Sod polyethylene glycol

Superoxide radical anion (O₂^•−) levels in brain blood vessels were measured by LC-MS–based detection of 2-OH-E⁺, which is the specific product of the reaction of O₂^•− with hydroethidine (HE; also known as dihydroethidium) (Invitrogen). Briefly, freshly isolated blood vessels from one mouse were equally divided into three parts and pretreated with saline, 50 μM L-NAME (Sigma-Aldrich), or SOD–polyethylene glycol (100 U/ml) (Sigma-Aldrich, S9549) in Krebs-Hepes buffer for 30 min. HE was then added to blood vessels to a final concentration of 50 μM in the buffer and incubated at 37°C for 30 min. After rinsing with ice-cold buffer, the vessels were centrifuged, collected, and kept at −80°C until analyses. Immediately before analyses, HE and its oxidation products were extracted from the vessels with ice-cold acetonitrile (50 μl/mg tissue) containing 0.1% formic acid and 3,8-diamino-6-phenylphenanthridine as an internal standard. After 30 min of shaking at 4°C, the samples were centrifuged (30 min × 20,000g at 4°C), and the supernatant (1 vol) was diluted with water containing 0.1% formic acid (3 vol). After additional centrifugation (15 min × 20,000g at 4°C), the supernatants were transferred into high-performance LC (HPLC) vials and analyzed by LC-MS, according to a published protocol (57 (link), 58 (link)).

Murine pulmonary arteries were carefully dissected free of surrounding tissue and cut into rings (1.8–2 mm in length). Vessel segments were mounted on a wire myograph in Krebs physiologic solution. Buffer solutions were continuously bubbled with 21% O₂, 5% CO₂, and 74% N₂ (PO₂¼ 17–19 kPa) 24, and stretched to a transmural pressure equivalent to 30 mm Hg. Contractility was recorded with an isometric force transducer and a displacement device coupled with a digitalization and data acquisition system (PowerLab). To confirm smooth muscle viability, arteries were first stimulated by raising the K⁺ concentration of the buffer to 80 mmoles/liter. Thereafter, concentration‐response curves to acetylcholine (10⁻⁹–10⁻⁵ moles/liter) and sodium nitroprusside (10⁻¹¹–10⁻⁵ moles/liter) were performed using cumulative addition to analyze the endothelium‐dependent and endothelium‐independent vasodilatation, respectively. For the reactive oxygen species (ROS) scavenging experiments, the concentration of superoxide dismutase (SOD)–polyethylene glycol (Sigma) used was 50 units/ml.