Abstract
Using bioinformatics analysis, we previously identified salusin-β, an endogenous bioactive peptide with diverse physiological activities. Salusin-β is abundantly expressed in the neuroendocrine system and in systemic endocrine cells/macrophages. Salusin-β acutely regulates hemodynamics and chronically induces atherosclerosis, but its unique physicochemical characteristics to tightly adhere to all types of plastic and glassware have prevented elucidation of its precise pathophysiological role. To quantitate plasma total salusin-β concentrations, we produced rabbit and chicken polyclonal antibodies against the C- and N-terminal end sequences, circumvented its sticky nature, and successfully established a sandwich enzyme-linked immunosorbent assay (ELISA). Salusin-β was abundantly present in the plasma of healthy volunteers, ranging from 1.9 to 6.6 nmol/L. Reverse phase-high performance liquid chromatography analysis showed that a single immunoreactive salusin-β peak coincided with synthetic authentic salusin-β. Plasma salusin-β concentrations were unaffected by postural changes and by potent vasopressin release stimuli, such as hypertonic saline infusion or smoking. However, salusin-β concentrations showed significant circadian variation; concentrations were high during the daytime and reached the lowest concentrations in the early morning. Plasma salusin-β levels in subjects with diabetes mellitus, coronary artery disease, and cerebrovascular disease showed distinctly higher levels than healthy controls. Patients with panhypopituitarism combined with complete central diabetes insipidus also showed significantly higher plasma salusin-β levels. Therefore, the ELISA system developed in this study will be useful for evaluating circulating total salusin-β levels and for confirming the presence of authentic salusin-β in human plasma. The obtained results suggest a limited contribution of the neuroendocrine system to peripheral total salusin-β concentrations and a role for plasma total salusin-β concentrations as an indicator of systemic vascular diseases.
Highlights
Immunoreactive salusin-β is localized to the neuroendocrine system in the brain and is present throughout systemic endocrine cells and certain hematopoietic cells, such as macrophages [1,2,3,4]
The enzyme-linked immunosorbent assay (ELISA) did not crossreact with other known bioactive peptides, such as salusin-α, angiotensin I, angiotensin II, oxytocin, and arginine vasopressin
Reverse-phase high performance liquid chromatography (RP-HPLC) fractionation of extracted human plasma and subsequent measurement of immunoreactive salusin-β showed a single peak coeluting with synthetic salusin-β peptide (Figure 2)
Summary
Immunoreactive salusin-β is localized to the neuroendocrine system in the brain and is present throughout systemic endocrine cells and certain hematopoietic cells, such as macrophages [1,2,3,4]. We used a novel strategy to produce polyclonal antiserum against such an amino acid sequence of low antigenicity [12] and successfully established a sandwich enzyme-linked immunosorbent assay (ELISA) suitable for detection of salusinβ in human plasma. This allowed us to investigate the physiological and pathophysiological significance of circulating salusin-β in humans
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