Chemiluminescence Measurement of Extracellular and Intracellular ROS

Chemiluminescence depends on the measurement of light emitted after reagents are added to a sample of human spermatozoa, causing a reaction. The chemiluminescence assay is one of the most commonly used techniques to measure seminal ROS levels in clinical andrology laboratories [7, 12, 29, 30]. The two major probes used to measure ROS generation in the chemiluminescence assay are luminol (5-amino-2,3-dihydro-1,4-phthalazinedione and 3-aminophthalic hydrazide) and lucigenin (N,N'-dimethyl-9,9'-biacridinium dinitrate). Both H2O2 and O2" are involved in luminol-dependent chemiluminescence because both catalase and SOD can disrupt the luminol signal very efficiently. The uncharged luminol molecule is membrane-permeant and can

Table 13.2 Tests for detection of reactive oxygen species (direct) or their oxidized products (indirect)

Assay Probe Extracellular/intracellular

Direct measurement Tetrazolium nitroblue Chemiluminescence

Indirect measurement Lipid peroxidation levels

Antioxidants, micronutrients, vitamins Ascorbate

Antioxidants enzymes

Chemokines

Antioxidant-pro-oxidant status

Ferricytochrome C

Luminol

Lucigenin

Thiobarbituric

High-performance liquid chromatography High-performance liquid chromatography Superoxide dismutase Catalase

Glutathione peroxidase Glutathione reductase ELISA

Total antioxidant

Extracellular Both

Extracellular

Measures oxidized component in the body fluids Serum and seminal plasma

Seminal plasma

Seminal plasma

Seminal plasma

Spermatozoa

Spermatozoa

Seminal plasma

Low chain-breaking levels react in the form of luminol or as a univalently oxidized luminol radical with a variety of ROS, including O2% H2O2, and Off [12, 30]. Luminol is sensitive to H2O2 and this sensitivity can be greatly increased by addition of horseradish peroxidase. The lumi-nol signal generated by human spermatozoa is initiated by a one-electron oxidative event that is mediated by H2O2. A luminescent signal is produced with luminol through a one-electron oxidative event mediated by H2O2 and either endogenous peroxidase or by addition of HRP [31]. Oxidation of luminol (one-electron) leads to the creation of a radical species which interacts with ground state oxygen to produce O2% which participates in the oxygenation of luminol radical species to create an unstable endoperoxide, which breaks down and leads to light emission. In this, the O2" is an essential intermediate for the luminol-dependent chemiluminescence. Also, the redox cycling activity associated with this probe allows the significant amplification of the signal and allows easy measurement of H2O2.

Lucigenin works through a one-electron reduction unlike luminol which requires one-electron oxidation that creates a radical from lucigenin; this radical gives up its electron to the grounds state oxygen to create O2", thus returning the lucigenin to its parent state [12, 30, 31]. The luminol probe is more advantageous than the lucigenin probe for several reasons. Luminol measures both intracellular and extracellular ROS such as hydrogen peroxide, superoxide anion, and hydroxyl radical; on the other hand, lucigenin measures only the extracellular ROS, and in particular, superoxide anion [22, 30]. Lucigenin is an excellent probe for evaluating O2" production as a nonspecific redox marker for the enhanced electron transfer activity associated with defective sperm function. Both the sensitivity and specificity of this probe are enhanced by its redox cycling activity [7, 29, 30].

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