Extraction of Adrenomedullin from various tissues

ADM protein levels from plasma and organs: Blood samples were collected in a polystyrol tube (stabilized with EDTA) and, after centrifugation, plasma was stored at 20°C. Livers, lungs, kidneys, hearts, and brains were removed and immediately frozen in liquid nitrogen and stored by 80°C. ADM levels from plasma and organs were measured from extracted samples. For extraction, 1 ml of plasma was used and 1 ml of buffer A [1% trifluoroacetic acid (TFA)] was added. After mixing and centrifugation at 10,000 g for 20 min at 4°C, samples were loaded on a equilibrated Sep-Pak C18 cartridge (Millipore).

For extraction from the different organs, tissues were homogenized in 5 ml/g lysis buffer (10 mM Tris, pH 7.4). After centrifugation (1,600 g for 15 min at 4°C) the supernatant was also loaded on a equilibrated SEP-Pak C18 cartridge (Millipore). Equilibration was performed by washing once with 1 ml of buffer B (60% acetonitril in 1% TFA) and three times with 3 ml of buffer A. Then the peptides were eluted with 3 ml of buffer B and collected in a 15-ml polystyrol tube, evaporated to dryness, and the residue was dissolved in 250 µl of RIA buffer. ADM levels were measured on the pre-extracted (Sep-Pak C18) plasma, and tissues were sampled by a sensitive RIA employing an antibody against rat ADM (Phoenix Pharmaceuticals). For the RIA, 100 µl of each sample were used.

Extraction of PVN from tissue samples

The concentrations of immunoreactive alpha-MSH was quantitated in the PVN, using commercially available 125 I RIA kit [alpha-MSH (acetylated alpha-MSH); Phoenix Pharmaceuticals].

For extraction of PVN peptides, 1 ml of 0.1 M acetic acid was added to each tissue sample, which was then transferred to a boiling water bath for 10 min. After cooling on ice, samples were homogenized in polypropylene tubes. The homogenates were centrifuged at 13,000 g for 15 min. The pellet was resuspended in 3 N NaOH and analyzed for total protein content. To assay for protein, 150 µl of supernatant were taken from each sample, and the remainder of supernatant was lyophilized and later used for alpha-MSH RIA. The RIA kit was validated before use with tissue extracts. Dose-response curves for PVN tissue extract, and increasing concentrations of the alpha-MSH added to a rat PVN tissue extract were parallel (P < 0.05) to the standard curve.

alpha-MSH (ranging from 1 to 32 pg) added to rat PVN tissue extract was consistently recovered from 100 µl of extract (90-100%). The assay sensitivity was 4 pg/tube. The cross-reactivity test, pro-vided by Phoenix Pharmaceuticals, indicated 0% cross-reactivity with opioid peptides, 0% cross-reactivity with Beta- and Gamma-MSH, and 0.02% cross-reactivity with ACTH for alpha-MSH assay.

Reference: Kim, Eun-Mee, Martha K. Grace, Catherine C. Welch,Charles J. Billington, and Allen S. Levine.  STZ-induced diabetes decreases and insulin normalizes POMC mRNA in arcuate nucleus and pituitary in rats.  Am. J. Physiol. (Regulatory Integrative Comp. Physiol. 45) 1999; 276: R1320-R1326.