MEASUREMENT OF ADENOSINE CONCENTRATION IN AQUEOUS AND VITREOUS

PURPOSE. The release of adenosine by the ischemic retina may be an ini tial signal in the development of ischemic macular edema and neovascul arization. The levels of adenosine have never been quantified in ocula r fluids. In this study, a technique was developed for in vivo measure ment of the concentration of adenosine in aqueous and vitreous. METHOD S. Aqueous and vitreous samples were obtained from bovine eyes after d eath and from live porcine eyes with the subject under general anesthe sia. Samples from live eyes were immediately incubated in the sampling syringe with pentoxifylline, erythro-9-(2-hydroxy-3-nonyl) adenine, a nd dipyridamole to prevent synthesis or degradation of adenosine durin g the collection procedure, fitered, and flash-frozen in liquid nitrog en. ALL samples were then filtered and purified on phenylboronate agar ose columns and incubated with chloroacetaldehyde to convert the adeno sine present in the sample to the fluorescent derivative 1,N-6-ethenoa denosine. The 1,N-6-ethenoadenosine was separated by high-pressure liq uid chromatography and then measured by fluorometry. RESULTS. Levels o f adenosine as low as 0.5 pmole could be detected with this procedure, compared with 20 pmoles by UV detection. By using this technique to m easure adenosine levels in the eyes of normal weanling domestic pigs, it was determined that the adenosine concentration in the aqueous was 321.3 +/- 164.9 nM and in the vitreous was 210.8 +/- 41.5 nM. CONCLUSI ONS. The conversion of adenine-containing compounds to fluorescent 1,N -6-etheno derivatives offers analytical advantages of selectivity and sensitivity for the quantitative determination of these compounds, wit h the fluorometric detection providing substantially greater sensitivi ty than direct detection by UV absorption. The levels obtained in vivo from anesthetized but otherwise healthy pigs presumably reflected bas al aqueous and vitreous adenosine levels under the described condition s. This method should be useful in investigating more directly the rol e of adenosine in models of retinal or ocular ischemia in vivo and in measuring adenosine levels in vitreous or aqueous samples from human p atients.