Presence of immunoreactive salusin-beta in human plasma and urine

Salusin-alpha and salusin-beta are multifunctional bioactive peptides originally identified using bioinformatics analyses. Salusin-beta has been shown to exert potent hypotensive, bradycardic, and pro-atherosclerotic effects. The form in which it exists in biological fluids remains undetermined due to technical difficulties originating from its unexpected physicochemical properties. Here we show that salusin-beta peptide adheres to polypropylene and polystyrene, so that the aliquoted peptide dissolved in distilled water may rapidly disappear from the solution. By circumventing these features and using an antibody against C-terminal portion of salusin-beta, we have successfully established a specific radioimmunoassay suitable for detection of immunoreactive human salusin-beta. We have characterized the molecular form of salusin-beta in human plasma and urine. The assay detected immunoreactive salusin-beta concentrations as low as 5 fmol/tube and the concentration required for 50% inhibition of binding was 122 fmol/tube. Cross-reactivities with salusin-alpha and other bioactive peptides were negligible. Reverse-phase high performance liquid chromatography coupled with the radioimmunoassay detection after extraction from plasma and urine and using an octyl-silica column, revealed a major immunoreactive component that co-eluted with authentic salusin-beta. Salusin-beta-like immunoreactivity in normal human urine ranged from 0.23 to 2.22 nmol/l (mean+/-SD, 1.16+/-0.84 nmol/l, n=10). These data present the first evidence that salusin-beta circulates and is excreted in its authentic form, thereby verifying the initially predicted processing sites for salusin-beta in humans.