April 2010
Volume 51, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2010
A Novel Method for the Detection and Quantification of Dideoxyosone Intermediates of AGEs in Human Lens Proteins
Author Affiliations & Notes
  • M. D. Linetsky
    Department of Ophthalmology,
    Case Western Reserve University, Cleveland, Ohio
  • K. Johar
    Iladevi Cataract and IOL Research Center, Ahmedabad, India
  • S. Padmanabha
    Department of Ophthalmology,
    Case Western Reserve University, Cleveland, Ohio
  • T. Parmar
    Iladevi Cataract and IOL Research Center, Ahmedabad, India
  • A. R. Vasavada
    Iladevi Cataract and IOL Research Center, Ahmedabad, India
  • R. H. Nagaraj
    Ophthalmology and Visual Sciences,
    Case Western Reserve University, Cleveland, Ohio
  • Footnotes
    Commercial Relationships  M.D. Linetsky, None; K. Johar, None; S. Padmanabha, None; T. Parmar, None; A.R. Vasavada, None; R.H. Nagaraj, None.
  • Footnotes
    Support  NIH grants R01EY-016219, R01EY-09912 and P30EY-11373, RPB and OLERF
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 4581. doi:
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      M. D. Linetsky, K. Johar, S. Padmanabha, T. Parmar, A. R. Vasavada, R. H. Nagaraj; A Novel Method for the Detection and Quantification of Dideoxyosone Intermediates of AGEs in Human Lens Proteins. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4581.

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

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Abstract
 
Purpose:
 

Dideoxyosones are intermediates in the synthesis of advanced glycation end products (AGEs) such as, pentosidine and glucosepane. Although the formation of pentosidine and glucosepane in the human lens has been firmly established, the formation of dideoxyosone has not been demonstrated. The purpose of this study was to develop a reliable assay to detect and quantify dideoxyosones in lens proteins.

 
Methods:
 

We synthesized a dideoxyosone trap, 3,4-diamino-N-(3-{[5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl) pentanoyl]amino} propyl)benzamide(BDAB) (structure shown below). BDAB was added at 300 µM concentration to one half of the freshly isolated lens and the tissue was homogenized add 1.0 ml 0.1M sodium phosphate buffer, pH 7.4 containing 0.1 M NaCl and 1 mM EDTA. The other half was homogenized similarly without BDAB. Lens homogenate was then lyophilized. The lyophilized sample was sonicated in HEPES buffer containing 1% Triton X-100. The sample was then passed through SDR-Hyper-D resin and the resulting protein was coated onto to ELISA plate wells at and 5 µg/well. The wells were blocked with 5% non-fat dry milk, incubated with avidin-conjugated to horseradish peroxidase (HRP) followed by incubation with a substrate for HRP. BSA (1 mg/ml) was glycated with 1 mM ribose in the presence or absence of 0-50 µM of BDAB for 7 days at 370C.

 
Results:
 

BSA incubated with ribose and BDAB showed high levels of dideoxyosones. Controls in which ribose or BDAB was omitted during incubation showed either no reaction or a 10-fold decrease in reaction. BDAB added cataractous human lens but not the controls (no BDAB added) showed high levels of dideoxyosones. The levels were 4.7, 4.1, 7.6 and 9 units per 5 µg protein in cataractous lenses from 40-50, 50-60, 60-70 and 70-85 year donors, respectively.

 
Conclusions:
 

Dideoxyosone intermediates are present in human lens proteins and they could be major intermediates for the synthesis protein crosslinking AGEs in cataractous lenses.  

 
Keywords: protein modifications-post translational • cataract • aging 
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