of ang and lf in tears from normal controls and des patients were compared quantitatively by elisa using a human lf detection kit ( bethyl laboratories , montgomery , tx , usa ) and a human ang elisa detection kit ( abcam , cambridge , england ) following the manufacturers ' instructions . in brief , standards or samples were added to 96-well plates and incubated for 1 hour at 4. the plates were washed four times , and a biotin antibody for ang or lf detection was added to each well .
the plates were covered , incubated for 1 hour at room temperature , and then washed four times .
a streptavidin solution for ang or an horseradish peroxidase solution was added to each well .
the plates were covered and incubated at room temperature for 45 minutes with ang or 30 minutes with lf .
after the washing step was repeated , the 3,3',5,5'-tetramethylbenzidine liquid substrate solution was added to each well .
the plates were incubated for 30 minutes at room temperature in the dark , and the stop solution was added to each well .
the optical density was measured at 450 nm on a microplate photometer spectramax 340pc using softmax pro 5.4.1 ( mds inc . ,
all data were analyzed with statistical software spss ver . 16.0 ( spss inc . ,
based on the normality of the data distribution , comparative analyses among individual samples of proteins were performed using the mann - whitney u - test , student 's t - test , and the chi - square test .
correlations between the protein concentrations and dry eye severity were assessed using spearman 's correlation test .
demographic data from the patients and healthy controls , including age , sex , and clinical test results , are summarized in table 1 .
the ang areas in des grades 3 and 4 patients were significantly lower than those of normal controls and des patients with lower grades .
however , there were no significant differences between normal controls and des patients with lower grades .
the ang blot densities were 508.15  60.80 in normal controls , 511.39  63.34 in des grade 1 patients , 460.40  35.72 in des grade 2 patients , 49.05  23.57 in des grade 3 patients , and 38.70  11.71 in des grade 4 patients .
the areas of bfgf , tgf-2 , and il-10 were significantly greater than those of normal controls only in grade 4 des patients , but these proteins did not correlate significantly with dry eye severity .
il-17 , angiostatin , stromal cell - derived factor-1 , fas ligand , and vascular endothelial growth factor did not differ significantly among participants , regardless of des grade .
1a and 1b and 2a-2e summarize the results of tear protein profiling from the immunodot blot assay .
the mean concentrations of ang in the tears of normal controls ( n = 29 ) and des grade 1 ( n = 10 ) , and 2 ( n = 10 ) patients were 206.65  100.23
the concentrations of ang in the tears of des grade 3 ( n = 22 ) and 4 ( n = 10 ) patients were 71.03  44.67 pg / ml and 41.16  33.83
the mean concentrations of lf in the tears of normal controls and des grade 1 , and 2 patients were 0.41  0.19 mg / ml , 0.27  0.11 mg / ml , and 0.27  0.19 mg / ml , respectively . the concentrations of lf in the tears of des grade 3 and 4 patients were 0.10  0.09 mg / ml and 0.06  0.04 mg / ml , respectively .
ang and lf concentrations in des patients were lower than those of normal controls and decreased significantly with increasing des severity , except in grades 1 and 2 .
the group - to - group differences in lf and ang were statistically significant ( p < 0.05 ) , except for the differences between des severity grades 1 and 2 . table 2 summarizes the mean concentrations of ang and lf in control and des tears . the ratios of ang and lf to total tear proteins were measured to supplement the concentration results in normal controls and des patients .
the percentage concentration of ang / total tear protein was 11.48  5.90 ( 10)% in the normal control group , 6.14  2.93 ( 10)% in des grade 1 , 5.74  2.12 ( 10)% in des grade 2 , 3.78  2.27 ( 10)% in des grade 3 , and 2.15  1.62 ( 10)% in des grade 4 patients .
the percentage concentration of lf / total tear protein was 21.69  7.58% in the normal control group , 13.65  6.70% in des grade 1 , 13.36  7.74% in des grade 2 , 5.54  4.68% in des grade 3 , and 3.05  2.31% in des grade 4 patients .
the group - to - group differences in the percentage concentrations of lf and ang were statistically significant ( p < 0.05 ) , except for those between des severity grades 1 and 2 . fig .
4 shows the significant correlation between the percentage concentrations of ang and lf in des patients [ lf ( % ) = 1.0  ang ( % )  10 + 4.7828 ] .
demographic data from the patients and healthy controls , including age , sex , and clinical test results , are summarized in table 1 .
the ang areas in des grades 3 and 4 patients were significantly lower than those of normal controls and des patients with lower grades .
however , there were no significant differences between normal controls and des patients with lower grades .
the ang blot densities were 508.15  60.80 in normal controls , 511.39  63.34 in des grade 1 patients , 460.40  35.72 in des grade 2 patients , 49.05  23.57 in des grade 3 patients , and 38.70  11.71 in des grade 4 patients .
the areas of bfgf , tgf-2 , and il-10 were significantly greater than those of normal controls only in grade 4 des patients , but these proteins did not correlate significantly with dry eye severity .
il-17 , angiostatin , stromal cell - derived factor-1 , fas ligand , and vascular endothelial growth factor did not differ significantly among participants , regardless of des grade .
1a and 1b and 2a-2e summarize the results of tear protein profiling from the immunodot blot assay .
the mean concentrations of ang in the tears of normal controls ( n = 29 ) and des grade 1 ( n = 10 ) , and 2 ( n = 10 ) patients were 206.65  100.23
pg / ml , 123.43  52.54 pg / ml , and 109.85  38.76 pg / ml , respectively .
the concentrations of ang in the tears of des grade 3 ( n = 22 ) and 4 ( n = 10 ) patients were 71.03  44.67 pg / ml and 41.16  33.83
the mean concentrations of lf in the tears of normal controls and des grade 1 , and 2 patients were 0.41  0.19 mg / ml , 0.27  0.11 mg / ml , and 0.27  0.19 mg / ml , respectively . the concentrations of lf in the tears of des grade 3 and 4 patients were 0.10  0.09 mg / ml and 0.06  0.04 mg / ml , respectively .
ang and lf concentrations in des patients were lower than those of normal controls and decreased significantly with increasing des severity , except in grades 1 and 2 .
the group - to - group differences in lf and ang were statistically significant ( p < 0.05 ) , except for the differences between des severity grades 1 and 2 . table 2 summarizes the mean concentrations of ang and lf in control and des tears . the ratios of ang and lf to total tear proteins were measured to supplement the concentration results in normal controls and des patients . the percentage concentration of ang / total tear protein was 11.48  5.90 ( 10)% in the normal control group , 6.14  2.93 ( 10)% in des grade 1 , 5.74  2.12 ( 10)% in des grade 2 , 3.78  2.27 ( 10)% in des grade 3 , and 2.15  1.62 ( 10)% in des grade 4 patients .
the percentage concentration of lf / total tear protein was 21.69  7.58% in the normal control group , 13.65  6.70% in des grade 1 , 13.36  7.74% in des grade 2 , 5.54  4.68% in des grade 3 , and 3.05  2.31% in des grade 4 patients .
the group - to - group differences in the percentage concentrations of lf and ang were statistically significant ( p < 0.05 ) , except for those between des severity grades 1 and 2 . fig .
4 shows the significant correlation between the percentage concentrations of ang and lf in des patients [ lf ( % ) = 1.0  ang ( % )  10 + 4.7828 ] .
several diagnostic tests for des , such as fluorescein staining , tbut measurement , schirmer 's test , rose bengal staining , impression cytology , and tear clearance rate measurement , have been widely used .
however , these commonly used diagnostic tests are reported to be poorly associated with patients ' symptoms , and no definite standards for measuring the therapeutic response to treatment in patients with des have been established .
recently , several studies have evaluated and graded des via proteomic analysis ; a proteomic approach is becoming more common as a novel diagnostic tool for des .
tear protein levels have been compared between evaporative dry eye patients and controls . in a previous study , lf and lipophilin a - c were lower in evaporative dry eye patients than in controls . on the other hand ,
as des is understood to be a multifactorial disorder and inflammation of the ocular surface is considered to be closely related with des , alterations in tear protein concentrations in dry eye patients may reflect the inflammatory status of the eye .
we hypothesized that the tear protein levels of des patients would differ from those of normal controls , and evaluated the correlation between the des severity and the concentrations of certain proteins . in the present study , concentrations of bfgf , tgf-2 , and
il-10 were significantly greater than those of normal controls only in grade 4 des patients , but these proteins did not correlate significantly with dry eye severity . as tgf-2 and il-10 are reported to be anti - inflammatory proteins secreted by cd4 + regulatory t cells upon inflammation of the ocular surface , and bfgf is reported to potentiate inflammatory mediator - induced leukocyte recruitment in chronic inflammation , we concluded that the results of our immunodot blot assay were similar to those of the previous studies .
however , the concentrations of bfgf , tgf-2 , tumor necrosis factor- , and il-10 were not significantly correlated with des severity . in contrast , ang was linearly associated with des severity , except between grades 1 and 2 .
we performed a further study of ang and lf by elisa to confirm that the ang levels in participants ' tears were inversely proportional to the disease severity grade in des patients , as lf has been reported to indicate reflect the severity of ocular surface damage due to des . in the present study ,
therefore , we concluded that both ang and lf are specific proteins that are significantly correlated with des severity , except between grades 1 and 2 .
this study has valuable implications for the practical use of proteomic analysis in diagnosing des severity .
although there have been few studies regarding ang in the field of ophthalmology , various biological functions have been disclosed .
we can infer the characteristics and functions of ang from studies that have investigated the effects of lf and the association between lf and des .
lf is a glycoprotein present in human breast milk and is secreted into tears by the lacrimal and meibomian glands .
the tear lf level has been reported to be an indicator of lacrimal secretory function .
lf has several functions , including anti - inflammatory effects that reduce tumor necrosis factor- and t - cell proliferation , increase il- 10 levels , and actions that promote cell growth and dna synthesis .
previous studies also have reported that tear lf levels correlated with the severity of conjunctivocorneal epithelial lesions in des patients . as des is a multifactorial disease of the tears and the ocular surface , including an inflammatory process that alters the tear protein composition
, it is worth considering lf as a therapeutic agent that may reduce des severity . indeed , dogru et al .  
reported that oral lf supplements improved tear stability and the state of the ocular surface epithelium , confirming the above - mentioned hypothesis .
possible interactions between ang and lf in the immune and inflammatory systems have been reported previously , and we found a significant positive correlation between ang and lf in the present study . although the roles of ang in inflammatory cascades are not clear , we could hypothesize that ang and lf interact with each other in des progression .
we also may suppose that the gradual decrease in ang levels with the increasing des severity reflects the progression of the inflammatory response , inferring from a previous study regarding the action of ang as a defensive agent in pro - inflammatory conditions .
together , these findings suggest that ang could be used as a diagnostic tool for grading des and its associated inflammatory symptoms . in addition , as ang is a natural protein present in the body , it is expected that an exogenous ang supplement may improve des symptoms , much like supplements of lf and albumin do .
it is necessary to investigate the possible actions of ang in the eye and the safety of its biological use through in vitro and in vivo studies of therapeutic exogenous supplements . in general , ang has been reported to induce the synthesis of ribosomal rna and neovascularization .
furthermore , there have been several reports regarding the action of ang in the posterior segments of the eye .
reported that ang may participate in pathologic angiogenesis in eyes with choroidal neovascularization . in contrast , marek et al .   postulated that ang is not associated with the pathogenesis of diabetic retinopathy , contrary to vascular endothelial growth factor , which is the main mediator responsible for pathological neovascularization in diabetic retinopathy . as the action of ang is still a matter of debate , further studies are needed regarding the action of ang in various regions of the eye .
first , ang levels did not differ significantly between the normal controls and des grade 1 and 2 patients in the immunodot blot assay .
these results do not support the conclusion of the present study that ang could be used for the diagnosis and grading of des . on the other hand ,
unlike the results of the immunodot blot assay , the elisa results indicated that the concentration of ang was negatively correlated with dry eye severity , and the group - to - group difference in ang was statistically significant , except between des grades 1 and 2 .
therefore , we believe that the present study is important and will be valuable because it is the first to discuss the correlation among ang , lf , and des severity .
second , we analyzed the participants ' clinical test results and the tear fluids collected from their right eyes , because it was uncertain whether both eyes would have the same des grade .
we recognize that this study is limited in evaluating the true state of dry eye severity , as only one eye was studied , and there may be variability in dry eye conditions between both eyes .
however , we thought that this methodology was appropriate , because the goal of this study was to evaluate the correlation between ang levels and dry eye severity by analyzing tear fluids and the diagnostic test results .
thirdly , although we tried to categorize the des patients based on completely consistent test results using the delphi classification , there were some discrepancies among the results of the tbut , schirmer 's test , and corneal staining .
in such cases , we placed priority on the corneal staining , as it is more reliable than both tbut and schirmer 's test . in conclusion ,
ang and lf concentrations were significantly lower in des patients than in normal controls , and were significantly correlated with des severity , except between grades 1 and 2 .
also , ang may be expected to act as a therapeutic agent in des patients , inferring from the results of previous and recent studies of lf .
further research should be carried out to evaluate the safety of ang for clinical use .
 purposeto investigate the properties of angiogenin ( ang ) as a potential tool for the diagnosis and grading of dry eye syndrome ( des ) by analyzing tear protein profiles.methodstear samples were collected with capillary tubes from 52 des patients and 29 normal individuals as controls . 
 tear protein profiles were analyzed with an immunodot blot assay as a screening test . to confirm that the tear ang levels were in inverse proportion to the disease severity grade , 
 the ang and lactoferrin ( lf ) tear contents of normal controls and des patients were compared in an enzyme - linked immunosorbent assay.resultsin the immunodot blot assay , the ang area was lower in patients with grades 3 and 4 des than in normal controls . 
 the areas of basic fibroblast growth factor , transforming growth factor 2 , and interleukin 10 were significantly greater than those of normal controls only in grade 4 des patients , but these proteins were not linearly correlated with dry eye severity . upon enzyme - linked immunosorbent assay analysis , 
 the mean concentrations of ang and lf decreased significantly as dry eye severity increased , except between grades 1 and 2 . 
 in addition , the ratios of ang and lf to total tear proteins were correlated significantly with des severity.conclusionsang level was significantly lower in des patients than in normal controls , and was significantly correlated with the worsening severity of des , except between grades 1 and 2 , as was lf . 
 therefore , ang may be a useful measure of des severity through proteomic analysis . 
Materials and Methods
Participants
Tear collection
Immunodot blot assay
Enzyme-linked immunosorbent assay
Statistical analysis
Results
Demographics of the participants
Immunodot blot assay
Enzyme-linked immunosorbent assay
Discussion