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The Measurement of Oxidative Stress in Semen and Use in Assisted Reproduction
Published in Nicolás Garrido, Rocio Rivera, A Practical Guide to Sperm Analysis, 2017
Ashok Agarwal, Joseph Vitale, Anthony Kashou
Lucigenin is another probe that can be used in chemiluminescence. It can react with a variety of reducing agents. For instance, it is especially sensitive in analyzing the enzymatic reaction producing H2O2 from O2− via SOD. The ability of SOD to enzymatically reduce O2− causes suppression of lucigenin-dependent cellular signals, and thus, provides a means to effectively measure O2−.22 Mechanistically, a one-electron reduction activates lucigenin. This results in the formation of a cation radical-form of lucigenin that rapidly couples with O2− to yield dioxetane.23 Dioxetane then decomposes into an excited N-methylacridone compound, which spontaneously emits blue light upon returning to its ground state.21 The intensity of light emitted can be used to measure the amount of O2− present.
Reconstituted Membrane Systems for Assaying Membrane Proteins in Controlled Lipid Environments
Published in Qiu-Xing Jiang, New Techniques for Studying Biomembranes, 2020
Lucigenin is a low-sensitivity fluorescent indicator for Cl−.59 It can be quenched via collision with Cl− and has been successfully used for glycine receptors and CFTR channels.60,61 It is thus suitable for monitoring the change of Cl− concentration inside proteoliposomes or in the extravesicular medium. We will describe a case for the latter with lucigenin in the outside (Figure 6.5). Reconstitute purified proteins into proteoliposomes using POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine) and POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phopo-1′-rac-glycerol) lipids in 3:1 weigh ratio and dialysis to remove detergents. Keep the salt (Cl− ion) concentration high (~400 mM) during reconstitution.Immediately before the flux assay, remove the extravesicular chloride ions using a size-exclusion column as described earlier. Dilute proteoliposomes (20 x) into a chloride-free buffer which has isethionate as a nonpermeable anion to maintain the ionic strength of the extravesicular solution.Add 0.1–0.5 µM lucigenin into the extravesicular buffer.Measure fluorescence by excitation at 455 nm and emission at 505 nm. Continued efflux of Cl− ions is initiated by adding 1.0 µM valinomycin to shunt charge accumulation due to chloride efflux through the channel (or an anion carrier, cholapod 3 as an example in Figure 6.6b).Once the fluorescence signal is stable, add 5.0 mM detergent (n-decyl-β-D maltopyranoside) to disrupt liposomes and release all chloride ions, reaching a maximal quenching of lucigenin signal.
Possible anti-inflammatory, antioxidant, and neuroprotective effects of apigenin in the setting of mild traumatic brain injury: an investigation*
Published in Immunopharmacology and Immunotoxicology, 2023
Pınar Kuru Bektaşoğlu, Dilan Demir, Türkan Koyuncuoğlu, Meral Yüksel, İrem Peker Eyüboğlu, Ayça Karagöz Köroğlu, Dilek Akakın, Alper Yıldırım, Erhan Çelikoğlu, Bora Gürer
Chemiluminescence (CL) is a direct, noninvasive method for the measurement of reactive oxygen radicals that utilizes luminol and lucigenin as enhancer probes. When added to in vitro biological systems, luminol and lucigenin produce high levels of excited products. Excited electrons from these compounds generate radiating light energy or CL that can be detected by a luminometer. Luminol detects radicals such as hydroxyl ions, hydrogen peroxide, and hydrochloric acid, whereas lucigenin is selective to superoxide anions [26]. Reactive oxygen species (ROS) were numerically measured after the addition of 0.2 mM of enhancers luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) and lucigenin (bis-N-methylacridinium nitrate) (Sigma Aldrich, USA). A purified luminol-hydrogen peroxide system was used for NO measurement. For this purpose, potassium carbonate (K2CO3) (0.4 mM), desferal (60 µM), H2O2 (4 mM), and purified luminol (3.6 M) were added to the tissues in 2 ml of phosphate-buffered saline (PBS) + HEPES buffer [27,28]. Counts were measured at room temperature using a luminometer (Junior LB 9509 luminometer; EG&G Berthold, Germany), and obtained at 1-min intervals for 5-min. The area under the curve was determined, and data were expressed as relative light units after counts were normalized to the weight of the brain tissue sample. Results were expressed as relative light units/mg tissue (rlu/mg).
Acute restraint stress increases blood pressure and oxidative stress in the cardiorenal system of rats: a role for AT1 receptors
Published in Stress, 2020
Gabriel T. do Vale, Drieli Leoni, Arthur H. Sousa, Natália A. Gonzaga, Daniela L. Uliana, Davi C. La Gata, Leonardo B. Resstel, Cláudia M. Padovan, Carlos R. Tirapelli
Lucigenin-derived luminescence was significantly greater in the LV from stressed rats (F3,33 = 26.35, p < 0.05), but treatment with losartan did not prevent this response (Figure 5(A)). On the other hand, acute restraint stress decreased the concentration of both H2O2 (F3,30 = 16.69, p < 0.05) and NOx (F3,37 = 36.85, p < 0.05) in the LV, but these responses were not affected by losartan (Figure 5(B,C)). Increased levels of TBARS were detected in the LV from stressed rats (F3,31 = 9.703, p < 0.05), and this response was not affected by losartan (Figure 5(D)). SOD activity in the LV was not affected by acute restraint stress (Figure 5(E)). On the other hand, catalase activity was significantly greater in the LV from stressed rats (F3,31 = 11.24, p < 0.05), but treatment with losartan did not prevent this response (Figure 5(F)).
Effects Of Endothelin-1 On Intracellular Tetrahydrobiopterin Levels In Vascular Tissue
Published in Scandinavian Cardiovascular Journal, 2018
Ruha Cerrato, Mark Crabtree, Charalambos Antoniades, Karolina Kublickiene, Ernesto L. Schiffrin, Keith M. Channon, Felix Böhm
Vascular superoxide production was measured in paired segments of IMA with the use of lucigenin-enhanced chemiluminescence [12]. The segments were divided into 4–6 rings, each approx 3 mm thick, depending on size, weight ranging from 6−20 mg/ring. The vessel segments were opened longitudinally to expose the endothelial surface and equilibrated in oxygenated (95% O2/5% CO2) Krebs-HEPES buffer (pH 7.4) at 37 °C. As a measure of eNOS coupling, we determined NOS-derived superoxide production in paired segments n = 12, which was estimated as the difference in superoxide production after 20 minutes of incubation with the NOS inhibitor NG-nitro-l-arginine methyl ester (L-NAME) alone [13] or followed by incubation with ET-1 (0.1nM) for 45 min. The vessel segment was then quickly transferred to a luminometer containing low-concentration lucigenin (5 µmol/L) in order to measure superoxide production.