Aging Aging

Analysis of Pentosidine 2152. Precipitate protein by addition of 440 mL of 20% trichloroacetic acid (TCA)while vortex-mixing. Centrifuge the sample at 1000g for 5 min (tabletop, clinicalcentrifuge) and discard the supernatant. Wash pellet once by suspension in 10%TCA and recentrifugation.3. Hydrolyze protein in 5 mL of 6 M HCl for 24 h at 110°C in screw cap tube under N 2 .4. Dry hydrolysate in vacuo and redissolve sample in 10 mL of deionized water andfilter through 0.22 µm luer-adaptable filters. Wash the filter with 1 mL of deionizedwater and pool with the filtrate.3.3.2. Solid-Phase Extraction with SP-Sephadex Minicolumns1. Swell SP-Sephadex overnight in deionized water at 4°C, according to themanufacturer’s instructions.2. Prepare minicolumns by filling small plastic columns with 3 mL of swollen gel.3. Wash minicolumns with 15 mL of deionized water, apply filtered samples, andwash with 20 mL of 0.1 M HCl. Elute with 7 mL of 1 M HCl. Pool and dry eluate(see Note 6).4. Redissolve eluate in 500 mL of 0.1% HFBA. Analyze a 200-µL aliquot byRP-HPLC, as described in Subheading 3.2. A typical chromatogram of a humanplasma sample is shown in Fig. 5A. Pentosidine may be expressed as µmoles ofpentosidine per milliliter plasma or per mole of lysine, measured separately byanalysis of the plasma sample or hydrolysate. The intraassay coefficient of variationis typically 8–10%.4. Notes1. The yield of pentosidine is typically ~1%, so the HPLC column could be overloadedby impurities during purification. The preliminary clean-up step isdesigned to eliminate excess reactants and salts that are not retarded by thecolumn, and brown products that are retained on the column after elution ofpentosidine. Column washing with 1% TFA is stopped when absorbance at~226 nm is less than 0.2 or has ceased to decrease. Switch to eluting solvent (1%TFA in 5% CH 3 CN) if absorbance at 326 nm begins to increase. Collectedfractions should be transparent or pale yellow. Brown color indicates overloadingof the minicolumn and calls for a decrease in the amount of sample applied. Atthis stage, sacrifice yield for purity.2. Dyer et al. (14) used radioactive lysine to calibrate their pentosidine standard. Selland Monnier (1) used a gravimetrically calibrated standard (Monnier, personalcommunication). They reported a molar extinction coefficient of 4195 at 326 nmin 0.1 M HCl. For determination of the extinction coefficient in the present study,we prepared a sample of pure N-acetyl,N'- hippuryl-pentosidine (hippuryl = benzoyl-glycine)from N a -hippuryl-lysine and N a -acetyl-arginine. The product waspurified by C-18 solid extraction and RP-HPLC using procedures similar to thosedescribed previously. Analysis by HPLC using a diode-array detector yielded asingle peak with an absorbance maximum at 326 nm (pentosidine) and secondarymaxima at 226 nm (amide carbonyl and carboxyl groups) and 278 nm (benzoyl