title: An Evaluation of Lysyl Oxidase-Derived Cross-Linking in Keratoconus by Liquid Chromatography/Mass Spectrometry.
abstract: OBJECTIVE
  Current 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).
  
  
  METHODS
  Surgical 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 110°C 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.
  
  
  RESULTS
  Cross-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.
  
  
  CONCLUSIONS
  Lower 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.
authors.affiliation: Department of Ophthalmology Columbia University College of Physicians and Surgeons, New York, New York, United States.
authors.forename: Anna
authors.initials: A
authors.lastname: Takaoka
authors.affiliation: Department of Ophthalmology Columbia University College of Physicians and Surgeons, New York, New York, United States.
authors.forename: Natasha
authors.initials: N
authors.lastname: Babar
authors.affiliation: Department of Ophthalmology Columbia University College of Physicians and Surgeons, New York, New York, United States.
authors.forename: Julia
authors.initials: J
authors.lastname: Hogan
authors.affiliation: Department of Ophthalmology Columbia University College of Physicians and Surgeons, New York, New York, United States.
authors.forename: MiJung
authors.initials: M
authors.lastname: Kim
authors.affiliation: The Cornea Research Foundation of America, Indianapolis, Indiana, United States.
authors.forename: Marianne O
authors.initials: MO
authors.lastname: Price
authors.affiliation: Price Vision Group, Indianapolis, Indiana, United States.
authors.forename: Francis W
authors.initials: FW
authors.lastname: Price
authors.affiliation: Department of Ophthalmology Columbia University College of Physicians and Surgeons, New York, New York, United States.
authors.forename: Stephen L
authors.initials: SL
authors.lastname: Trokel
authors.affiliation: Department of Ophthalmology Columbia University College of Physicians and Surgeons, New York, New York, United States.
authors.forename: David C
authors.initials: DC
authors.lastname: Paik
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003315:Cornea / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D007640:Keratoconus / Q000378:metabolism* / Q000473:pathology; D013058:Mass Spectrometry / Q000379:methods*; D008875:Middle Aged; D008249:Protein-Lysine 6-Oxidase / Q000378:metabolism*; D055815:Young Adult
chemical_list: D008249:Protein-Lysine 6-Oxidase