The Chemical Principles Underpinning Chemiluminescence

The two major probes that have been used to assess ROS generation by human spermatozoa are luminol (5 amino-2,3-dihydro-1,4-phthalazinedione) and lucigenin (10,10'-dimethyl-9,9'-biacridinium dinitrate). Because lucigenin carries a positive ionic charge, it is generally thought to be membrane-impermeant and to respond to ROS, particularly superoxide (O2-0 anion in the extracellular space. In contrast, the uncharged luminol is thought to be relatively membrane-permeant and to react with a variety of ROS including O2-\ H2O2 . and OH* . both in the intra- and extracellular spaces. However, the sensitivity of this probe towards extracellular H2O2 can be greatly accentuated by the addition of horseradish peroxidase [31, 32]. While these probes are extremely sensitive and convenient for diagnostic purposes, the data they generate have to be interpreted with care and should be underpinned by knowledge of the chemical principles responsible for their reactivity. As emphasized above, a variety of confounding factors can influence the chemiluminescence responses observed with these probes in addition to the inevitable presence of contaminating leukocytes including incubation time, medium composition, pH, presence of seminal plasma, and albumin source, all of which can have a profound impact on the signals obtained [29],

14.2.2.1 Luminol

It has been presumed that both H2O2 and O.- are involved in luminol-dependent chemiluminescence because both catalase and superoxide dismutase (SOD) can disrupt the signal with great efficiency (Fig. 14.4a-c). The luminol signal generated by human spermatozoa is initiated by a one-electron oxidative event mediated by H2O2 and either endogenous peroxidase [32-34] or, in order to sensitize the assay for extracellular H2O2, by the addition of horse radish peroxidase to the medium [32, 35]. The one-electron oxidation of luminol leads to the creation of a radical species (L2). The latter interacts with ground state oxygen to produce O2- that then participates in the oxygenation of L2 to create an unstable endoperoxide, which breaks down with the release of light (Fig. 14.4a). According to this scheme, O.- is an essential intermediate in the manifestation of luminol-dependent chemilumines-cence and it is for this reason that SOD is such an effective inhibitor of this probe. However, the biochemical activity of SOD should never be taken to indicate the primary production of O2- by human spermatozoa, as sometimes suggested; O2- is simply an artificially created intermediate that is essential for luminol-dependent chemiluminescence [32]. Indeed, any univalent oxidant has the potential to generate O2-\ and hence chemiluminescence, in the presence of luminol, including ferricya-nide, persulfate, hypochlorite, ONOO-, and xanthine oxidase (Fig. 14.4a).

H2O2 lies upstream of O2- in the reaction scheme depicted in Fig. 14.4a and its involvement in the initial oxidation of luminol partly accounts for the inhibitory effects of catalase (Fig. 14.4b). In addition, H2O2 will also react directly with the azaquinone (L+) and thereby contribute to the formation of excited aminophthalic acid, the chemiluminescent species [36] . In some species (rat and mouse but not human), secondary radical species are created in the presence spermatozoa and luminol/peroxidase that generate very intense chemiluminescent signals over prolonged periods of time. These responses may reflect the nonenzymatic generation of NO and ONOO as a consequence of H2O2-mediated attacks on arginine [37].

One of the most important points to emphasize about Fig. 14.4 is the opportunity this chemiluminescence detection system presents for redox cycling. All that is needed is a source of H2 O2 (or alternative oxidizing species) to initiate the one-electron oxidation of luminol, and an azaquinone reductase, such as diaphorase, to reduce L+ back to the parent luminol (L). The remaining elements of the chemiluminescent cascade can be generated by the detection system itself (O2- by the interaction of L* with ground state oxygen, H.O2 by the SOD-induced dismutation of O2-\ L+ by the dismutation of L*).

Fig. 14.4 Luminol-dependent chemiluminescence. (a) Schematic representation of the underlying chemistry; L=luminol; L'=a luminol radical created by the one-electron oxidation of L. L+ = azaquinone formed by the further one-electron oxidation of L^ by oxygen, generating O2- as a by-product. The reaction of L' with O2- or L+ with H2O2 generates an unstable endoperoxide, the decomposition of which leads to production of the chemiluminescence species, an electronically excited aminophthalate. Redox cycling of the probe could result if human spermatozoa possessed an appropriate reductase to convert L+ back to the parent L. Any reactant that can achieve the univalent oxidation of luminol will generate chemiluminescence in this assay including H2O ^peroxidase and ONOO-. (b) PMA-induced chemiluminescence quenched by catalase. (c) PMA-induced chemiluminescence quenched by SOD. Reproduced from Aitken et al. [29], with kind permission from American Society of Andrology

Fig. 14.4 Luminol-dependent chemiluminescence. (a) Schematic representation of the underlying chemistry; L=luminol; L'=a luminol radical created by the one-electron oxidation of L. L+ = azaquinone formed by the further one-electron oxidation of L^ by oxygen, generating O2- as a by-product. The reaction of L' with O2- or L+ with H2O2 generates an unstable endoperoxide, the decomposition of which leads to production of the chemiluminescence species, an electronically excited aminophthalate. Redox cycling of the probe could result if human spermatozoa possessed an appropriate reductase to convert L+ back to the parent L. Any reactant that can achieve the univalent oxidation of luminol will generate chemiluminescence in this assay including H2O ^peroxidase and ONOO-. (b) PMA-induced chemiluminescence quenched by catalase. (c) PMA-induced chemiluminescence quenched by SOD. Reproduced from Aitken et al. [29], with kind permission from American Society of Andrology

While the scheme presented in Fig. 14.4a is a major simplification of the chemistry involved in luminol-dependent chemiluminescence, from a diagnostic andrology point of view there are several points to emerge from this analysis that should be emphasized: (1) the redox cycling activity characteristic of this probe will lead to a significant amplification of the signal and may explain why alternative methods for measuring H2 O2 have failed to detect this oxidant in purified suspensions of human spermatozoa, even though such activity has been clearly established in other species; (2) the complexity of this redox chemistry is such that we cannot state with certainty what is being measured with the luminol assay, it is certainly not just H+O2 [29]; (3) in particular, luminol assays may be measuring redox activity characterized by the cellular generation of oxidizing species capable of creating U; (4) notwithstanding the reservations that might be expressed concerning the specificity of this probe, the luminol assay is robust and generates results that are highly correlated with sperm function (see below).

14.2.2.2 Lucigenin

This probe is thought to be sensitive to the cellular generation of O2- largely because of the ability of SOD to suppress lucigenin-dependent cellular signals [32]. However, the reservations that apply to the use of luminol to detect specific ROS also apply to lucigenin. In the case of lucigenin, activation of the probe requires a one-electron reduction, rather than the one-electron oxidation associated with luminol-dependent chemiluminescence [33]. This one-electron reduction creates a radical (LucH+0 from lucigenin (Luc2+) that rapidly gives up its electron to ground state oxygen to create O2- and return the lucigenin to its parent state (Fig. 14.5). The LucH+^ generated from the one-electron reduction of lucigenin then combines with O2- to produce the dioxetane (Fig. 14.5), which in turn decomposes with the generation of light (chemiluminescence). The O2- involved in the last reaction could come from an independent cellular source, such as an NADPH oxidase, in which case the chemiluminescence recorded would reflect the generation of O2-\ as originally proposed for both leukocytes [38] and spermatozoa [32, 39] . However, an unknown proportion of the O2- involved in this reaction is an artifact created by the reaction between LucH+^ and ground state oxygen. Chemiluminescence created by the cellular generation of O2- or the redox cycling of lucigenin cannot be readily distinguished, since both sources of ROS are suppressible by SOD. When an agonist such as PMA (12-myristate, 13-acetate phorbol ester) is used to stimulate chemilumines-cence through the activation of protein kinase C, then it is probable that the cellular production of O2- is being measured (Fig. 14.5b). However, when chemiluminescence is generated by the addition of NAD(P)H, then the system may also be detecting the presence of any oxidoreductase capable of effecting the one-electron reduction of lucigenin [40]. In the case of spermatozoa, we have clearly demonstrated that lucigenin chemiluminescence in the presence of NADH or NADPH does not represent O2- production, but rather the respective abilities of cytochrome b5 reductase and cytochrome P450 reductase to reduce Luc2+ to LucH+^ and artificially trigger a redox cycle that generates O2- as a by-product [41, 42].

Fig. 14.5 Lucigenin-dependent chemiluminescence. (a) Schematic representation of the underlying chemistry; Luc2+ = lucigenin; LH+" = a lucigenin radical created by the one-electron reduction of Luc2+. The reaction of LH+" with oxygen generates O2-\ The latter then participates in an oxygenation reaction with LH+ generating a dioxetane that decomposes with the generation of chemiluminescence. Any entity that can affect the one-electron reduction of lucigenin will, in the presence of oxygen, create a redox cycle that produces high levels of and chemiluminescence. It is impossible to distinguish the relative contribution of such probe-dependent and cell-dependent chemiluminescence. (b) PMA-induced signal is likely to involve the primary cellular production of O2-\ (c) In contrast, the chemiluminescence generated by the addition of exogenous NADH (or NADPH) is likely to involve secondary O2" production following the univalent reduction of the probe by NAD(P)H-dependent oxidoreductases. Reproduced from Aitken et al. [29], with kind permission from American Society of Andrology

Fig. 14.5 Lucigenin-dependent chemiluminescence. (a) Schematic representation of the underlying chemistry; Luc2+ = lucigenin; LH+" = a lucigenin radical created by the one-electron reduction of Luc2+. The reaction of LH+" with oxygen generates O2-\ The latter then participates in an oxygenation reaction with LH+ generating a dioxetane that decomposes with the generation of chemiluminescence. Any entity that can affect the one-electron reduction of lucigenin will, in the presence of oxygen, create a redox cycle that produces high levels of and chemiluminescence. It is impossible to distinguish the relative contribution of such probe-dependent and cell-dependent chemiluminescence. (b) PMA-induced signal is likely to involve the primary cellular production of O2-\ (c) In contrast, the chemiluminescence generated by the addition of exogenous NADH (or NADPH) is likely to involve secondary O2" production following the univalent reduction of the probe by NAD(P)H-dependent oxidoreductases. Reproduced from Aitken et al. [29], with kind permission from American Society of Andrology

Notwithstanding the deficiencies in lucigenin as a probe for evaluating O2- production, it does have value as a nonspecific redox marker for the enhanced electron transfer activity associated with defective sperm function [43]. In many ways, the sensitivity and diagnostic value of the probe is enhanced by its redox cycling activity, rather than diminished. If specific quantification of O2- production is required, then there are alternative chemiluminescent probes that do not redox cycle including the Cypridina luciferin analog, MCLA [44], and coelenterazine [45].

Pregnancy Guide

Pregnancy Guide

A Beginner's Guide to Healthy Pregnancy. If you suspect, or know, that you are pregnant, we ho pe you have already visited your doctor. Presuming that you have confirmed your suspicions and that this is your first child, or that you wish to take better care of yourself d uring pregnancy than you did during your other pregnancies; you have come to the right place.

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