Detection of adenosine in vivo in intraocular fluids

PURPOSE: Adenosine is known to be released from ischemic tissue and may play an important role in the regulation of ocular neovascularization. Although adenosine levels have been quantified in tissues such as brain, lung, and heart, adenosine has not been quantified in intraocular fluids. In this study we describe a sensitive method for detection and quantification of adenosine in aqueous and vitreous fluids. METHODS: Bovine aqueous and vitreous samples were collected from enucleated eyes. 200 μl samples were diluted to 10 ml with 250 mM ammonium acetate (AA), pH 9.0 and purified using a phenyl boronate agarose (PBA-60) column. Adenosine was eluted from the column with AA, pH 4.5. The sample adenosine was converted to a fluorescent derivative, 1-N⁶-ethenoadenosine (Σ-ado), by incubating the sample with chloroacetaldehyde at 60°C for 12 hours. After conversion to Σ-ado, the samples were again purified using a PBA-60 column. Σ-ado was separated from interfering compounds by C₁₈ reverse phase HPLC and quantified by fluorometry. ¹⁴C adenosine was used to validate the assay. RESULTS: Using this assay, we were able to detect and quantify adenosine in both aqueous and vitreous fluids. The detection limit of this assay for Σ-ado was 0.5 pmole. CONCLUSIONS: This assay is a simple and highly sensitive assay for the quantification of adenosine in intraocular fluids. This method will be useful for the quantification of adenosine levels in intraocular fluids of an animal model of retinal ischemia.