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David C Paik, Anna Takaoka, Stephen L Trokel; Native enzymatic collagen cross-linking in human cornea as determined by simultaneous fluorescence and mass spectometry (LC/MS). Invest. Ophthalmol. Vis. Sci. 2014;55(13):4210.
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© ARVO (1962-2015); The Authors (2016-present)
Even while corneal tissue cross-linking using riboflavin photochemistry (CXL) is gaining widespread clinical use, we still know very little regarding the native enzymatic cross-links that “pre-exist” in the human cornea. Thus, the present study was undertaken in order to examine levels of enzymatic collagen cross-links in human corneal tissue by traditional (HPLC/DAD/FLD) and modern methods (LC/MS).
Human corneas (n=11) were obtained from the NY Eyebank from donor ages 18-58. The samples were pulverized, defatted (folch), reduced (NaBH4), hydrolyzed (6N HCl at 110oC 18hrs), and cellulose enriched prior to analysis by C8 LC/MS (Agilent 1100 system) equipped with DAD, FLD, and MSD in SIM mode (20mM heptafluorobutyric acid/MeOH 70:30 isocratic at 1mL/min). A post-column flow splitter allowed for simultaneous fluorescence and mass detection. Acetylated pyridinoline (AcPYD) was used as an internal standard. Nine different cross-links were measured and included enzymatic collagen di- [DHLNL (m/z=308.2), HLNL (m/z=292.2), LNL (m/z=276.1)] and tri-functional [HHL (m/z=445.2), PYD (m/z=429.2), DPYD (m/z=413)] cross-links, elastin [desmosines (m/z=526.3)] cross-links, and a non-enzymatic age-related glycation [pentosidine (m/z =379.2)] cross-link. Cross-link density (mol/mol) was expressed relative to collagen (MW=300kD) content (determined colorimetrically).
Average cross-link levels (mol/mol collagen) were as follows: DHLNL=0.048±0.01, HLNL=0.043±0.002, LNL=0.19±0.02, and HHL=0.042±0.01. When stratified by age older (n=6) or younger (n=5) than 45y/o, the levels were DHLNL [young=0.033±0.01 vs. old=0.062±0.008, (p=0.045)]; HLNL [young=0.041±0.003 vs. old=0.045±0.001, (p=0.24)]; LNL [young=0.185±0.025 vs. old=0.194±0.024, (p=0.80)]; and HHL [young=0.054±0.016 vs. old=0.032±0.009 (p=0.264)]. PYD, DPYD, desmosines, and pentosidine were not detected. Collagen content (based on a 14% hydroxyproline calculation) was 56±5% in young vs. 50±2% in controls (p=0.35).
The human cornea contains both di- and tri-functional histidine (HHL) based collagen cross-links, with LNL being the major difunctional. In addition, no tri-functional PYDs and elastin were detected by cross-link analysis. These studies are relevant to our understanding of the human cornea in aging, disease, and therapy.
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