Endocrine Hypertension, Adrenal Steroids and Development of a Saliva Based Aldosterone Assay as a Potential Screening Method

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.