ROS (Reactive Oxygen Species)-Generating Systems in Mitochondria, Microsomes and Peroxisomes

Volume 4 • Issue 6 • 1000242 Pharm Anal Acta ISSN: 2153-2435 PAA, an open access journal *Corresponding author: Hideharu Shintani, Chuo University, School of Science, 1-13-27, Kasuga Bunkyo 112-0003 Tokyo, Japan, Tel: +81425922336; E-mail: shintani@mail.hinocatv.ne.jp Received May 14, 2013; Accepted June 24, 2013; Published June 28, 2013 Citation: Shintani H (2013) ROS (Reactive Oxygen Species)-Generating Systems in Mitochondria, Microsomes and Peroxisomes. Pharm Anal Acta 4: 242. doi:10.4172/2153-2435.1000242 Copyright: © 2013 Shintani H. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ROS (Reactive Oxygen Species)-Generating Systems in Mitochondria, Microsomes and Peroxisomes Hideharu Shintani* Chuo University, School of Science, 1-13-27, Kasuga Bunkyo 112-0003 Tokyo, Japan


Remove fat and endothelium, cut into 2-3 cm cubes and keep
on ice in a vinyl bag until mincing.
3. Mince with an electric food cutter for ca 1 min and keep on ice in a vinyl bag. It is possible to stop experiments by freezing the mince in a vinyl bag at this step. For quick freezing and thawing, the mince should be spread thinly in the bag. Usually, 1-2 kg of the mince is used for the preparation of SMP. 4. Suspend in ice-cold 0.25 M sucrose-0.185% K 2 HP0 4 (3 L/kg mince). 5. Adjust pH to ca 7.5 quickly by adding 6 M KOH (8-10 mL/ kg). 6. Homogenize with a food mixer at maximum power for 2 min. The homogenates should be kept on ice until centrifugation.
8. Decant the supernatant and filter through four layers of gauze.

Results and calculations
The SOD inhibitable reduction of epinephrine is calculated as the reduction by . O 2 - (Figure 1). The absorbance coefficient for adrenochrome is 2.96 mM/cm.

Microsomes
Preparation of acetylated cytochrome-c.
The reduction of cytochrome-c by endogenous electron-transfer systems in microsomes is reduced to below 10% by acetylation of cytochrome-c.

Protocol
AII procedures should be performed at temperatures below 4°C.
4. Allow the reaction to continue for 60 min.
5. Dialyse twice for 12 h in 2 L water at 4°C.
6. Determine the concentration and store at -20°C.
Measure the increase in absorbance at 550-540 nm in Hepes-NaOH (pH 7.5, 50 mM) after reduction by addition of a small amount of powdered Na 2 S 2 0 4 . The millimolar absorption coefficient is 19.1 mM/ cm. Acetylated cytochrome-c is stable to several freeze thaw cycles. 11. Store at -20 o C.

Results
Approximately 50 mg microsomes are obtained from 10 g wet weight liver. 14. Store at -20°C.

Results and Calculations
The SOD-inhibitable part is calculated as the reduction of acetylated cytochrome-c by . O 2 - (Figure 2).

Measurement of the production of hydrogen peroxide (H 2 O 2 )
H 2 O 2 results from the dismutation of . O 2 in mitochondria and microsomes. In peroxisomes H 2 O 2 is produced by acyl coenzyme A , an oxidase which is involved in the beta-oxidation of fatty acids.

Lipid peroxidation
Ferric salts are reduced to the ferrous state by the mitochondrial or microsomal electron transport systems, and ferrous salts are more active in redox reactions leading to lipid peroxidation. Malondialdehyde (MDA), a degradation product resulting from lipid peroxidation reactions, is a widely used quantitative marker for lipid peroxidation.