Table 2.
Methods of measuring reactive nitrogen species (RNS).
| Method(s) | Type | Principle | Advantage(s) | Reference(s) |
|---|---|---|---|---|
| Cytochrome C reduction test | Direct | Reduction of ferricytochrome C to ferrocytochrome C is used to detect superoxide formation. | Gold standard for measuring extracellular superoxide anions. | [87] |
| Electron spin resonance (ESR), electron paramagnetic resonance (EPR) | Direct | (i) Magnetic properties of unpaired electrons in free radicals enable them to absorb electromagnetic radiation on application of external magnetic field; this then generates absorption spectra utilizing the energy of the electron spin state, which is measured using ESR spectrophotometers. (ii) Provide direct detection of the “instantaneous” presence of free radical species in a sample. (iii) Play a major role in the assessment of most of the oxidants characterized by very short half-life (nanoseconds to microseconds) usually by using stabilizing molecules called spin-traps/probes. |
(i) Used to measure oxidative stress on proteins and lipids. Simple, and have high sensitivity and specificity. (ii) Detects free radicals and paramagnetic molecules. The magnetic field-based EPR detection enables nondestructive (in vitro) and noninvasive (in vivo) measurements of biological samples. EPR spectroscopy, coupled with the use of paramagnetic probes, is a potential technique for accurate and precise determination of ROS concentrations in a variety of biological samples. |
[86,90] |
| Xylenol orange-based assay | Direct | (i) Uses automated analyzer. (ii) ROS in semen oxidizes ferrous to ferric ion and this forms a colored complex with xylenol orange in an acidic medium, the color intensity of which can be measured spectrophotometrically. (iii) Results are expressed in μmol H O2 2 equiv./L. |
It is rapid, easy, stable, inexpensive, reliable, and sensitive. | [104] |
| ROS measurement via chemiluminescence | Direct | (i) Measures real-time production of ROS. (ii) Uses two probes—luminol and lucigenin. (iii) Luminol measures global ROS levels, both extracellular and intracellular (superoxide anion, hydrogen peroxide, and hydroxyl radical). (iv) Lucigenin is specific for superoxide anion and hydroxyl radical. |
Chemiluminescence is a robust, sensitive, and specific method. | [105,106,107,108,109,110,111] |
| Flow cytometry | Direct | (i) ROS measurement of hydrogen peroxide and superoxide anion via flow cytometry. (ii) Dihydroethidium measures intracellular superoxide anion and dichlorofluoroscein diacetate for intracellular hydrogen peroxide. |
Requires very low amounts of spermatozoa, and high-specificity intracellular ROS in spermatozoa. | [102,112,113,114] |
| Endtz test | Indirect | (i) ROS is mainly generated by leukocytes. (ii) Myeloperoxidase is used to stain polymorphonuclear granulocytes, but does not provide any information regarding ROS generation by spermatozoa. |
Indirect indicator of excessive ROS generation by leukocytes in semen. | [91,102,104] |
| Redox potential GSH/GSSG | Indirect | (i) Balance of reduced glutathione and its oxidized form (GSSG) gives an indication of ROS levels in vivo. (ii) GSH/GSSG levels are measured biochemically or using high-performance liquid chromatography. |
Can be used to measure oxidative stress in vitro and in vivo. | [92,93] |
| Thiobarbituric acid assay (TBARS) | Indirect | (i) Measures lipid peroxidation. (ii) Detects malondialdehyde (MDA-TBA) adduct by colorimetry or fluoroscopy. |
Simple but non-specific. | [103,104] |
| Oxidation-reduction potential | Indirect | (i) Measures the redox balance in a given biological system. (ii) It measures all known and unknown oxidants and antioxidants in a given sample. |
Can be measured both in seminal ejaculates and in seminal plasma (both fresh and frozen). | [98,99,100] |