Short communicationIdentification of an Adrenomedullin precursor fragment in plasma of sepsis patients
Introduction
Adrenomedullin (ADM) is a 52 aminoacids comprising peptide with multiple functions, the most prominent being its strong vasodilatory acitivity [2], [6]. ADM is derived from a larger precursor (preproADM, 185 aminoacids) by posttranslational processing, presumably following a scheme which is generally known for the maturation of various biologically active peptides (Fig. 1) [7]. Besides ADM, another biologically active peptide termed PAMP (proadrenomedullin N-terminal 20 peptide) is generated from preproADM. Similar to ADM, albeit by a different mechanism, PAMP has a strong hypotensive effect [2].
From the processing scheme of preproADM two other peptides can be predicted to be produced, that is the region located between PAMP and ADM, and the region flanking ADM at its C-terminus (Fig. 1). These peptides, termed proADM 45–92 and proADM 153–185 (also known as Adrenotensin), have not been described in the blood circulation so far, but have been synthesized chemically and assessed for their potential function in vitro: while Adrenotensin was found to be vasoconstrictive, no effect of proADM 45–92 was observed [3].
Quantification of ADM gene products would be helpful in the diagnosis, monitoring and prognosis of various cardiovascular diseases and sepsis, for which elevated plasma concentrations of ADM have been described [8], [13]. However, the reliable measurement of ADM is hampered by the existence of a binding protein [12], a short half-life time and extreme physical properties [9]. From their described biological functions it can be inferred, that the accurate quantification of PAMP and Adrenotensin might not be readily achievable as well.
No information exists so far on the fate of endogenous proADM 45–92, whether or not it is subject to further processing steps, whether or not it is rapidly cleared, whether or not it is present in the blood circulation. If present, it could be a highly interesting diagnostic target reflecting the actually released levels of the ADM gene products. Thus, we set out to determine its presence in plasma of septic shock patients.
Section snippets
Immunoassay
Three preproADM-related peptides were purchased from JERINI AG, Berlin, Germany. The peptides covered positions 68–86 (SPCD19), 83–94 (PSR13) and 45–92 (proADM 45–92) of preproADM, the first two with an additional N-terminal cystein residue. Sheep antibodies against SPCD19 and PSR13 were generated and purified as described [10]. A chemiluminescence label-/coated tube-based sandwichimmunoassay was set up as described [10], employing the anti-SPCD19 antibody as tracer, and anti-PSR13 as
Results
A sandwichimmunoassay for a mid-regional part of preproADM was developed employing antibodies which had been generated against peptides representing positions 68–86 and 83–94 of preproADM (Fig. 1). With this assay immunoreactivity could be detected in plasma samples from patients with septic shock, which was much higher than in normal controls (Fig. 2). After reversed phase HPLC of various plasma samples immunoanalysis revealed that the analyte was homogenous and uniform among septic shock
Discussion
A processing pathway for preproADM leading to ADM and PAMP has been proposed based on a sequence comparison with other peptide hormone precursors [7]. What happens in vivo to the other two peptide regions predicted to be derived from proADM, has been unknown so far.
We have identified now one of these peptides – proADM 45–92 – in plasma of patients with septic shock. This finding is in concordance with the proposed processing scheme. The molecule is apparently stable, since no smaller peptides
Acknowledgements
We like to thank Dr. Christian Scheler (Proteome Factory AG) and Dr. Monika Ühlein (B·R·A·H·M·S Aktiengesellschaft) for consultancy about mass spectrometric analyses, and Dr. Martina Strebelow, Sonja Tietz (LAD GmbH), Christine Alonso, Marko Talke and Detlef Hintzen (B·R·A·H·M·S Aktiengesellschaft) for work on the immunoassay.
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