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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)
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.
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.
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.
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.
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