Studies on the human ovarian renin-angiotensin system: optimization of assay methodology and effects of follicular stimulants

Abstract

The renal and genital tracts share a common embryological origin; it is thus not surprising that tissues from both can synthesize renin. Preliminary studies showed extremely high concentrations of renin in follicular fluid (FRC) following ovarian stimulation for in-vitro fertilization. This necessitated complete revalidation of the renin assays and showed that data obtained using commercial kits were invalid. An assay protocol was developed using a 1:2 dilution of follicular fluid taken into EDTA (0.3 mol/l) and o-phenanthroline (0.05 mol/l). The assay was performed at pH 7.5 in the presence of excess exogenous (sheep) renin substrate, with incubation periods of 5, 10 and 15 min at 37 degrees C. This protocol resulted in the linear generation of angiotensin I (AI). Activation of inactive renin was performed using eightfold more trypsin than was required for plasma samples. Follicular renin substrate concentrations (FRS) were measured using the same assay methodology as used for measurement of plasma renin substrate concentrations (PRS). Storage of samples at -18 degrees C for up to 2 months was found not to affect the FRC, although repeated freeze-thaw cycles did. FRC and plasma renin concentrations (PRC) were very similar in 25 unstimulated control women, studied in the follicular phase of the menstrual cycle. Trypsin activation increased follicular total renin concentration (FTRC) more than plasma total renin concentration (PTRC) (P less than 0.0001). FRS was slightly higher than PRS (P less than 0.02). Ovarian stimulation with clomiphene citrate (CC; six women) was without effect on these parameters.(ABSTRACT TRUNCATED AT 250 WORDS)