A sensitive method for differential determination of kininase I, II and neutral endopeptidase (NEP) in human urine.

In order to clarify the significance of NEP in human renal kallikrein-kinin system, an assay system was developed for the simultaneous determination of kininase I, II and NEP activities in human. Each kininase activity was determined by measuring the hydrolysis of bradykinin in the presence of specific inhibitors of kininase I (2-mercaptomethyl-3-guanidinoethylthiopropanoic acid), kininase II (captopril) and NEP (phosphoramidon) in 8 normal subjects. The effects of the different assay buffers on kininase activities were also investigated by using a phosphate buffer. Total kininase, kininase I, II and NEP activities were 499 +/- 65 ng/min/ml (mean +/- S.E.), 55 +/- 8, 141 +/- 21 and 299 +/- 42, respectively in our method using a tris buffer, while a phosphate buffer brought about activities of 358 +/- 43, 45 +/- 5, 156 +/- 21 and 135 +/- 25 ng/min/ml. The relative contributions of kininase I, II and NEP to total kininase activity were 11, 29 and 59% in our assay system, while they were 13, 44 and 35% when a phosphate buffer was used. From these results it was suggested that 1) phosphate may inhibit urinary NEP activity, so that a tris buffer should be used as the incubation buffer, 2) NEP is the major component of human urinary kininases, and 3) NEP may play an important role in the renal kallikrein-kinin system.