Assay of carboxypeptidase N activity in serum by liquid-chromatographic determination of hippuric acid.
We describe conditions for determining carboxypeptidase N (EC 3.4.17.3) activity by liquid chromatography. Serum (10 microL) is mixed with the artificial substrates hippuryl-L-arginine (30 mmol/L) and hippuryl-L-lysine (100 mmol/L) in 50 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer solution at pH 8.2 and 7.8, respectively. The hippuric acid product is separated from the substrate in less than 2 min by reversed-phase "high-performance" liquid chromatography and measured spectrophotometrically. o-Methyl hippuric acid is used as internal standard. By this method, optimized for activity and sensitivity of detection, carboxypeptidase N activities are 60-fold greater than those by another procedure (J Chromatogr 266:173-177, 1983). The mean value for 80 normal control subjects was 74.8 (SD 10.3) nmol of hippuric acid released per milliliter of serum per minute for hippuryl-L-arginine substrate, 378 (SD 55) for hippuryl-L-lysine substrate. The sensitivity and precision of the method make it suitable both for routine clinical determinations and as a reference procedure.
D000293:Adolescent; D000328:Adult; D000368:Aged; D001120:Arginine / Q000031:analogs & derivatives; D002268:Carboxypeptidases / Q000097:blood*; D002851:Chromatography, High Pressure Liquid; D006626:Hippurates / Q000097:blood*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000031:analogs & derivatives; D001121:Lysine Carboxypeptidase / Q000097:blood*; D008875:Middle Aged; D012016:Reference Values; D013696:Temperature
D006626:Hippurates; C007945:hippuryl-L-lysine; C001650:hippuryl-L-arginine; D001120:Arginine; D002268:Carboxypeptidases; D001121:Lysine Carboxypeptidase; D008239:Lysine; C030514:hippuric acid