Improved micromethod for assay of serum angiotensin converting enzyme

We describe conditions for determining angiotensin converting enzyme (EC 3.4.15.1) in serum, by liquid chromatography. Serum (10 microL) is assayed with the artificial substrate hippuryl-glycyl-glycine (30 mmol/L) in a 50 mmol/L "HEPES" buffer solution with a high salt content (300 mmol of NaCl and 400 mmol of Na2SO4 per liter), at pH 8.0. The resulting enzymic activity is ninefold that of the currently popular spectrophotometric assay of Cushman and Cheung as modified by Lieberman (Am. J. Med. 59: 365-372, 1975). The hippuric acid end product is separated from the substrate by reversed-phase liquid chromatography and measured spectrophotometrically at 228 nm. o-Methyl hippuric acid is used as internal standard. The mean value for 100 normal control subjects was 317 (SD 96) nmol of hippuric acid released per milliliter of serum per minute. The enzyme activity is greater in newborns (p less than 0.05) and has a tendency to decrease with age. This partly automated method, which is optimized with regard to activity and detection, can be used in clinical routine.