human donor control corneas ( age 1858 years ) were obtained from the eye - bank for sight restoration , new york , new york .
surgical kc specimens ( age 2268 years ) were obtained at the time of corneal transplant from patients undergoing anterior lamellar keratoplasty ( alk ) , deep anterior lamellar keratoplasty ( dalk ) , and penetrating keratoplasty ( pkp ) through an ongoing clinical collaboration and with informed consent .
samples were divided into two age groups : 40 years and > 40 years . sample numbers ( n ) for control  40 was n = 5 ; control > 40 , n = 6 ; kc  40 , n = 8 ; and kc > 40 , n = 9 .
liquid chromatography / ms  grade methanol was purchased from vwr ( randor , pa , usa ) .
( st . louis , mo , usa ) : ion chromatography  grade heptafluorobutyric acid ( hfba ) , analytic - grade 6n hydrochloric acid ( hcl ) , sodium borohydride ( nabh4 ) , sodium hydroxide , hplc - grade butanol , hplc - grade 1-propanol , sodium acetate trihydrate , citric acid monohydrate , glacial acetic acid ( acoh ) , chloramine - t , dimethylaminobenzaldehyde , acs - grade perchloric acid , hplc - grade acetonitrile ( acn ) , and l - hydroxyproline ( oh - pro ) .
( san diego , ca , usa ) and pent was purchased from cayman chemical ( ann arbor , mi , usa ) .
the elastin cross - links desmosine ( des ) and isodesmosine ( ide ) were also commercially available from mp biomedicals ( santa ana , ca , usa ) .
we also obtained as a kind gift from simon p. robins ( university of aberdeen , aberdeen , scotland , uk ) , the major trifunctional corneal cross - link hhl as well as the difunctional cross - link hydroxylysinonorleucine ( hlnl ) .
a 6-mm trephine punch provided approximately 20 mg human cornea , which was sufficient mass for dsc analysis .
the weight of the kc specimens varied between 5 to 20 mg , depending on the size and the thickness .
( amounts below 2 mg have smaller signal - to - noise ratios and can complicate thermogram peak analysis . )
the samples were rinsed in bathing solution containing antiprotease , antimicrobial solution containing 1 mm edta and penicillin / streptomycin .
tissue samples were carefully blotted in a standardized , repetitive manner on a double - folded paper towel to remove excess solution / dulbecco 's phosphate - buffered saline and transferred to preweighed 50-l aluminum pans .
( residual water in the pan can shift the thermogram downward , since water has a high heat capacity . )
the samples were flattened onto the bottom of the pan to ensure uniform heat transfer from the pan to the tissue and reduce the margin of error in the readings .
the pans were immediately hermetically sealed by using a dsc pan - sealing press ( perkin - elmer universal crimper press ; perkin - elmer , waltham , ma , usa ) , which is used to prevent tissue dehydration from evaporation , and loaded into the dsc autosampler .
thermal denaturation temperature of all samples was measured by using a perkin - elmer dsc 6000 autosampler and pyris software ( version 11.0 ) .
the samples were heated from 40c to 70c at a scan speed of 1c / min , which was determined to be an optimal scan speed from preliminary runs .
the dsc heat flow endotherm data were then analyzed by interpolation of a segment of the curve containing the major endotherm .
the maximal transition temperature point was identified by using the pyris data analysis peak search function .
the instrument has an expected margin of error of 0.3c , based on calibration runs , and is consistent with the manufacturer 's specifications .
post dsc analysis , the tissue was recovered and dried , and dry - weight was recorded . to stabilize the schiff bases contained in the difunctional cross - links , dried samples were reduced with nabh4 ( reagent / sample weight ratio of 1:30 ) for 1 hour at room temperature .
the reduced samples were then washed with water twice , dried , and hydrolyzed with 6n hcl in vacuo for 18 hours at 110c after argon sparging .
the hydrolysates were dried to evaporate hcl , reconstituted with h2o , and re - dried to remove residual hcl .
post hydrolysis , cross - link enrichment was carried out by using a cellulose mini - column method ( bond - elut cellulose agilent part no .
the cellulose column was equilibrated with organic mobile phase ( acn : acoh : h2o [ 8:1:1 ] ) .
the dried hydrolysate was dissolved in acn : acoh ( 6:1 ) mixture and applied on equilibrated cellulose column .
the column was flushed with 12 ml organic mobile phase to remove most of the free amino acids and residual acid .
the cross - links were then eluted with water , collected , and dried in preparation for hplc analysis .
an internal standard , acetyl - pyd , was added post hydrolysis and pre cellulose in order to account for any losses during the enrichment step . having these standards
allowed us to build accurate , reproducible , calibration curves and we recalibrated on a weekly basis .
calibration curves were prepared by injecting standards at seven concentrations ranging from 0 to 180 pmol dhlnl , 0 to 130 pmol hlnl , 0 to 200 pmol lnl , 0 to 116 pmol hhl , 0 to 24 pmol pyd , 0 to 10.7 pmol dpyd , 0 to 209 pmol des / ide , and 0 to 146 pmol pent .
the calibration curve of each compound was described by the linear equation , with coefficient ( r ) of the curve > 0.995 for all the compounds .
cellulose - enriched cross - links were analyzed on a lc / ms system consisting of an agilent 1100 hplc with uv / visible diode array / fluorescence / mass ( dad / fld / msd ) detectors ( chemstation rev.b.03.01 ) .
the mobile phase was an isocratic , degassed 20 mm hfba / meoh mixture ( 70:30 ) , with a flow rate of 1 ml / min .
eclipse xdb - c8 column ( 4.6 mm  250 mm ) was used with operating temperature of 25c using the column oven .
a tee fitting was introduced after the dad in order to direct 80% flow to the fld and 20% to the msd .
thus , a pump flow rate of 1 ml / min resulted in a 0.2 ml / min flow rate to the msd . using selected ion monitoring allowed for greater sensitivity , and the following target ions
were measured : dhlnl ( m / z = 308.2 ) , hlnl ( m / z = 292.2 ) , lnl ( m / z = 276.1 ) , hhl ( m / z = 445.2 ) , pyd ( m / z = 429.2 ) , dpyd ( m / z = 413 ) , des / ide ( m / z = 526.3 ) , and pent ( m / z = 379.2 ) . running the fld in parallel to the msd allowed for an additional quality control measure , since the fluorescence signals are well characterized for collagen cross - links such as the pyridinolines ( ex295/em395 ) and the nonenzymatic glycation and age - associated cross - link pent ( ex335/em385 ) , and as such ,
the relevant chromatograms were extracted by using chemstation software and the abundance measurements were converted into picomoles by using the programmed calibration table .
the cross - link densities were then determined for each cross - link relative to collagen content , which is determined colorimetrically by the method described in the hydroxyproline analysis assay section , and expressed in mol / mol quantity .
each sample was injected three times and we obtained a collagen cross - link profile from every sample .
summary of analysis of kc versus control for all ages combined , age  40 years and > 40 years the following was adopted from previously reported procedures .
stock buffer ( 10x ) : 50 g citric acid monohydrate , 12 ml glacial acoh , 120 g sodium acetate trihydrate , 34 g naoh were made up to a final volume of 1l distilled water .
the ph was adjusted to 6 and the buffer was stored at 4c until use .
the following reagents were prepared freshly on the day of the experiment : oh - pro standard , chloramine - t reagent , and 4-(dimethylamino ) benzaldehyde ( dmba ) reagent .
oh - pro with assay buffer to the appropriate concentrations ( 010 g / ml ) .
chloramine - t reagent was prepared by dissolving 0.3525 g chloramine - t powder in 5.175 ml h2o , with 6.5 ml n - propanol and 13.325 ml stock buffer .
the dmba mixture was made by dissolving 3.75 g dmba in 15 ml n - propanol and 6.5 ml perchloric acid .
appropriate dilutions of standard oh - pro solution ( 010 g / ml ) and hydrolysate ( 1:50 , 1:100 , or 1:250 ) were prepared by using assay buffer .
after aliquoting 200 l standard oh - pro solution and diluted hydrolysate into tubes , 100 l chloramine - t reagent was added , gently pipetted to mix , and incubated at room temperature for 20 minutes .
one hundred microliters of dmba reagent was then added to each sample , gently mixed , and the chromophore was developed by incubating the sample at 60c for 15 minutes .
immediately after the incubation , absorbance of each sample was measured at 540 nm by using a spectrophotometer ( elx800 ; bio - tek instruments , inc . ,
the total collagen content for each sample was calculated by assuming 14% oh - pro content per collagen by weight and using 300 kda for the molecular weight of the collagen molecule .
statistical analysis was carried out with the assistance of the biostatistics consulting service of the irving center for clinical trials at columbia university medical center .
all quantitative data ( mean  sd ) including all ages combined were assessed for significance initially by using student 's t - test .
this was followed by a stratified analysis comparison within the age groups , 40 years and > 40 years ( a stratified analysis ) .
the results from both t - tests and wilcoxon rank sum tests were in good agreement .
for the skewed data , t - tests were done with log - transformed values .
statistical significance of all tests was based on an  value of 0.05 ( p < 0.05 ) . owing to the small sample numbers , multiple comparison adjustments were not considered relevant for this study .
data from the lc / ms cross - link analysis were correlated with tm by using data as reported in the accompanying report in order to assess whether individual or total cross - link levels correlated with fibril thermal stability .
correlation coefficients for each cross - link as a function of tm were determined by using microsoft excel ( version 14.0 ; microsoft corporation , redmond , wa , usa ) .
human donor control corneas ( age 1858 years ) were obtained from the eye - bank for sight restoration , new york , new york .
surgical kc specimens ( age 2268 years ) were obtained at the time of corneal transplant from patients undergoing anterior lamellar keratoplasty ( alk ) , deep anterior lamellar keratoplasty ( dalk ) , and penetrating keratoplasty ( pkp ) through an ongoing clinical collaboration and with informed consent .
samples were divided into two age groups : 40 years and > 40 years . sample numbers ( n ) for control  40 was n = 5 ; control > 40 , n = 6 ; kc  40 , n = 8 ; and kc > 40 , n = 9 .
liquid chromatography / ms  grade methanol was purchased from vwr ( randor , pa , usa ) . the following reagents were purchased from sigma - aldrich corp .
( st . louis , mo , usa ) : ion chromatography  grade heptafluorobutyric acid ( hfba ) , analytic - grade 6n hydrochloric acid ( hcl ) , sodium borohydride ( nabh4 ) , sodium hydroxide , hplc - grade butanol , hplc - grade 1-propanol , sodium acetate trihydrate , citric acid monohydrate , glacial acetic acid ( acoh ) , chloramine - t , dimethylaminobenzaldehyde , acs - grade perchloric acid , hplc - grade acetonitrile ( acn ) , and l - hydroxyproline ( oh - pro ) .
( san diego , ca , usa ) and pent was purchased from cayman chemical ( ann arbor , mi , usa ) .
the elastin cross - links desmosine ( des ) and isodesmosine ( ide ) were also commercially available from mp biomedicals ( santa ana , ca , usa ) .
we also obtained as a kind gift from simon p. robins ( university of aberdeen , aberdeen , scotland , uk ) , the major trifunctional corneal cross - link hhl as well as the difunctional cross - link hydroxylysinonorleucine ( hlnl ) .
a 6-mm trephine punch provided approximately 20 mg human cornea , which was sufficient mass for dsc analysis . the weight of the kc specimens varied between 5 to 20 mg , depending on the size and the thickness .
( amounts below 2 mg have smaller signal - to - noise ratios and can complicate thermogram peak analysis . )
the samples were rinsed in bathing solution containing antiprotease , antimicrobial solution containing 1 mm edta and penicillin / streptomycin .
tissue samples were carefully blotted in a standardized , repetitive manner on a double - folded paper towel to remove excess solution / dulbecco 's phosphate - buffered saline and transferred to preweighed 50-l aluminum pans .
( residual water in the pan can shift the thermogram downward , since water has a high heat capacity . )
the samples were flattened onto the bottom of the pan to ensure uniform heat transfer from the pan to the tissue and reduce the margin of error in the readings .
the pans were immediately hermetically sealed by using a dsc pan - sealing press ( perkin - elmer universal crimper press ; perkin - elmer , waltham , ma , usa ) , which is used to prevent tissue dehydration from evaporation , and loaded into the dsc autosampler .
thermal denaturation temperature of all samples was measured by using a perkin - elmer dsc 6000 autosampler and pyris software ( version 11.0 ) .
the samples were heated from 40c to 70c at a scan speed of 1c / min , which was determined to be an optimal scan speed from preliminary runs .
the dsc heat flow endotherm data were then analyzed by interpolation of a segment of the curve containing the major endotherm .
the maximal transition temperature point was identified by using the pyris data analysis peak search function .
the instrument has an expected margin of error of 0.3c , based on calibration runs , and is consistent with the manufacturer 's specifications .
post dsc analysis , the tissue was recovered and dried , and dry - weight was recorded . to stabilize the schiff bases contained in the difunctional cross - links , dried samples were reduced with nabh4 ( reagent / sample weight ratio of 1:30 ) for 1 hour at room temperature .
the reduced samples were then washed with water twice , dried , and hydrolyzed with 6n hcl in vacuo for 18 hours at 110c after argon sparging .
the hydrolysates were dried to evaporate hcl , reconstituted with h2o , and re - dried to remove residual hcl . after the last washing step
post hydrolysis , cross - link enrichment was carried out by using a cellulose mini - column method ( bond - elut cellulose agilent part no . 168726 ; agilent technologies , inc . , santa clara , ca , usa ) .
the cellulose column was equilibrated with organic mobile phase ( acn : acoh : h2o [ 8:1:1 ] ) .
the dried hydrolysate was dissolved in acn : acoh ( 6:1 ) mixture and applied on equilibrated cellulose column .
the column was flushed with 12 ml organic mobile phase to remove most of the free amino acids and residual acid .
the cross - links were then eluted with water , collected , and dried in preparation for hplc analysis .
an internal standard , acetyl - pyd , was added post hydrolysis and pre cellulose in order to account for any losses during the enrichment step .
having these standards allowed us to build accurate , reproducible , calibration curves and we recalibrated on a weekly basis .
calibration curves were prepared by injecting standards at seven concentrations ranging from 0 to 180 pmol dhlnl , 0 to 130 pmol hlnl , 0 to 200 pmol lnl , 0 to 116 pmol hhl , 0 to 24 pmol pyd , 0 to 10.7 pmol dpyd , 0 to 209 pmol des / ide , and 0 to 146 pmol pent .
the calibration curve of each compound was described by the linear equation , with coefficient ( r ) of the curve > 0.995 for all the compounds .
cellulose - enriched cross - links were analyzed on a lc / ms system consisting of an agilent 1100 hplc with uv / visible diode array / fluorescence / mass ( dad / fld / msd ) detectors ( chemstation rev.b.03.01 ) .
the mobile phase was an isocratic , degassed 20 mm hfba / meoh mixture ( 70:30 ) , with a flow rate of 1 ml / min .
eclipse xdb - c8 column ( 4.6 mm  250 mm ) was used with operating temperature of 25c using the column oven .
a tee fitting was introduced after the dad in order to direct 80% flow to the fld and 20% to the msd .
thus , a pump flow rate of 1 ml / min resulted in a 0.2 ml / min flow rate to the msd . using selected ion monitoring allowed for greater sensitivity , and the following target ions
were measured : dhlnl ( m / z = 308.2 ) , hlnl ( m / z = 292.2 ) , lnl ( m / z = 276.1 ) , hhl ( m / z = 445.2 ) , pyd ( m / z = 429.2 ) , dpyd ( m / z = 413 ) , des / ide ( m / z = 526.3 ) , and pent ( m / z = 379.2 ) . running the fld in parallel to the msd allowed for an additional quality control measure , since the fluorescence signals are well characterized for collagen cross - links such as the pyridinolines ( ex295/em395 ) and the nonenzymatic glycation and age - associated cross - link pent ( ex335/em385 ) , and as such , can be readily compared to the expected msd signal abundances . the relevant chromatograms were extracted by using chemstation software and the abundance measurements were converted into picomoles by using the programmed calibration table .
the cross - link densities were then determined for each cross - link relative to collagen content , which is determined colorimetrically by the method described in the hydroxyproline analysis assay section , and expressed in mol / mol quantity .
each sample was injected three times and we obtained a collagen cross - link profile from every sample .
summary of analysis of kc versus control for all ages combined , age  40 years and > 40 years
stock buffer ( 10x ) : 50 g citric acid monohydrate , 12 ml glacial acoh , 120 g sodium acetate trihydrate , 34 g naoh were made up to a final volume of 1l distilled water .
the ph was adjusted to 6 and the buffer was stored at 4c until use .
the following reagents were prepared freshly on the day of the experiment : oh - pro standard , chloramine - t reagent , and 4-(dimethylamino ) benzaldehyde ( dmba ) reagent .
oh - pro with assay buffer to the appropriate concentrations ( 010 g / ml ) .
chloramine - t reagent was prepared by dissolving 0.3525 g chloramine - t powder in 5.175 ml h2o , with 6.5 ml n - propanol and 13.325 ml stock buffer .
the dmba mixture was made by dissolving 3.75 g dmba in 15 ml n - propanol and 6.5 ml perchloric acid .
appropriate dilutions of standard oh - pro solution ( 010 g / ml ) and hydrolysate ( 1:50 , 1:100 , or 1:250 ) were prepared by using assay buffer .
after aliquoting 200 l standard oh - pro solution and diluted hydrolysate into tubes , 100 l chloramine - t reagent was added , gently pipetted to mix , and incubated at room temperature for 20 minutes .
one hundred microliters of dmba reagent was then added to each sample , gently mixed , and the chromophore was developed by incubating the sample at 60c for 15 minutes . immediately after the incubation , absorbance of each sample was measured at 540 nm by using a spectrophotometer ( elx800 ; bio - tek instruments , inc . ,
the total collagen content for each sample was calculated by assuming 14% oh - pro content per collagen by weight and using 300 kda for the molecular weight of the collagen molecule .
statistical analysis was carried out with the assistance of the biostatistics consulting service of the irving center for clinical trials at columbia university medical center . all quantitative data ( mean  sd ) including all ages combined were assessed for significance initially by using student 's t - test .
this was followed by a stratified analysis comparison within the age groups , 40 years and > 40 years ( a stratified analysis ) .
the results from both t - tests and wilcoxon rank sum tests were in good agreement . for the skewed data , t - tests were done with log - transformed values .
statistical significance of all tests was based on an  value of 0.05 ( p < 0.05 ) .
owing to the small sample numbers , multiple comparison adjustments were not considered relevant for this study .
data from the lc / ms cross - link analysis were correlated with tm by using data as reported in the accompanying report in order to assess whether individual or total cross - link levels correlated with fibril thermal stability .
correlation coefficients for each cross - link as a function of tm were determined by using microsoft excel ( version 14.0 ; microsoft corporation , redmond , wa , usa ) .
the results for hydration , collagen content , tm for all groups and for age  40 years and age > 40 years are shown in the table . before the lc / ms sample preparation , tm , water content , and collagen content of each sample were determined .
water content was measured to determine the hydration status of the tissue , and collagen content was measured colorimetrically ( oh - pro assay ) , in order to express the cross - link levels on a mole / mole basis .
thermal denaturation temperature of each sample was measured by using dsc and the results of thermal denaturation testing are shown in figure 2a .
the average tm of controls and kc corneas did not show significant difference ( 60.08  1.54 vs. 59.21  1.16 , p = 0.12 ) .
since kc progression is known to be more severe in the younger population , the samples were divided into two groups : age  40 years and age > 40 years .
compared with the control , the kc  40 group had lower tm values , indicating less fibril stability ( 61.40  0.56 , 58.94  0.73 , p = 0.0002 ) .
this amount of change in tm can be achieved by using the cxl methods as well as other chemical topical approaches .
interestingly , the older control samples had a significant decrease in tm as compared to the younger control samples .
such a trend has also been reported previously albeit it has not been well studied . with regard to tm for many other tissues ,
the tm generally increases with aging and this is generally believed to be as a result of age - related nonenzymatic cross - linking .
this is true for tendon , skin , cartilage , blood vessel , and other tissues .
comparison of human kc versus eye - bank controls for ( a ) average tm , ( b ) average tissue hydration ( % water content ) , and ( c ) average collagen content ( % ) .
black dots represent the mean values , and gray dots represent the actual values measured . in each case
, comparisons were made between controls and kc by using student 's t - test .
hydration status of corneal samples was determined by calculating percentage water content in each sample ( fig .
the hydration status of controls and kc corneas was not significantly different ( 75%  3% vs. 72%  12% , p = 0.39 ) .
however , kc  40 samples were slightly more hydrated ( swollen ) as compared to those of controls  40 ( 82%  4% vs. 73%  3% , p = 0.0013 ) . on the other hand , kc > 40 samples were slightly more dehydrated than those of controls ( 64%  11% vs. 76%  3% , p = 0.01 ) .
water content of control  40 and control > 40 samples were 73%  3% and 76%  3% , respectively , which was in good agreement with previously reported hydration status of corneal tissue .
considering that the older kc samples were relatively dehydrated compared to the older control samples , the observed tm of older kc corneas could have been slightly higher than it was at normal hydration status .
the kc  40 samples were slightly more hydrated than control  40 samples , which could mean that the observed tm was artificially lowered owing to the extra water in the sample .
however , in our experience , this level of difference in hydration will not produce a significant shift in tm .
it is not clear whether the hydration could be responsible for the observed 2c shift in the thermal denaturation temperature between the kc  40 and control corneas .
the levels varied from approximately 35% to 59% , which was lower than values reported in the literature .
there was no notable difference in average collagen content between controls and kc corneas ( 45%  5% vs. 47%  7% , p = 0.48 ) .
collagen content of younger populations in control and kc was not particularly different ( 48%  4% vs. 45%  8% , p = 0.43 ) .
thus , our results regarding collagen content are essentially in agreement with previous reports and indicate that collagen content per se likely is not a key consideration in kc pathogenesis .
figures 1a and 1b show the nine cross - link compounds that were measured , and the elution profile of the standard cross - link compounds , by using lc / ms .
selective ion monitoring mode ( details described in the materials and methods section ) was used in order to optimize signal strength .
of all the nine cross - links , dhlnl , hlnl , lnl , and hhl were detected in both kc and control corneas .
figure 3a summarizes the detected cross - link density expressed relative to the collagen content of each sample ( mol / mol collagen ) .
one of the major findings of this analysis was that lnl was the dominant difunctional cross - link in human cornea .
furthermore , compared to the controls , kc corneas contained fewer lnl molecules with the average values of 0.132  0.063 vs. 0.190  0.055 ( p = 0.02 ) .
the level of dhlnl on the other hand was slightly increased in kc ( 0.074  0.031 vs. 0.048  0.024 , p = 0.03 ) .
when the samples were divided by age groups , it was found that lnl had the same trend in kc  40 versus control ( 0.114  0.052 vs. 0.185  0.057 , p = 0.04 ) .
for dhlnl , however , the trend was not seen in either age group ( table ) .
( a ) levels of specific di- and trifunctional corneal ctp cross - links in samples detected by lc / ms and ( b ) the sum of all the cross - links detected in each groups .
statistically significant difference was detected in dhlnl and lnl levels ( p < 0.05 ) .
regarding hlnl and hhl , the levels were not significantly different between kc and control ( hlnl : 0.045  0.015 vs. 0.043  0.005 , p = 0.68 ; hhl : 0.047  0.030 vs. 0.042  0.030 , p = 0.70 ) . of note , hhl has been shown to be derived from an unreduced form of hlnl ( i.e. , deh - hlnl ) in skin and cornea .
this deh - hlnl matures into a trifunctional stable cross - link , hhl , by reacting with histidine ; therefore , hlnl and hhl tend to have an inverse relationship , with hlnl being higher when hhl is lower , and vice versa . in our study
, we did not observe such a relationship . to the best of our knowledge ,
however , because the chromatographic and detection methods were quite different , it is difficult to draw direct comparisons . regarding other cross - links included in the analysis ,
we did not detect levels of either pyd or dpyd in any of our samples .
this was consistent with previous reports showing that both of these trifunctional collagen cross - links are absent in corneal tissue .
the tetrafunctional cross - links des and ide result from a spontaneous condensation of two adjacent lnl difunctional cross - links in the elastin molecule , which has a unique primary sequence involving adjacent lysine residues that allow for intermolecular difunctional lnl formation followed by the desmosine - resulting condensation .
desmosines have not been previously detected in corneal tissue and our findings are in agreement with this .
previous studies using a two - step hplc method have found the nonenzymatic glycosylation cross - link pent to be increased in the diabetic human cornea .
we did not detect pent , however , in either kc samples or controls , and the reason for this discrepancy with prior studies is not completely clear but may relate to the differences in the analytic techniques used .
figure 3b shows the total cross - link density ( sum of all the cross - links ) of control and kc samples .
there was no significant difference between kc and control ( 0.297  0.104 vs. 0.323  0.088 , p = 0.5 ) .
the younger populations that are considered to have the severe forms of kc did not differ in their total cross - link density when compared to the controls .
this suggests that rather than the overall cross - link density , there might be a particular cross - link ( such as lnl ) that holds the key to fibril instability . to determine if there was a direct correlation between fibril stability and either total cross - link density or any specific cross - links , we created scatterplots of cross - link density as a function of tm ( fig .
there was not a statistically significant correlation between tm and total cross - link density ( fig .
4 ) or the density of individual specific cross - links ( data not shown ) . thus ,
the fibril thermal stability changes were not directly attributable to any of the individual cross - links examined or in total .
this suggests that other causes of fibril instability may exist , including as yet unidentified cross - links .
analysis of total cross - link density ( mol / mol collagen ) as a function of tm for control ( black circle ) and kc ( open circle ) samples .
the trend lines are expressed with a solid line ( control ) and a dotted line ( kc ) .
there was no statistical significant correlation between fibril stability and the cross - link density .
thermal denaturation temperature of each sample was measured by using dsc and the results of thermal denaturation testing are shown in figure 2a .
the average tm of controls and kc corneas did not show significant difference ( 60.08  1.54 vs. 59.21  1.16 , p = 0.12 ) .
since kc progression is known to be more severe in the younger population , the samples were divided into two groups : age  40 years and age > 40 years .
compared with the control , the kc  40 group had lower tm values , indicating less fibril stability ( 61.40  0.56 , 58.94  0.73 , p = 0.0002 ) .
this amount of change in tm can be achieved by using the cxl methods as well as other chemical topical approaches .
interestingly , the older control samples had a significant decrease in tm as compared to the younger control samples .
such a trend has also been reported previously albeit it has not been well studied . with regard to tm for many other tissues ,
the tm generally increases with aging and this is generally believed to be as a result of age - related nonenzymatic cross - linking .
this is true for tendon , skin , cartilage , blood vessel , and other tissues .
comparison of human kc versus eye - bank controls for ( a ) average tm , ( b ) average tissue hydration ( % water content ) , and ( c ) average collagen content ( % ) .
black dots represent the mean values , and gray dots represent the actual values measured . in each case
, comparisons were made between controls and kc by using student 's t - test .
hydration status of corneal samples was determined by calculating percentage water content in each sample ( fig .
the hydration status of controls and kc corneas was not significantly different ( 75%  3% vs. 72%  12% , p = 0.39 ) .
however , kc  40 samples were slightly more hydrated ( swollen ) as compared to those of controls  40 ( 82%  4% vs. 73%  3% , p = 0.0013 ) . on the other hand , kc
> 40 samples were slightly more dehydrated than those of controls ( 64%  11% vs. 76%  3% , p = 0.01 ) .
water content of control  40 and control > 40 samples were 73%  3% and 76%  3% , respectively , which was in good agreement with previously reported hydration status of corneal tissue .
considering that the older kc samples were relatively dehydrated compared to the older control samples , the observed tm of older kc corneas could have been slightly higher than it was at normal hydration status .
the kc  40 samples were slightly more hydrated than control  40 samples , which could mean that the observed tm was artificially lowered owing to the extra water in the sample .
however , in our experience , this level of difference in hydration will not produce a significant shift in tm .
it is not clear whether the hydration could be responsible for the observed 2c shift in the thermal denaturation temperature between the kc  40 and control corneas .
the levels varied from approximately 35% to 59% , which was lower than values reported in the literature .
there was no notable difference in average collagen content between controls and kc corneas ( 45%  5% vs. 47%  7% , p = 0.48 ) .
collagen content of younger populations in control and kc was not particularly different ( 48%  4% vs. 45%  8% , p = 0.43 ) .
thus , our results regarding collagen content are essentially in agreement with previous reports and indicate that collagen content per se likely is not a key consideration in kc pathogenesis .
figures 1a and 1b show the nine cross - link compounds that were measured , and the elution profile of the standard cross - link compounds , by using lc / ms .
selective ion monitoring mode ( details described in the materials and methods section ) was used in order to optimize signal strength .
of all the nine cross - links , dhlnl , hlnl , lnl , and hhl were detected in both kc and control corneas .
figure 3a summarizes the detected cross - link density expressed relative to the collagen content of each sample ( mol / mol collagen ) .
one of the major findings of this analysis was that lnl was the dominant difunctional cross - link in human cornea .
furthermore , compared to the controls , kc corneas contained fewer lnl molecules with the average values of 0.132  0.063 vs. 0.190  0.055 ( p = 0.02 ) .
the level of dhlnl on the other hand was slightly increased in kc ( 0.074  0.031 vs. 0.048  0.024 , p = 0.03 ) .
when the samples were divided by age groups , it was found that lnl had the same trend in kc  40 versus control ( 0.114  0.052 vs. 0.185
( a ) levels of specific di- and trifunctional corneal ctp cross - links in samples detected by lc / ms and ( b ) the sum of all the cross - links detected in each groups .
statistically significant difference was detected in dhlnl and lnl levels ( p < 0.05 ) .
regarding hlnl and hhl , the levels were not significantly different between kc and control ( hlnl : 0.045  0.015 vs. 0.043  0.005 , p = 0.68 ; hhl : 0.047  0.030 vs. 0.042  0.030 , p = 0.70 ) . of note
, hhl has been shown to be derived from an unreduced form of hlnl ( i.e. , deh - hlnl ) in skin and cornea .
this deh - hlnl matures into a trifunctional stable cross - link , hhl , by reacting with histidine ; therefore , hlnl and hhl tend to have an inverse relationship , with hlnl being higher when hhl is lower , and vice versa . in our study
, we did not observe such a relationship . to the best of our knowledge ,
however , because the chromatographic and detection methods were quite different , it is difficult to draw direct comparisons .
regarding other cross - links included in the analysis , we did not detect levels of either pyd or dpyd in any of our samples .
this was consistent with previous reports showing that both of these trifunctional collagen cross - links are absent in corneal tissue .
the tetrafunctional cross - links des and ide result from a spontaneous condensation of two adjacent lnl difunctional cross - links in the elastin molecule , which has a unique primary sequence involving adjacent lysine residues that allow for intermolecular difunctional lnl formation followed by the desmosine - resulting condensation .
desmosines have not been previously detected in corneal tissue and our findings are in agreement with this .
previous studies using a two - step hplc method have found the nonenzymatic glycosylation cross - link pent to be increased in the diabetic human cornea .
we did not detect pent , however , in either kc samples or controls , and the reason for this discrepancy with prior studies is not completely clear but may relate to the differences in the analytic techniques used .
figure 3b shows the total cross - link density ( sum of all the cross - links ) of control and kc samples .
there was no significant difference between kc and control ( 0.297  0.104 vs. 0.323  0.088 , p = 0.5 ) .
the younger populations that are considered to have the severe forms of kc did not differ in their total cross - link density when compared to the controls .
this suggests that rather than the overall cross - link density , there might be a particular cross - link ( such as lnl ) that holds the key to fibril instability .
to determine if there was a direct correlation between fibril stability and either total cross - link density or any specific cross - links , we created scatterplots of cross - link density as a function of tm ( fig .
there was not a statistically significant correlation between tm and total cross - link density ( fig .
4 ) or the density of individual specific cross - links ( data not shown ) . thus , the fibril thermal stability changes were not directly attributable to any of the individual cross - links examined or in total .
this suggests that other causes of fibril instability may exist , including as yet unidentified cross - links .
analysis of total cross - link density ( mol / mol collagen ) as a function of tm for control ( black circle ) and kc ( open circle ) samples .
the trend lines are expressed with a solid line ( control ) and a dotted line ( kc ) .
there was no statistical significant correlation between fibril stability and the cross - link density .
thermal denaturation temperature ( also known as thermal shrinkage temperature ) , has been used in the evaluation of a variety of collagenous tissues for many decades .
it has been a mainstay in the biomaterials industry along with various mechanical testing modalities .
evaluation of tm is well suited to collagenous tissues , since collagen molecules in a fibril undergo a largely  cooperative  or coordinated denaturation , producing a prominent , well - defined , endotherm .
although this technique has been used in many other tissues , very little published literature exists on thermal denaturation of the cornea , and only two reports have considered age - related changes in tm of human cornea : one by stringer and parr in 1964 and the other by chang et al . in 1996 . both have found that tm decreases with increasing age . in general , the opposite effect is seen with other tissues such as tendon and skin , in which tm increases with aging .
this is thought to be due to the accumulation of nonenzymatic cross - links with age .
however , we did not detect the age - related nonenzymatic cross - link pent in our samples , suggesting that age - related corneal cross - linking was not significant in our samples .
because corneal tissue is so largely made up of collagenous proteins , the major peak is a valid measure of collagen fibril denaturation that occurs during the unwinding of molecular triple helices .
we have previously used a related , albeit cruder , method of measuring the same properties , known as thermal shrinkage temperature analysis .
thermal denaturation can be used to determine differences in the degree of induced cross - linking in tissues treated with cxl or chemical agents ( i.e. , glutaraldehyde , formaldehyde , genipin , glyceraldehyde , formaldehyde releasers ) and has been used to study changes in cross - linking in studies related to aging as well as modulation of enzymatic cross - linking . of note , thermal denaturation of lens capsule tissue
is unaffected by proteoglycan removal using guanidine but is disrupted by collagenase digestion , indicating that the primary determinant of the tm is not proteoglycans but collagenous structures .
difunctional cross - links in human kc tissue have been measured previously in three separate reports . unfortunately , because the methods used at that time are quite different from that of the present study using lc / ms , it is not possible to draw direct comparisons .
quantitation in those studies uses scintillation counting and is reported as a rate in counts per minute , making unit conversions into mol / mol not possible .
the hhl densities in mol / mol units have been reported by yamauchi et al . in bovine skin and cornea .
again , it is difficult to make direct comparisons because the detection methods were dissimilar ( i.e. , chemical derivatization and uv / visible light detection ) .
the initial attempt to measure difunctional enzymatic cross - links in kc was reported in 1978 by cannon and foster , at which time the investigators suggested that kc patients ' corneas contain higher levels of lnl . however , opposite findings have been reported in follow - up studies carried out in 1985 and 1988 ; hhl , a major nonreducible trifunctional cross - link , was not included in any of these studies , as the methods for detection were not available at that time .
we were particularly interested in seeing how levels of hhl , a major trifunctional cross - link found in cornea and skin but not in sclera or tendon , may be altered in kc .
because hhl is derived from hlnl , a functional impairment or deficiency of lox could result in lower levels of hlnl and by extension , hhl . on the premise that lox or loxl enzymes are altered in kc , we expected to see such changes , but did not . in our study
the determinant of whether the collagen fibrils form dhlnl or lnl is governed by lox and/or lysyl hydroxylase .
our findings support the possibility that the activity of lox and/or lysyl hydroxylase is involved in the pathogenesis of kc .
lysinonorleucine is an important difunctional connective tissue protein cross - link that is found not only in collagenous proteins but also in elastin .
although previously considered to be absent from corneal tissue , recent evidence from kamma - lorger and others suggests that an extensive network of elastin - resembling microfibrils exists in the human cornea and is particularly conspicuous in the posterior cornea .
that is , elastin or a similar protein ( i.e. , oxytalan , fibrillin ) is present , particularly in the posterior corneal layers .
lysinonorleucine is a difunctional cross - link that is a condensation of two lysine moieties without hydroxylation .
a deficiency in lnl in kc tissue thus could represent diminished cross - linking either in fibrillar collagen or possibly an elastic precursor protein such as oxytalan ( also known as fibrillin ) .
there are three major possibilities for cross - linking defects in the fibrillin molecule : disulfide linkages ( not enzyme mediated)fibrillin is the most cysteine - rich protein known .
its initial assembly is initiated by disulfide bond formation ; transglutaminase and formation of -glutamyl - lysine cross - links 
in addition to the findings by qian and glanville from 1997 that alignment of fibrillin molecules in elastic microfibrils is defined by transglutaminase - derived cross - links , a tropoelastin - fibrillin - transglutaminase  mediated cross - link has been identified as being a critical interaction ; and lox and loxl - derived lnl precursor aldimine schiff base and desmosine  although the appearance of desmosines from the formation of two adjacent lnl schiff base precursors has been well described for elastin , the identification of difunctional lnl schiff base precursors specifically in fibrillin microfibrillar protein is not well known , to the best of our knowledge .
cross - linking for both collagen and elastin processes are mediated by lox and its related subtypes , namely , loxl-1 to loxl-4 .
the precise substrate specificity for lox and the four loxl enzymes is not completely known .
however , lox and loxl-1 are known to be involved in elastin cross - linking .
in addition , hydroxylation can also play a key role in pathogenesis by determining which difunctional cross - links form preferentially .
although the findings from this study support the genetic and immunohistochemical study findings that a lox deficiency underlies pathogenesis , we acknowledge several limitations .
first , we compared specimens obtained from three types of lamellar keratoplasty ( alk , dalk , and pkp ) to full - thickness corneal buttons obtained post mortem from the eye bank .
most of our surgical tissue samples were from alk or dalk ; alk generally includes the anterior 25% to 50% of the cornea , while dalk includes 90% to 95% of the corneal thickness .
there are known differences in mechanical properties between anterior and posterior corneal lamellae , including increased interweaving anteriorly with a more orthogonal orientation posteriorly .
depth - dependent differences in enzymatic cross - link levels could be present and represent a limitation of this study . in future studies
, we will address this potentially important issue by determining regional and depth - dependent differences in cross - link levels .
second , as these were surgical specimens , they represented the most advanced kc cases and therefore may not reflect well the cross - links present during the early progression of disease .
that is why we chose to focus on the younger group ( age  40 years ) for associations related to pathogenesis because these younger patients would be more likely to show an earlier state of disease .
many of the patients had developed scarring from contact lens wear , thus clearly entering into a damage - repair cycle that may not fully reflect native collagen protein cross - link chemistry . in the present study we evaluated chemical collagen and elastin cross - links as well as the tm of surgical kc specimens .
our results indicate that fibril stability was reduced in kc samples below the age of 40 years .
in addition , the difunctional cross - link lnl is the primary difunctional cross - link in the human cornea , and the levels were reduced in kc samples as compared to controls .
 purposecurrent literature contains scant information regarding the extent of enzymatic collagen cross - linking in the keratoconus ( kc ) cornea . 
 the aim of the present study was to examine levels of enzymatic lysyl oxidase  derived cross - links in stromal collagen in kc tissue , and to correlate the cross - link levels with collagen fibril stability as determined by thermal denaturation temperature ( tm).methodssurgical kc samples ( n = 17 ) and eye - bank control ( n = 11 ) corneas of age 18 to 68 years were analyzed . 
 the samples were defatted , reduced ( nabh4 ) , hydrolyzed ( 6n hcl at 110c for 18 hours ) , and cellulose enriched before analysis by c8 high - performance liquid chromatography equipped with parallel fluorescent and mass detectors in selective ion monitoring mode ( 20 mm heptafluorobutyric acid / methanol 70:30 isocratic at 1 ml / min ) . 
 nine different cross - links were measured , and the cross - link density was determined relative to collagen content ( determined colorimetrically ) . 
 the tm was determined by differential scanning calorimetry.resultscross-links detected were dihydroxylysinonorleucine ( dhlnl ) , hydroxylysinonorleucine , lysinonorleucine ( lnl ) , and histidinohydroxylysinonorleucine in both control and kc samples . 
 higher dhlnl levels were detected in kc , whereas the dominant cross - link , lnl , was decreased in kc samples . 
 decreased lnl levels were observed among kc  40 corneas . 
 there was no difference in total cross - link density between kc samples and the controls . 
 pyridinolines , desmosines , and pentosidine were not detected . 
 there was no notable correlation between cross - link levels with fibril instability as determined by tm.conclusionslower levels of lnl in the kc cornea suggest that there might be a cross - linking defect either in fibrillar collagen or the microfibrillar elastic network composed of fibrillin . 
Materials and Methods
Corneal Samples
Chemical Reagents
Differential Scanning Calorimetry
Acid Hydrolysis of Corneal Samples
Cross-Link Enrichment and LC/MS
Hydroxyproline Analysis Assay
Statistical Analysis
Analysis of LC/MS Data With T
Results
Thermal Denaturation Temperature
Tissue Hydration
Collagen Content
Cross-Linking Evaluation
Cross-Link Density Versus T
Discussion