Abstract

Recent evidence has shown the increased incidence of PA in approximately 15% of the hypertensive population, making a non-invasive and simple screening method for the measurement of aldosterone levels necessary. The use of saliva for determination of steroid hormones is now widely used and accepted and salivary aldosterone concentrations have previously been reported at around 30% of those seen in plasma. Furthermore, there is a current lack of longitudinal and systematic studies addressing the involvement of aldosterone in the regulation of the RAAS in rodents due to sample volume restrictions and the lack of sensitivity to detect the very low aldosterone concentrations in commercially available assays. We developed a non-isotopic, competitive immunoassay for the determination of aldosterone levels in saliva, as well as in human and mouse plasma samples. The assay employs an aldosterone-biotin conjugate as the tracer and end-point determination through time-resolved fluorescence (TR-FIA) with Streptavidin-Europium as the detectable label. No pretreatment or purification of saliva is necessary while a simple extraction step is incorporated for the assessment of plasma levels. A polyclonal antibody was used for the development of the saliva assay giving a lower limit of detection of 19 pg/ml for each 50µl sample. Similarly, a highly specific monoclonal antibody against aldosterone, exhibiting a more sensitive linear working range starting from 8 pg/ml is used to detect aldosterone in 50µl of plasma. The monoclonal antibody could potentially also be used for the determination of salivary aldosterone levels, however this was not sufficiently evaluated in the studies shown here and further investigation of the exact assay conditions is needed. Inter- and intra-assay coefficients of variation, mean recoveries, accuracy and linearity were validated for both assays and the assay results correlated significantly with a commercially available radioimmunoassay for plasma in both settings. Overall, salivary aldosterone was found to correspond to approximately 28% of the concentrations seen in plasma and reflected the changes seen with posture and ACTH stimulation accurately. The assay presents the additional possibility of using salivary aldosterone levels, in combination with salivary cortisol, as a diagnostic tool in a clinical setting to screen suspected cases of PA and exclude healthy subjects. The salivary aldosterone to cortisol ratio remains elevated in PA persons due to autonomous hypersecretion of aldosterone throughout the day, alongside decreasing levels of cortisol, and can be clearly distinguished from healthy persons above a cut-off level of 0.1. Furthermore, as aldosterone concentrations are acutely affected by ACTH it was determined that sampling for this test should be carried out in the evening to avoid stress factors as well as diurnal fluctuations. In addition, as basal aldosterone values and those after suppression and stimulation under different conditions were found within the linear range of the assay, it is proposed that the assay could be especially useful to monitor adrenocortical function in pharmacological and dietary intervention studies in rodent models where repeated sampling and volumes collected are limited and measurement of multiple blood parameters is desirable.