For purine determinations, fibroblasts were detached by trypsinization, counted in a Neubauer chamber, and resuspended in 0.4 N PCA. For urine samples, 150 µl of supernatant were used and 5 µl of perchloric acid (PCA) 60% (6 N) were added to obtain a final concentration of 0.2 N PCA. The samples were kept on ice for 15 min and then centrifuged at 12,000×g for 5 min at 4 °C. Pellet obtained from fibroblast extraction was stored at − 20 °C for later protein quantification and supernatant from fibroblasts or urines was neutralized with 5 M potassium carbonate (Sigma-Aldrich, 209619) and filtered through PVDF micro spin filters (Thermo Scientific, F2517-5) via centrifugation at 10,000×g for 10 min at 4 °C. The supernatants filtered were kept at − 80 °C for HPLC determination.
HPLC coupled to an UV detector was used for purine determinations. Analytes were separated using reverse-phase ion-pair chromatography on an Atlantis T3 column (Waters, 186003729). The optimized method for nucleotides separation and quantification consist on this sequence of stepped gradient of buffer A (10 mM of ammonium acetate (Sigma-Aldrich, A1542) and 2 mM of tetrabutylammonium phosphate monobasic solution (Sigma-Aldrich, 268100) pH5) and buffer B (10 mM of ammonium phosphate (Sigma-Aldrich, 09709), 2 mM of tetrabutylammonium phosphate, and 25% of acetonitrile (J.T. Baker, 76045) pH7) as follows: 100% of buffer A for 10 min, a linear gradient of buffer B up to 75% over 10 min, 9 min at 75% buffer B, a linear gradient to 100% buffer B over 3 min, 8 min at 100% buffer B, a linear gradient to 100% A in 1 min and finally 10 min maintained at 100% buffer A. The identification of purines was made by comparing their retention times to known standards and were quantified at 254 nm in a deuterium lamp. Sample analysis was performed by EZ Chrom Elite/ELITE UV–VIS software.
To determine intracellular concentration of nucleotides, skin fibroblasts single cell volume (2 × 10–6 μl) was calculated empirically.
HPLC coupled to an UV detector was used for purine determinations. Analytes were separated using reverse-phase ion-pair chromatography on an Atlantis T3 column (Waters, 186003729). The optimized method for nucleotides separation and quantification consist on this sequence of stepped gradient of buffer A (10 mM of ammonium acetate (Sigma-Aldrich, A1542) and 2 mM of tetrabutylammonium phosphate monobasic solution (Sigma-Aldrich, 268100) pH5) and buffer B (10 mM of ammonium phosphate (Sigma-Aldrich, 09709), 2 mM of tetrabutylammonium phosphate, and 25% of acetonitrile (J.T. Baker, 76045) pH7) as follows: 100% of buffer A for 10 min, a linear gradient of buffer B up to 75% over 10 min, 9 min at 75% buffer B, a linear gradient to 100% buffer B over 3 min, 8 min at 100% buffer B, a linear gradient to 100% A in 1 min and finally 10 min maintained at 100% buffer A. The identification of purines was made by comparing their retention times to known standards and were quantified at 254 nm in a deuterium lamp. Sample analysis was performed by EZ Chrom Elite/ELITE UV–VIS software.
To determine intracellular concentration of nucleotides, skin fibroblasts single cell volume (2 × 10–6 μl) was calculated empirically.
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