Measurement of ROS homeostasis in isolated mitochondria

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

In this chapter, we describe the currently most advanced methods applied for the quantitative assessment of ROS homeostasis inside the mitochondrion. These techniques are of particular interest in the field of oxidative stress. After discussing the importance of quantifying mitochondrial ROS homeostasis, three major aspects of this phenomenon and the pertinent methodologies for detection are delineated in detail. First the most important methods, based on fluorimetric or spectrophotometric approaches, for the detection of mitochondrial ROS are described. Elimination of ROS generated inside the mitochondrion is another crucial mechanism that also needs to be quantified accurately to estimate the antioxidant capacity of mitochondria under specific conditions. Since ROS generation and elimination manifest in concert, there needs to exist independent methods for the estimation of the net effect. Such a sensitive biochemical marker in the mitochondrion is aconitase, a citric acid cycle enzyme which is greatly sensitive to ROS. We describe two procedures for the precise determination of aconitase activity. A few auxiliary techniques and good practices having relevance in the successful accomplishment of the more delicate approaches are also mentioned. All other relevant technical considerations including advantages/disadvantages of the various methods and the most common artifacts are also discussed.

Original languageEnglish
Pages (from-to)199-223
Number of pages25
JournalMethods in Enzymology
Volume547
Issue numberC
DOIs
Publication statusPublished - 2014

Fingerprint

Mitochondria
Homeostasis
Aconitate Hydratase
Oxidative stress
Citric Acid Cycle
Antioxidants
Artifacts
Oxidative Stress
Biomarkers
Enzymes

Keywords

  • Aconitase
  • Amplex
  • Mitochondria
  • Oxidative stress
  • Reactive oxygen species
  • Red
  • ROS elimination

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Medicine(all)

Cite this

Measurement of ROS homeostasis in isolated mitochondria. / Tretter, L.; Ambrus, A.

In: Methods in Enzymology, Vol. 547, No. C, 2014, p. 199-223.

Research output: Contribution to journalArticle

@article{a5da7b353dd748f8aa3d8fc9a10a8e6b,
title = "Measurement of ROS homeostasis in isolated mitochondria",
abstract = "In this chapter, we describe the currently most advanced methods applied for the quantitative assessment of ROS homeostasis inside the mitochondrion. These techniques are of particular interest in the field of oxidative stress. After discussing the importance of quantifying mitochondrial ROS homeostasis, three major aspects of this phenomenon and the pertinent methodologies for detection are delineated in detail. First the most important methods, based on fluorimetric or spectrophotometric approaches, for the detection of mitochondrial ROS are described. Elimination of ROS generated inside the mitochondrion is another crucial mechanism that also needs to be quantified accurately to estimate the antioxidant capacity of mitochondria under specific conditions. Since ROS generation and elimination manifest in concert, there needs to exist independent methods for the estimation of the net effect. Such a sensitive biochemical marker in the mitochondrion is aconitase, a citric acid cycle enzyme which is greatly sensitive to ROS. We describe two procedures for the precise determination of aconitase activity. A few auxiliary techniques and good practices having relevance in the successful accomplishment of the more delicate approaches are also mentioned. All other relevant technical considerations including advantages/disadvantages of the various methods and the most common artifacts are also discussed.",
keywords = "Aconitase, Amplex, Mitochondria, Oxidative stress, Reactive oxygen species, Red, ROS elimination",
author = "L. Tretter and A. Ambrus",
year = "2014",
doi = "10.1016/B978-0-12-801415-8.00012-6",
language = "English",
volume = "547",
pages = "199--223",
journal = "Methods",
issn = "0076-6879",
publisher = "Academic Press Inc.",
number = "C",

}

TY - JOUR

T1 - Measurement of ROS homeostasis in isolated mitochondria

AU - Tretter, L.

AU - Ambrus, A.

PY - 2014

Y1 - 2014

N2 - In this chapter, we describe the currently most advanced methods applied for the quantitative assessment of ROS homeostasis inside the mitochondrion. These techniques are of particular interest in the field of oxidative stress. After discussing the importance of quantifying mitochondrial ROS homeostasis, three major aspects of this phenomenon and the pertinent methodologies for detection are delineated in detail. First the most important methods, based on fluorimetric or spectrophotometric approaches, for the detection of mitochondrial ROS are described. Elimination of ROS generated inside the mitochondrion is another crucial mechanism that also needs to be quantified accurately to estimate the antioxidant capacity of mitochondria under specific conditions. Since ROS generation and elimination manifest in concert, there needs to exist independent methods for the estimation of the net effect. Such a sensitive biochemical marker in the mitochondrion is aconitase, a citric acid cycle enzyme which is greatly sensitive to ROS. We describe two procedures for the precise determination of aconitase activity. A few auxiliary techniques and good practices having relevance in the successful accomplishment of the more delicate approaches are also mentioned. All other relevant technical considerations including advantages/disadvantages of the various methods and the most common artifacts are also discussed.

AB - In this chapter, we describe the currently most advanced methods applied for the quantitative assessment of ROS homeostasis inside the mitochondrion. These techniques are of particular interest in the field of oxidative stress. After discussing the importance of quantifying mitochondrial ROS homeostasis, three major aspects of this phenomenon and the pertinent methodologies for detection are delineated in detail. First the most important methods, based on fluorimetric or spectrophotometric approaches, for the detection of mitochondrial ROS are described. Elimination of ROS generated inside the mitochondrion is another crucial mechanism that also needs to be quantified accurately to estimate the antioxidant capacity of mitochondria under specific conditions. Since ROS generation and elimination manifest in concert, there needs to exist independent methods for the estimation of the net effect. Such a sensitive biochemical marker in the mitochondrion is aconitase, a citric acid cycle enzyme which is greatly sensitive to ROS. We describe two procedures for the precise determination of aconitase activity. A few auxiliary techniques and good practices having relevance in the successful accomplishment of the more delicate approaches are also mentioned. All other relevant technical considerations including advantages/disadvantages of the various methods and the most common artifacts are also discussed.

KW - Aconitase

KW - Amplex

KW - Mitochondria

KW - Oxidative stress

KW - Reactive oxygen species

KW - Red

KW - ROS elimination

UR - http://www.scopus.com/inward/record.url?scp=84925388552&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84925388552&partnerID=8YFLogxK

U2 - 10.1016/B978-0-12-801415-8.00012-6

DO - 10.1016/B978-0-12-801415-8.00012-6

M3 - Article

C2 - 25416360

AN - SCOPUS:84925388552

VL - 547

SP - 199

EP - 223

JO - Methods

JF - Methods

SN - 0076-6879

IS - C

ER -