Trypsin Activation of Inactive Renin in Human Plasma an Assessment of Some Methodological Aspects

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Abstract
The following methodological aspects of the use of trypsin as an activator of inactive renin in human plasma were studied: the effect of SBTI [soybean trypsin inhibitor] on renin activity and angiotensin; the reaction velocity of trypsin on inactive renin; the optimum trypsin concentration; and the ability of human plasma to neutralize exogenous trypsin. Some commercially available SBTI may exert an angiotensinase-like effect which can be abolished by PMSF [phenylmethylsulfonyl fluoride]. At 4.degree. C activation of inactive renin reached a maximum within the first 2 min; then no further activation could be demonstrated. Trypsin 2 mg/ml yielded more inactive renin than trypsin 1 or 0.5 mg/ml. A higher concentration (3 mg/ml) gave substantially equivalent activation as with trypsin 2 mg/ml, whereas when using a still higher concentration (4 mg/ml) a degradation of the renin system components was noted. Endogenous trypsin inhibitors can eventually inactivate exogenous trypsin up to 3 mg/ml. Approximately 20% of renin is destroyed by trypsin 4 mg/ml within 2 min at 4.degree. C, while an additional 40% is lost during the incubation at 37.degree. C if no SBTI is added. To active inactive renin at 4.degree. C pH 7.0 with trypsin, 2 mg/ml for 2 min are used. SBTI is then added and the generation of angiotensin I at 37.degree. C pH 5.7 in the presence of PMSF is measured by radioimmunoassay. Active renin is measured using a parallel procedure which employs dilutents only instead of trypsin and SBTI. Inactive renin is calculated as a difference between trypsin-activated and active renin. The within-assay coefficient of variation is 6.1% and the between-assay variability 8.1%. In 26 normal subjects and 34 essential hypertensive patients on a normal Na diet, inactive renin was more than 4-fold higher than active plasma renin.