December 2002
Volume 43, Issue 13
Free
ARVO Annual Meeting Abstract  |   December 2002
Rigidification of Corneas Treated In Vitro With Glyceraldehyde: Characterization of Two Novel Crosslinks and Two Chromophores
Author Affiliations & Notes
  • FJ Tessier
    Chemical Engineering CALTECH Pasadena CA
  • G Tae
    Chemical Engineering CALTECH Pasadena CA
  • VM Monnier
    Institute of Pathology CWRU Cleveland OH
  • JA Kornfield
    Chemical Engineering CALTECH Pasadena CA
  • Footnotes
    Commercial Relationships   F.J. Tessier, None; G. Tae, None; V.M. Monnier, None; J.A. Kornfield, None. Grant Identification: Support: NEI Grant 097099 & EY-11373
Investigative Ophthalmology & Visual Science December 2002, Vol.43, 3234. doi:
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      FJ Tessier, G Tae, VM Monnier, JA Kornfield; Rigidification of Corneas Treated In Vitro With Glyceraldehyde: Characterization of Two Novel Crosslinks and Two Chromophores . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3234.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Abstract: : Purpose: Keratoconus is a degenerative corneal disease that was recently proposed to be stabilized or compensated by nonsurgical increase in stiffness. Therefore, we characterize new adducts involved in crosslinking the cornea during glyceraldehyde treatment. Methods and Results: Since the primary sites of glycation are lysine and arginine residues, we incubate Na-acetyl-lysine, with or without Na-acetyl-arginine, with glyceraldehyde at 37°C in phosphate buffer (pH7.5). By HPLC chromatography, five prominent chromophores were detected from these incubation mixtures. The structures of the new adducts were solved by mass spectrometry and NMR, including two new crosslinks that are both fluorophores (lys-lys, "Lys-hydroxy-triosidine", LHT; and arg-lys "Arg-hydroxy-triosidine", AHT), and two single lysine adducts (Trihydroxy-triosidine and Triosidine carbaldehyde). All new Maillard products were formed with two molecules of glyceraldehyde. The fifth major adduct detected was Argpyrimidine, a well-known fluorescent arginine adduct. On porcine corneas (60 mg) treated in vitro with 0.3 or 0.6 mg of glyceraldehyde for 2 and 6 days, the change of modulus was measured on intact tissue and the three fluorophores were quantified after acid hydrolysis. After 2 days of incubation, the concentration of LHT, AHT and Argpyrimidine increased linearly with the concentration of glyceraldehyde and reached 2515, 206 and 6011 pmol/mg of protein, respectively, corresponding with a 5-fold increase in shear modulus. No further changes in concentration or modulus were detected between 2 and 6 days of incubation. Comparison of fluorophore concentrations and change in cornea modulus indicated a positive correlation (r2= 0.99, 0.97, 0.99 for LHT, AHT and Argpyrimidine, respectively). Finally, the solved adducts and Argpyrimidine accounted for ∼40% of the conventional total fluorescence of a treated cornea or ∼80% of the two-photon total fluorescence (lex = 800nm). Conclusion: We have identified four new glyceraldehyde-specific Maillard products, among which two are crosslinks and fluorophores. A strong association with tissue stiffening suggests the significance of these crosslinks during the treatment of corneas to enhance their mechanical integrity.

Keywords: 525 protein modifications-post translational • 370 cornea: basic science • 450 keratoconus 
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