Abstract
We recently discovered that the vascular responsiveness to adrenomedullin (AM), a potent vasoactive peptide, decreased during sepsis and hemorrhage in the rat and was markedly improved by its novel binding protein (AMBP-1). Moreover, AM/AMBP-1 appears to be one of the leading candidates for further development to treat sepsis and hemorrhage. However, the extremely high cost of commercial AMBP-1 limits the development of human AM and AMBP-1 as therapeutic agents. The purpose of this study was to isolate and purify AMBP-1 from normal human serum and test its stability and biological activity under in vitro and in vivo conditions. AMBP-1 was isolated and purified from normal human serum with a yield of about 3.0 mg per 100 mL and purity of >99%. The purified AMBP-1 has a AM-binding capacity similar to that of the commercial AMBP-1. Human AM and human AMBP-1 in combination significantly inhibited lipopolysaccharide-induced tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 production from macrophages. The biological activity of the purified human AMBP-1 was well preserved when stored at 45 degrees C for 5 d in solution or at 100 degrees C for 1 h in powder. Moreover, administration of AM and purified AMBP-1 to hemorrhaged rats attenuated tissue injury and neutrophil accumulation. Purified AMBP-1 in combination with AM also suppressed the hemorrhage-induced rise in serum cytokines TNF-alpha and IL-6. Thus, we have successfully purified biologically active AMBP-1 from human normal serum and demonstrated the stability of purified human AMBP-1. This technique will enable us to further develop human AM/AMBP-1 as a novel treatment for safe and effective therapy of patients with hemorrhagic shock, sepsis, and ischemic injury.
Highlights
Adrenomedullin (AM) is a potent vasoactive peptide [1,2] originally identified in extracts of human pheochromocytoma in 1993 [3]
AMBP-1 was first concentrated from human serum by polyethylene glycol 4000 (PEG-4000) precipitation followed by three chromatography procedures
The isolated protein was identified as human AMBP-1 with more than 95% confidence using the Mascot database search algorithm
Summary
Adrenomedullin (AM) is a potent vasoactive peptide [1,2] originally identified in extracts of human pheochromocytoma in 1993 [3]. Administration of AM can cause hyperdynamic responses, including increased cardiac output and decreased afterload [4,5]. Circulating levels of AM increase in patients with sepsis and systemic inflammatory response syndrome and following major surgery, hypoxia, hemorrhagic and cardiogenic shock, or ischemia-reperfusion injury. A study by Elsasser et al [6] demonstrated the presence of a specific AM binding protein in mammalian blood. It was discovered in part by the specific binding of [125I]AM to a 120-kDa band on a blot obtained from a nonreducing, electrophoretic gel separation of serum proteins from several species including humans [6]. Pio et al purified this binding protein, named it AMBP-1, and discovered that AMBP-1 is identical to complement factor H [7]
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