A direct radioimmunoassay for human renin substrate and identification of multiple substrate types in plasma.

Abstract

Plasma renin substrate, a widely measured parameter of the renin reaction, is quantitated indirectly by the measurement of liberated angiotensin I upon exhaustive incubation of plasma with added renin. To overcome methodological problems of this assay system, we have developed a direct radioimmunoassay for this plasma protein using renin substrate purified from pooled plasma of normotensive subjects as the antigen. Comparison of substrate quantitated by the two assay systems (direct and indirect) indicates a 1:1 correlation with the exception of certain subjects with elevated substrate levels induced by estrogen therapy. To study the possibility of multiple substrate forms, we have made a comparison of substrate quantitated by both radioimmunoassays in conjunction with electrophoresis of plasma on polyacrylamide gel. One major form of substrate with a retardation factor (Rf) = 0.60 was found in normotensive and essential hypertensive subjects which gave a 1:1 correspondence on quantitation by the two methods. In contrast, six of 16 women on oral contraceptives demonstrated three forms of substrate (Rf = 0.16, 0.35, and 0.60) on electrophoresis. Substrate with Rf = 0.16 and 0.35 did not cross-react with the antiserum prepared against substrate from normotensive subjects, implying structural differences in these proteins.

Multiple forms of human plasma renin substrate.

“…Plasma renin substrate was quantitated by two methods: (a) direct, a detailed description of this method has previously been reported by Eggena et al (2). Briefly, '25I-labeled renin substrate and its specific antiserum (produced in rabbit) were added to a diluted plasma sample and equilibrated at 4°C for 18 h. The bound and free antigens are separated after a second equilibration with goat anti-rabbit gamma globulin by centrifugation.…”

“…Received for publication 14 November 1977 and in revised form 27 March 1978. say underestimated the renin substrate concentration suggesting that other forms of renin substrate were present in these plasmas which could be cleaved by renin to generate angiotensin I, but did not crossreact with the antiserum produced against the renin substrate of normal plasma (2). The following study examines the various forms of plasma renin substrate in several clinical states associated with hypertension and in patients receiving agents known to stimulate renin substrate synthesis.…”

“…We have previously described the purification of renin substrate from normal human plasma (1) and the development of a direct radioimmunoassay for this protein (2). The direct assay for renin substrate in normal human plasma was validated by demonstrating a 1:1 correlation to substrate measured by the generation of angiotensin I by prolonged incubation with exogenous homologous renin (indirect method).…”

Relation between blood pressure and renin, renin substrate, angiotensin II, aldosterone and urinary sodium and potassium in 574 ambulatory subjects.

“…"• u Previous studies on substrate in essential hypertension have failed to identify such marked elevations of substrate as are reported here. 15 ' 17 The majority of these studies represent observations on relatively small numbers of subjects. The coefficient of variation associated with substrate measurement was 40% in our study population.…”

Studies on angiotensinogen of plasma and cerebrospinal fluid in normal and hypertensive human subjects.

“…7 '" A direct radioimmunoassay developed for plasma angiotensinogen was also utilized as previously described; 9 no interference was evident when this assay was employed using CSF samples.…”