An Evaluation of Lysyl Oxidase-Derived Cross-Linking in Keratoconus by Liquid Chromatography/Mass Spectrometry

Investigative Ophthalmology & Visual Science
Anna TakaokaDavid C Paik

Abstract

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). 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. Cross-links detected were dihydroxylysinonorleucine (DHLNL), hydroxylysinonorleucine, lysinonorleucine (LNL), and histidinohydroxylysinonorleucine in both control and KC samples. Higher DHLNL levels were detected in KC...Continue Reading

Citations

Sep 10, 2019·Mass Spectrometry Reviews·Nick A van HuizenTheo M Luider
Oct 17, 2019·Cells·Tina B McKayDimitrios Karamichos
Oct 4, 2017·Scientific Reports·Rabab SharifDimitrios Karamichos
Dec 17, 2020·Asia-Pacific Journal of Ophthalmology·Rohit ShettySwaminathan Sethu
Nov 11, 2020·Experimental Eye Research·Marcony R Santhiago, J Bradley Randleman

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Methods Mentioned

BETA
deamination
differential scanning calorimetry
Assay
glycosylation

Software Mentioned

Pyris
Chemstation
Excel

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