In line flow through gamma detector

Metabolic products of ¹²⁵I-hAng-(1–12) by CPA and rhChymase enzymes were analyzed by HPLC. Briefly, in a 200 μL reaction volume highly purified radiolabeled human ¹²⁵I-Ang-(1–12) substrate (~50 fmoles, specific activity 3900 cpm/fmol, purity ≥98%) was incubated with CPA (0.325 μg/mL), rhChymase (0.325 μg/mL) or a combination of CPA + rhChymase (1:1 or 1:⅓ ratio) in 50 mM Tris-HCl buffer solution containing 150 mM NaCl (pH 8.0) for 15 min (5 min for combination experiments) at 37 °C. The enzymatic re-actions were stopped by adding an equal volume of 1% phosphoric acid and centrifuged at 28,000 g for 10 min. The ¹²⁵I-hAng-(1–12) products were separated by HPLC on a C18 column using a linear gradient from 10% to 50% mobile phase B at a flow rate of 0.35 mL/min at 32 °C. The solvent system consisted of 0.1% phosphoric acid (mobile phase A) and 80% acetonitrile/0.1% phosphoric acid (mobile phase B). The eluted ¹²⁵I-hAng-(1–12) products were monitored by an in-line flow-through gamma detector (BioScan Inc., Washington, DC). Products were identified by comparison of retention times of synthetic (¹²⁵I) standard Ang peptides and the data were analyzed with Shimadzu LCSolution (Kyoto, Japan) acquisition software.

Metabolic products of ¹²⁵I-Ang-(1-12) and ¹²⁵I-Ang I by PMs were analyzed by reverse-phase high-performance liquid chromatography (HPLC) under different combinations of RAS inhibitor cocktails as described in Table 1. Briefly, the PMs (50–100 µg per reaction mixture) were pre-incubated with various combinations of RAS and peptidase inhibitors (50 µM each) for 15 min, then a highly purified ¹²⁵I-Ang-(1-12) or ¹²⁵I-Ang I (1 nmol/l) substrate was added to the reaction mixtures and incubated for an additional 60 min (for chymase) or 120 min (for ACE) at 37° C. The enzymatic reactions were stopped by adding an equal volume of 1% phosphoric acid, mixing well and centrifuging at 28,000 g for 20 min to remove the PMs. The clear supernatants were filtered and ¹²⁵I-Ang products were separated by HPLC on a C-18 column. A linear gradient from 10% to 50% mobile phase B at a flow rate of 0.35 mL/min at 32° C was used for the HPLC analyses. The solvent system consisted of 0.1% phosphoric acid (mobile phase A) and 80% acetonitrile/0.1% phosphoric acid (mobile phase B). The eluted ¹²⁵IAng products were monitored by an in-line flow-through gamma detector (BioScan Inc., Washington, DC). Products were identified by comparison of retention times of synthetic [¹²⁵I] standard Ang peptides and the data were analyzed with Shimadzu LCSolution (Kyoto, Japan) acquisition software.