Aging Aging

Analysis of Pentosidine 211Fig. 2. Effects of age and diabetes on concentration of pentosidine in human skincollagen (3).2. Materials1. N a -Acetyl-lysine, N a -acetyl-arginine, D-ribose, trifluoroacetic acid (TFA) andheptafluorobutyric acid (HFBA) are available from Aldrich (St. Louis, MO,USA); SP-Sephadex C-25 from Pharmacia (Bromma, Sweden); C-18 solid phaseextraction columns (Sep-Paks) from Waters (Milford, MA, USA); and 0.22 µMluer-adaptable filters from Millipore (Bedford, MA, USA).2. HPLC system: Two-solvent gradient system, equipped with fluorescence anddiode array detectors. Reverse-phase, C-18 column, 15 × 0.46 cm, Vydac 218TP,300A Pore Size; HPLC grade acetonitrile (CH 3 CN).3. Speed-Vac Centrifugal evaporator system (Savant Instruments, Farmingdale, NY,USA).4. Heating block for heating 13 × 100 mm screw-cap test tubes at 65°C and 110°C.5. Source of nitrogen gas (10 psi pressure).3. Methods3.1. Preparation of Pentosidine Standard1. Dissolve N a -acetyl-lysine (18.8 mg, 0.1 mmol), N a -acetyl-arginine (21.6 mg,0.1 mmol), and D-ribose (15 mg, 0.15 mmol) in 1 mL of 0.2 M sodium phosphatebuffer, pH 9.0, in a 13 × 100 mm screw-cap tube with Teflon-lined cap. Adjustto pH 9 with 0.1 M NaOH.2. Place in heating block at 65°C. Monitor pH, initially at hourly intervals, readjustingto pH 9.0, as needed.