*4.2. Luminol*/*HRP*

Luminol (5-amino-2,3-dihydro-1,4-phthalazine-dione) present the advantage of having a high sensitivity and the capacity to detect both intra- and extracellular ROS [82,118]. To react with O2•<sup>−</sup>, luminol is first converted into an intermediate radical by a one-electron oxidation normally mediated by H2O2 [126,127] and enhanced by the addition of horseradish peroxidase (HRP) [82,128] (Figure 4). One major limitation is the fact that the luminol radical reacts not only with O2•<sup>−</sup> but also with various compounds capable of donating an electron [126,127], thus showing indiscriminate recognition of several free radicals. In addition, other complex and di fficult-to-control factors, such as the formation of O2•<sup>−</sup> by the luminol radical, may influence the chemiluminescence of this probe [118,127,129]. Therefore, according to Zhang and colleagues [118], it is "*unwise to monitor the dynamics of free radical generation in cells or systems with this probe alone*".

**Figure 4.** Chemical reactions responsible for luminol chemiluminescence. Luminol is first oxidized by many radicals (e.g., •OH and CO3•<sup>−</sup>, except O2•<sup>−</sup>) and peroxidases, forming the luminol radical (L•<sup>−</sup>) (1). L•<sup>−</sup> then reacts with O2•<sup>−</sup>, forming the short-lived intermediate hydroperoxide (2). Molecular oxygen may be reduced to O2•<sup>−</sup> by L•<sup>−</sup> (3), with a rate around seven orders of magnitude lower than that for reaction (2), resulting in the production of 5-aminophthalazine-1,4-dione. The latter may also form the intermediate hydroperoxide by the addiction of hydrogen peroxide anions (4). The intermediate hydroperoxide is quickly decomposed to 3-aminophyhalane in an excited state (5), which emits light on relaxation to the ground state (6).

Previous studies have used the luminol-based technique to sugges<sup>t</sup> that pathological spermatozoa (e.g., amorphous heads, damaged acrosomes and retained cytoplasmic droplets) generate higher amounts of ROS than their normal counterparts [130,131]. Nevertheless, one possible interpretation for these data is that luminol–HRP reacts with sperm containing luminol-reactive metabolites not ye<sup>t</sup> specified. This is reinforced by the fact that the retention of an excess of residual cytoplasm, a common feature of abnormal sperm, is associated with higher ROS measurements [132]. It is important to note that the excess of residual cytoplasm may contain higher amounts of the metabolites responsible for luminol signal, therefore not directly related to ROS production.
