April 2010
Volume 51, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2010
A Simple Method for Measuring Aliphatic β-Nitroalcohols Using the Griess Colorimetric Nitrite Assay
Author Affiliations & Notes
  • Q. Wen
    Ophthalmology,
    Columbia University, New York, New York
  • Y. Yoe
    Ophthalmology,
    Columbia University, New York, New York
  • M. R. Solomon
    Biomedical Engineering,
    Columbia University, New York, New York
  • D. C. Paik
    Ophthalmology,
    Columbia University, New York, New York
    Surgery, Christiana Hospital, Newark, Delaware
  • Footnotes
    Commercial Relationships  Q. Wen, None; Y. Yoe, None; M.R. Solomon, None; D.C. Paik, None.
  • Footnotes
    Support  Research to Prevent Blindness, NIH/NCRR UL1RR024156, NIH/NEI R21EY018937 (dcp)
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 3473. doi:https://doi.org/
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      Q. Wen, Y. Yoe, M. R. Solomon, D. C. Paik; A Simple Method for Measuring Aliphatic β-Nitroalcohols Using the Griess Colorimetric Nitrite Assay. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3473. doi: https://doi.org/.

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

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Abstract

Purpose: : The ability to selectively enhance the biomechanical properties of a given tissue in vivo could have widespread clinical utility. Our recent studies suggest that aliphatic β-nitro alcohols (BNAs) may represent a useful class of compounds for use as in vivo therapeutic corneoscleral cross-linking agents. However, current assay methods for these compounds involve the use of chromatographic instrumentation. Thus, the following study was undertaken in order to standardize a simple method for BNA quantitation based on denitration.

Methods: : The optimal conditions necessary for denitration were studied with respect to pH, heating temperature, and heating time using the colorimetric Griess nitrite assay (max=550nm). Standard curves were then created for two mono-nitroalcohols [2-nitroethanol (2ne) and 2-nitro-1-propanol (2nprop)], a nitro-diol [2-methyl-2-nitro-1,3-propanediol (MNPD)], and a nitro-triol [2-hydroxymethyl-2-nitro-1,3-propanediol (HNPD)]. Finally, recovery studies were conducted using tissue homogenates (10mg/ml and 30mg/ml wet weight) from rabbit cornea and sclera.

Results: : Optimal conditions for denitration included a pH of 7-8, 100oC, and 2 hours heating time. Standard curves of the 4 nitroalcohols at pH 7.4 showed excellent linearity and reproducibility in the 100-500µM range with R2 values >0.98. The equations were as follows: y=(0.000979*x)-0.04335 for 2ne, y=(0.00209*x)-0.0553 for 2nprop, y=(0.00211*x)-0.09319 for MNPD, and y=(0.000991*x)-0.02126 for HNPD. The lower limit of detection was ~20 µM for all 4 compounds. Recovery in tissue homogenates were variable. Interestingly, the free methyl group on the nitro-containing carbon in 2nprop and the nitro-diol exhibited a denitration promoting effect.

Conclusions: : The Griess colorimetric nitrite assay can be successfully used for the quantitative determination of BNA content in solution and is simpler than conventional chromatographic methods. The assay takes advantage of a denitration that occurs under alkaline pH and can be accelerated by heating. Future work will use this method in studies related to therapeutic tissue cross-linking.

Keywords: keratoconus • drug toxicity/drug effects • extracellular matrix 
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