Adrenomedullin(1–52) measured in human plasma by radioimmunoassay: plasma concentration, adsorption, and storage Lynley K. Lewis, * Mary W. Smith, Timothy G. Yandle, A. Mark Richards, and M. Gary Nicholls We describe a specific and sensitive RIA for human adrenomedullin (AM)(1–52). The detection limit and the concentration required for 50% inhibition of binding were 0.1 and 1.2 fmol/tube, respectively. Cross-reactivi- ties with AM(1–12), AM(13–52), calcitonin gene-related peptide, amylin, and other vasoactive hormones were negligible. AM immunoreactivity in normal subjects ranged from 2.7 to 10.1 pmol/L (n  44). We investigated factors influencing the recovery and measurement of AM in the assay. Recovery of labeled AM (>80%) was markedly higher than that of unlabeled AM (56%). Immunoreactivity of exogenous AM added to plasma decreased up to 70% over four freeze–thaw cycles, whereas endogenous AM was stable. Alkali-treated ca- sein (1 g/L) reduced adsorption of AM to surfaces and significantly increased assay precision compared with bovine serum albumin (P <0.0001). HPLC separation of extracted plasma verified the presence of AM(1–52). We suggest that considerable care is needed to ensure that accurate and reproducible results are obtained from studies quantifying this peptide. Adrenomedullin (AM) 1 is a vasodilator peptide first iso- lated from human pheochromocytoma tissue in 1993 [1]. It has natriuretic and diuretic properties [2, 3] and may act in an autocrine or paracrine manner, although a physio- logical role for circulating AM remains possible [4]. Ob- taining accurate measurements of the concentration of AM in plasma is therefore vital. In normal volunteers, mean plasma AM concentrations have been variably reported as 19 pmol/L [1], 2– 4 pmol/L [5– 8], or 8 pmol/L [9], and urine concentrations were found to be six times higher than those in plasma [8]. Plasma concentrations are reportedly increased in various disease states, including congestive heart failure [6, 10, 11], chronic renal failure [8, 12], sepsis [9], and acute myocardial infarction [13]. AM has structural homology with amylin, an extremely “sticky” peptide [14], and thus may prove difficult to measure accurately in plasma. A number of assay systems for AM have been published in brief, but in our labora- tory, most extraction methods gave low and inconsistent results. We address the difficulties associated with mea- suring AM in human plasma and present the steps we took to resolve them. Materials and Methods assay reagents Human AM(1–52), AM(1–12), AM(13–52), Tyr 0 -C-type natriuretic peptide 22 (Tyr 0 -CNP-22), and calcitonin gene- related peptide were purchased from Peninsula Laboratories. Alkali-treated casein (ATC) was prepared according to the method of Livesey and Donald [15]. Bovine serum albumin (BSA), casein, aminopeptidase M, and carboxypeptidase A were obtained from Sigma Chemical Co. The buffer (PATC) used in the assay to dilute standard- sand antibodies and to reconstitute extracts was 0.05 mol/L phosphate buffer, pH 7.4, with 1 g/L ATC, 1 mL/L Triton X-100, 0.1 g/L Na 2 EDTA, and 0.2 g/L sodium azide. Working standards of AM(1–52) were prepared by serial dilution of a stock standard with PATC buffer; aliquots were stored at -20 °C until the day of assay. Antisera were prepared as follows. Human AM(1–52) (2 mg) was conjugated to BSA (4.4 mg) with use of 6.6 mg of carbodiimide [16] and then dialyzed and emulsified Cardioendocrine Research Group, Christchurch Hospital, Christchurch, New Zealand. * Address correspondence to this author at: Department of Medicine, Christchurch Hospital, PO Box 4345, Christchurch, New Zealand. Fax 64 3 3640 818; e-mail llewis@chmeds.ac.nz. 1 Nonstandard abbreviations: AM, adrenomedullin; ATC, alkali-treated casein; PATC, ATC buffer (see text for composition); BSA, bovine serum albumin; TFA, trifluoroacetic acid; IC 50 , median inhibiting concentration; irAM, immunoreactive adrenomedullin; and NSB, nonspecific binding. Received August 26, 1997; revision accepted December 8, 1997. Clinical Chemistry 44:3 571–577 (1998) Endocrinology and Metabolism 571 Downloaded from https://academic.oup.com/clinchem/article/44/3/571/5642558 by guest on 16 September 2021