**5. Microbicidal combustion and chemiluminescence**

Reactions of 1 O2\* with singlet multiplicity substrates (1 Sub) are spin-allowed and highly exergonic. The exergonicities of most biochemical reactions are sufficient for rotational and vibrational excitation, but not electronic excitation. Dioxygenation reactions are sufficiently exergonic for electronic excitation. Oxygenations producing singlet multiplicity endoperoxide and dioxetane intermediates are excellent candidates for luminescence [51]. The disintegrations of such intermediates generate **nπ\*** electronically excited products, that is, an electron from the nonbonding (**n**) orbital of oxygen populates the pi antibonding (**π\***) orbital of the carbonyl. Singlet multiplicity **nπ\*** excited molecules have short lifetimes. Electronic transition from the **π\*** of the carbonyl to the **n** of oxygen with photon emission is spin-allowed.

In addition to direct reaction of 1 O2\* with 1 Sub, other singlet multiplicity reactants such as 1 OCl<sup>−</sup> can react with <sup>1</sup> Sub to yield chloramine products (1 Sub-Cl) or dehydrogenated products (1 Sub-2RE). Such products can in turn react with <sup>1</sup> H2O2 yielding endoperoxide or dioxetane intermediates with subsequent disintegration to **nπ\***-excited carbonyl products relaxing by photon emission [52, 53]. The fundamental principle is that all reactants and products are singlet multiplicity nonradicals.

Dioxygenations yielding intermediate endoperoxide and dioxetanes disintegrate yielding an **nπ\*** electronically excited carbonyl. **Figure 7** illustrates the energy and orbital differences that characterize the carbonyl states. Physical generation of a **nπ\*** electronically excited carbonyl occurs when a fluorescent compound in its ground state absorbs a photon of appropriate energy. Because the ground state of the carbonyl is singlet, an electronically excited singlet multiplicity carbonyl undergoes rapid spin-allowed relaxation to ground state with a lifetime of less than 10<sup>−</sup><sup>8</sup> second [51]. Fluorescence describes photon-generated excitation followed by photon emission. Chemiluminescence or luminescence describes chemically generated electronic excitation followed by photon emission.

The metabolic changes of the respiratory burst describe the movement of RE required to change the spin multiplicity of <sup>3</sup> O2 from triplet to doublet (2 HO2), and ultimately to singlet, that is, <sup>1</sup> H2O2 and 1 O2\*. MPO catalyzes the 2RE oxidation of 1 Cl<sup>−</sup> to 1 HOCl followed by chemical reaction with a second <sup>1</sup> H2O2 to generate 1 O2\*. Changing the bi-fermionic 3 O2 to bosonic <sup>1</sup> O2\*eliminates the spin barrier to direct dioxygenation of bosonic singlet multiplicity biological molecules. If intermediate endoperoxides and dioxetanes are generated, their disintegration yields electronically excited **nπ\*** carbonyl functions that relax by photon emission. By changing the spin multiplicity of oxygen, neutrophil leukocytes realize its electronegative potential for combustive microbicidal action. Such combustion generates electronically excited products emitting light in the visible range of the spectrum.

**73**

globulins [56].

**Figure 7.**

neutrophil activity measured.

*Essence of Reducing Equivalent Transfer Powering Neutrophil Oxidative Microbicidal Action…*

The native chemiluminescence of neutrophils is proportional to respiratory burst activity [4, 54]. Since the luminescence resulting from microbicidal combustion is proportional to dioxygenations, especially those yielding endoperoxide and dioxetane intermediates, it follows that native neutrophil luminescence is influenced by the molecular composition of the microbe combusted. Native luminescence from phagocytosing neutrophils can be detected using less than a million neutrophils. For perspective, a milliliter of normal human blood contains about 4 million neutrophils. The native luminescence product of neutrophil combustive action is of low intensity. However, electronic excitation and the resultant luminescence is unambiguous evidence of neutrophil combustive dioxygenation action. Native luminescence has been usefully applied to measurement of neutrophil metabolic defects, e.g., chronic granulomatous disease [54, 55], and neutrophil responsiveness to humoral immune factors, such as complement and immuno-

*Orbital diagram plot depicting the* **nπ\*** *electronically excited singlet state and the singlet ground state of a carbonyl. The gray dashed brackets indicate the carbonyl with the participating carbon and oxygen atoms shown on to the left and right, respectively. In the carbonyl diagram on the left, the* **nπ\*** *notation indicates that an electron of the nonbonding (***n***) orbital of the carbonyl oxygen atom has been excited to the pi antibonding (***π\****) orbital of the carbonyl. Although excited, the electrons remained paired and the excited state is singlet* 

Inclusion of high quantum yield chemiluminigenic substrates as probes (CLP)

Phagocytic or chemical activation of neutrophil respiratory burst metabolism can be tested using the dye nitro-blue tetrazolium (NBT) [59]. The NBT reaction measures neutrophil reduction activity, not neutrophil oxidation activity. A positive NBT result requires neutrophil respiratory burst activity resulting in reduction of

of neutrophil dioxygenation activities greatly increases the sensitivity and, to some degree, the specificity for detecting such activities [52, 57, 58]. With regard to increasing sensitivity, a CLP must be susceptible to neutrophil dioxygenation activities. This is achieved when endoperoxide or dioxetane intermediate are produced. The breakdown of such intermediates yields electronically excited **nπ\*** carbonyl functions that relax by light emission. Use of a CLP typically increases the sensitivity for detecting dioxygenation activity by several orders of magnitude. Selecting a CLP with reactive specificity also provides information with regard to the nature of

**6.1 Probing reductive oxygenation activity with lucigenin**

*DOI: http://dx.doi.org/10.5772/intechopen.81543*

**6. Chemiluminigenic probes**

*multiplicity. Electron relaxation from* **π\****-to-***n** *yields photon emission.*

*Essence of Reducing Equivalent Transfer Powering Neutrophil Oxidative Microbicidal Action… DOI: http://dx.doi.org/10.5772/intechopen.81543*

**Figure 7.**

*Neutrophils*

Reactions of 1

tants such as 1

nonradicals.

10<sup>−</sup><sup>8</sup>

1 Cl<sup>−</sup> to 1

In addition to direct reaction of 1

dehydrogenated products (1

OCl<sup>−</sup> can react with <sup>1</sup>

ated electronic excitation followed by photon emission.

required to change the spin multiplicity of <sup>3</sup>

ultimately to singlet, that is, <sup>1</sup>

Changing the bi-fermionic 3

Specificity of MPO binding results in specificity of microbicidal action. Binding specificity allows synergistic MPO-LAB interaction and suppression of pathogens. It also suggests a role for MPO in the selection and maintenance of LAB in the normal flora [48]. Healthy human adults release about a hundred billion MPO-rich neutrophils into the circulating blood each day. The circulating lifetime of the neutrophil is reportedly less than a day. The neutrophils then leave the blood and enter a tissue and body cavity phase lasting a few days [36]. Migration of MPO-rich neutrophils into the mouth and vagina is well-known [49, 50]. When quantified, the neutrophil count of the mouth is proportional to the blood neutrophil count. These spaces typically provide an acidic milieu. Neutrophil disintegration with MPO release may provide LAB with a selective advantage in such body spaces.

**5. Microbicidal combustion and chemiluminescence**

O2\* with singlet multiplicity substrates (1

highly exergonic. The exergonicities of most biochemical reactions are sufficient for rotational and vibrational excitation, but not electronic excitation. Dioxygenation reactions are sufficiently exergonic for electronic excitation. Oxygenations producing singlet multiplicity endoperoxide and dioxetane intermediates are excellent candidates for luminescence [51]. The disintegrations of such intermediates generate **nπ\*** electronically excited products, that is, an electron from the nonbonding (**n**) orbital of oxygen populates the pi antibonding (**π\***) orbital of the carbonyl. Singlet multiplicity **nπ\*** excited molecules have short lifetimes. Electronic transition from the **π\*** of the carbonyl to the **n** of oxygen with photon emission is spin-allowed.

O2\* with 1

yielding endoperoxide or dioxetane intermediates with subsequent disintegration to **nπ\***-excited carbonyl products relaxing by photon emission [52, 53]. The fundamental principle is that all reactants and products are singlet multiplicity

yielding an **nπ\*** electronically excited carbonyl. **Figure 7** illustrates the energy and orbital differences that characterize the carbonyl states. Physical generation of a **nπ\*** electronically excited carbonyl occurs when a fluorescent compound in its ground state absorbs a photon of appropriate energy. Because the ground state of the carbonyl is singlet, an electronically excited singlet multiplicity carbonyl undergoes rapid spin-allowed relaxation to ground state with a lifetime of less than

Dioxygenations yielding intermediate endoperoxide and dioxetanes disintegrate

 second [51]. Fluorescence describes photon-generated excitation followed by photon emission. Chemiluminescence or luminescence describes chemically gener-

The metabolic changes of the respiratory burst describe the movement of RE

dioxygenation of bosonic singlet multiplicity biological molecules. If intermediate endoperoxides and dioxetanes are generated, their disintegration yields electronically excited **nπ\*** carbonyl functions that relax by photon emission. By changing the spin multiplicity of oxygen, neutrophil leukocytes realize its electronegative potential for combustive microbicidal action. Such combustion generates electroni-

H2O2 and 1

O2 to bosonic <sup>1</sup>

cally excited products emitting light in the visible range of the spectrum.

HOCl followed by chemical reaction with a second 1

Sub) are spin-allowed and

Sub-Cl) or

H2O2

HO2), and

O2\*.

Sub, other singlet multiplicity reac-

Sub to yield chloramine products (1

O2 from triplet to doublet (2

O2\*. MPO catalyzes the 2RE oxidation of

O2\*eliminates the spin barrier to direct

H2O2 to generate 1

Sub-2RE). Such products can in turn react with <sup>1</sup>

**72**

*Orbital diagram plot depicting the* **nπ\*** *electronically excited singlet state and the singlet ground state of a carbonyl. The gray dashed brackets indicate the carbonyl with the participating carbon and oxygen atoms shown on to the left and right, respectively. In the carbonyl diagram on the left, the* **nπ\*** *notation indicates that an electron of the nonbonding (***n***) orbital of the carbonyl oxygen atom has been excited to the pi antibonding (***π\****) orbital of the carbonyl. Although excited, the electrons remained paired and the excited state is singlet multiplicity. Electron relaxation from* **π\****-to-***n** *yields photon emission.*

## **6. Chemiluminigenic probes**

The native chemiluminescence of neutrophils is proportional to respiratory burst activity [4, 54]. Since the luminescence resulting from microbicidal combustion is proportional to dioxygenations, especially those yielding endoperoxide and dioxetane intermediates, it follows that native neutrophil luminescence is influenced by the molecular composition of the microbe combusted. Native luminescence from phagocytosing neutrophils can be detected using less than a million neutrophils. For perspective, a milliliter of normal human blood contains about 4 million neutrophils. The native luminescence product of neutrophil combustive action is of low intensity. However, electronic excitation and the resultant luminescence is unambiguous evidence of neutrophil combustive dioxygenation action. Native luminescence has been usefully applied to measurement of neutrophil metabolic defects, e.g., chronic granulomatous disease [54, 55], and neutrophil responsiveness to humoral immune factors, such as complement and immunoglobulins [56].

Inclusion of high quantum yield chemiluminigenic substrates as probes (CLP) of neutrophil dioxygenation activities greatly increases the sensitivity and, to some degree, the specificity for detecting such activities [52, 57, 58]. With regard to increasing sensitivity, a CLP must be susceptible to neutrophil dioxygenation activities. This is achieved when endoperoxide or dioxetane intermediate are produced. The breakdown of such intermediates yields electronically excited **nπ\*** carbonyl functions that relax by light emission. Use of a CLP typically increases the sensitivity for detecting dioxygenation activity by several orders of magnitude. Selecting a CLP with reactive specificity also provides information with regard to the nature of neutrophil activity measured.

#### **6.1 Probing reductive oxygenation activity with lucigenin**

Phagocytic or chemical activation of neutrophil respiratory burst metabolism can be tested using the dye nitro-blue tetrazolium (NBT) [59]. The NBT reaction measures neutrophil reduction activity, not neutrophil oxidation activity. A positive NBT result requires neutrophil respiratory burst activity resulting in reduction of

the tetrazolium ring of the dye to a dark blue water-insoluble formazan precipitate. NBT is a large complex nitrogen heterocyclic compound with abundant resonance and electron delocalization possibilities. That NBT reduction might be linked to neutrophil univalent reduction of molecular oxygen was considered, and we observed that adding a small grain of potassium superoxide (KO2) to a solution of NBT resulted in immediate reduction of the dye to a dark blue formazan precipitate [15]. Normal neutrophils reduce NBT upon activation of NADPH oxidase. The neutrophils of chronic granulomatous disease patients have defective NADPH oxidase, and as such, are incapable of NBT reduction [60].

Lucigenin (aka, bis-*N*-methylacridinium nitrate, or dimethyl biacridinium nitrate (1 DBA+2)) is a heterocyclic organic compound known to generate chemiluminescence as a product of base-catalyzed peroxidation [61]. If sufficiently alkaline, singlet multiplicity <sup>1</sup> lucigenin reacts with the conjugate base of peroxide (1 HO2 <sup>−</sup>) producing a dioxetane (1 lucigenin-dioxetane) intermediate that disintegrates to a **nπ\***-excited carbonyl function that relaxes to ground state by **π\***-to-**n** transition with photon emission. The p*K*a of <sup>1</sup> H2O2 is 11.7. As previously considered, <sup>1</sup> H2O2 is the sum product of two RE reductions of <sup>3</sup> O2. Consequently, lucigenin chemiluminescence is the product of reductive dioxygenation. Both lucigenin and peroxide are singlet multiplicity reactants. Spin restriction is not a problem. Alkalinity favors the formation of <sup>1</sup> HO2 <sup>−</sup> and dioxygenation yielding a dioxetane.

Lucigenin is a heterocyclic compound with resonance and electron delocalization possibilities, and can undergo one RE reduction yielding a doublet multiplicity product (2 lucigenin+RE+ ). Such reduction may involve 2 O2 <sup>−</sup> or some other 1RE reductant. The product radical, <sup>2</sup> lucigenin+RE+ , can now react with 2 O2 <sup>−</sup> by SOMO-SOMO overlap, that is, a doublet-doublet annihilation, producing a singlet multiplicity product, the <sup>1</sup> lucigenin-dioxetane intermediate. As depicted in **Figure 8**, the disintegration of this unstable dioxetane yields chemiluminescence [52, 58, 62, 63].

Reduction of lucigenin by 2RE, that is, by a bosonic orbital electron couple, maintains singlet multiplicity. Such a reduced 1 lucigenin+2RE can react with <sup>1</sup> O2\*, but not 3 O2, to produce chemiluminescence [64]. As shown in **Figure 8**, the state of lucigenin reduction determines the deoxygenating agent required. All reactions shown satisfy the spin conservation rules.

The radical product of 1RE reduction of lucigenin, <sup>2</sup> lucigenin+RE+ , can react with the radical product of NADPH oxidase, <sup>2</sup> O2 <sup>−</sup>, resulting in intermediate dioxetane formation with breakdown to a **nπ\*** electronically excited carbonyl with relaxation by light emission, and as such, lucigenin can be applied as a chemiluminigenic probe for measurement of NADPH oxidase activity [52, 58, 63]. MPO haloperoxidase activity does not yield lucigenin-luminescence.

Chicken blood phagocytes, that is, heterophil leukocytes, have oxidase activity, but are deficient in haloperoxidase. Chemical or phagocytic stimulation of these heterophil leukocytes results in lucigenin-dependent luminescence responses comparable to those observed from human neutrophils under similar test conditions and using similar stimuli [58, 65]. However, the luminol-dependent luminescence responses of MPO-deficient chicken heterophils are a hundredfold lower than those observed from MPO-rich human neutrophils. In addition, azide (N3 <sup>−</sup>), a known inhibitor of MPO, inhibits the luminol-dependent luminescence responses of MPOrich human neutrophil. Azide shows no inhibitory action against the luminol or the lucigenin luminescence responses of MPO-deficient chicken heterophils [66]. These chicken heterophil results plus the previously described macrophage results [57] experimentally support the position that luminol provides a very sensitive measure of MPO activity. However, the weaker luminol-luminescence measured is evidence for haloperoxidase-independent oxidase activity.

**75**

*Essence of Reducing Equivalent Transfer Powering Neutrophil Oxidative Microbicidal Action…*

*DOI: http://dx.doi.org/10.5772/intechopen.81543*

**6.2 Probing oxygenation activities with cyclic hydrazides**

*that precedes the reactant, and 1RE indicates one reducing equivalent.*

O2—X→ <sup>1</sup>

N2, and ultimately, ground state <sup>1</sup>

O2 <sup>∗</sup> → <sup>1</sup>

Lucigenin-luminescence is a reductive dioxygenation.

H2O2 → 2<sup>1</sup>

luminol with <sup>3</sup>

Like lucigenin, alkalinity and 1

luminol + <sup>3</sup>

luminol + <sup>1</sup>

lucigenin + <sup>1</sup>

addition of molecular oxygen plus 2RE, that is, <sup>1</sup>

dioxygenation:

thalate\* plus 1

with <sup>1</sup>

**Figure 8.**

<sup>1</sup>

The reaction of <sup>1</sup>

<sup>1</sup>

<sup>1</sup>

Luminol chemiluminescence is a well-established phenomenon, but the mechanisms responsible for luminol-luminescence are diverse [67]. Luminol (5-amino-2,3-dihydrophthalazine-1,4-dione) is a nonradical, cyclic hydrazide [68]. Luminol dioxygenation is thought to involve an intermediate endoperoxide with disintegration yielding the **nπ\*** electronically excited aminophthalate that relaxes by photon emission. Albrecht first described the blood-catalyzed luminol-luminescence [69].

*Oxygenating reactions yielding lucigenin chemiluminescence. Spin multiplicity is shown by the superscript value* 

additional requirement for a catalyst, for example, blood or peroxidase. To appreciate how these CLS differ, compare, and contrast the net reactions responsible for luminol-luminescence and lucigenin-luminescence. Luminol-luminescence is a

O2\* (Eq. (15)) is spin allowed producing **nπ\*** electronically excited 1

As per Eq. (16), lucigenin-luminescence requires the spin-allowed reactive

aminophthalate + <sup>1</sup>

aminophthalate + <sup>1</sup>

H2O2 are required, but luminol-luminescence has an

O2 (Eq. (14)) is not spin allowed, but reaction

aminophthalate plus a photon.

N−methylacrodone + Photon (16)

N2 + Photon (14)

N2 + Photon (15)

H2O2. The product of this reductive

aminoph-

*Essence of Reducing Equivalent Transfer Powering Neutrophil Oxidative Microbicidal Action… DOI: http://dx.doi.org/10.5772/intechopen.81543*

**Figure 8.**

*Neutrophils*

nitrate (1

oxide (1

considered, <sup>1</sup>

dioxetane.

ity product (2

not 3

plicity product, the <sup>1</sup>

alkaline, singlet multiplicity <sup>1</sup>

HO2

the tetrazolium ring of the dye to a dark blue water-insoluble formazan precipitate. NBT is a large complex nitrogen heterocyclic compound with abundant resonance and electron delocalization possibilities. That NBT reduction might be linked to neutrophil univalent reduction of molecular oxygen was considered, and we observed that adding a small grain of potassium superoxide (KO2) to a solution of NBT resulted in immediate reduction of the dye to a dark blue formazan precipitate [15]. Normal neutrophils reduce NBT upon activation of NADPH oxidase. The neutrophils of chronic granulomatous disease patients have defective NADPH oxidase,

Lucigenin (aka, bis-*N*-methylacridinium nitrate, or dimethyl biacridinium

luminescence as a product of base-catalyzed peroxidation [61]. If sufficiently

disintegrates to a **nπ\***-excited carbonyl function that relaxes to ground state by

H2O2 is the sum product of two RE reductions of <sup>3</sup>

lucigenin chemiluminescence is the product of reductive dioxygenation. Both lucigenin and peroxide are singlet multiplicity reactants. Spin restriction is not a

Lucigenin is a heterocyclic compound with resonance and electron delocalization possibilities, and can undergo one RE reduction yielding a doublet multiplic-

). Such reduction may involve 2

lucigenin+RE+

SOMO overlap, that is, a doublet-doublet annihilation, producing a singlet multi-

disintegration of this unstable dioxetane yields chemiluminescence [52, 58, 62, 63]. Reduction of lucigenin by 2RE, that is, by a bosonic orbital electron couple,

O2

formation with breakdown to a **nπ\*** electronically excited carbonyl with relaxation by light emission, and as such, lucigenin can be applied as a chemiluminigenic probe for measurement of NADPH oxidase activity [52, 58, 63]. MPO haloperoxi-

Chicken blood phagocytes, that is, heterophil leukocytes, have oxidase activity, but are deficient in haloperoxidase. Chemical or phagocytic stimulation of these heterophil leukocytes results in lucigenin-dependent luminescence responses comparable to those observed from human neutrophils under similar test conditions and using similar stimuli [58, 65]. However, the luminol-dependent luminescence responses of MPO-deficient chicken heterophils are a hundredfold lower than those

inhibitor of MPO, inhibits the luminol-dependent luminescence responses of MPOrich human neutrophil. Azide shows no inhibitory action against the luminol or the lucigenin luminescence responses of MPO-deficient chicken heterophils [66]. These chicken heterophil results plus the previously described macrophage results [57] experimentally support the position that luminol provides a very sensitive measure of MPO activity. However, the weaker luminol-luminescence measured is evidence

O2, to produce chemiluminescence [64]. As shown in **Figure 8**, the state of lucigenin reduction determines the deoxygenating agent required. All reactions shown

DBA+2)) is a heterocyclic organic compound known to generate chemi-

HO2

lucigenin reacts with the conjugate base of per-

lucigenin-dioxetane) intermediate that

O2

lucigenin+2RE can react with <sup>1</sup>

lucigenin+RE+

<sup>−</sup>, resulting in intermediate dioxetane

, can now react with 2

lucigenin-dioxetane intermediate. As depicted in **Figure 8**, the

H2O2 is 11.7. As previously

<sup>−</sup> or some other 1RE

O2

<sup>−</sup> and dioxygenation yielding a

O2. Consequently,

<sup>−</sup> by SOMO-

, can react with

<sup>−</sup>), a known

O2\*, but

and as such, are incapable of NBT reduction [60].

<sup>−</sup>) producing a dioxetane (1

problem. Alkalinity favors the formation of <sup>1</sup>

maintains singlet multiplicity. Such a reduced 1

The radical product of 1RE reduction of lucigenin, <sup>2</sup>

observed from MPO-rich human neutrophils. In addition, azide (N3

dase activity does not yield lucigenin-luminescence.

for haloperoxidase-independent oxidase activity.

lucigenin+RE+

reductant. The product radical, <sup>2</sup>

satisfy the spin conservation rules.

the radical product of NADPH oxidase, <sup>2</sup>

**π\***-to-**n** transition with photon emission. The p*K*a of <sup>1</sup>

**74**

*Oxygenating reactions yielding lucigenin chemiluminescence. Spin multiplicity is shown by the superscript value that precedes the reactant, and 1RE indicates one reducing equivalent.*

### **6.2 Probing oxygenation activities with cyclic hydrazides**

Luminol chemiluminescence is a well-established phenomenon, but the mechanisms responsible for luminol-luminescence are diverse [67]. Luminol (5-amino-2,3-dihydrophthalazine-1,4-dione) is a nonradical, cyclic hydrazide [68]. Luminol dioxygenation is thought to involve an intermediate endoperoxide with disintegration yielding the **nπ\*** electronically excited aminophthalate that relaxes by photon emission. Albrecht first described the blood-catalyzed luminol-luminescence [69]. Like lucigenin, alkalinity and <sup>1</sup> H2O2 are required, but luminol-luminescence has an additional requirement for a catalyst, for example, blood or peroxidase. To appreciate how these CLS differ, compare, and contrast the net reactions responsible for luminol-luminescence and lucigenin-luminescence. Luminol-luminescence is a dioxygenation:

 <sup>1</sup> luminol + <sup>3</sup> O2—X→ <sup>1</sup> aminophthalate + <sup>1</sup> N2 + Photon (14)

The reaction of <sup>1</sup> luminol with <sup>3</sup> O2 (Eq. (14)) is not spin allowed, but reaction with <sup>1</sup> O2\* (Eq. (15)) is spin allowed producing **nπ\*** electronically excited <sup>1</sup> aminophthalate\* plus 1 N2, and ultimately, ground state <sup>1</sup> aminophthalate plus a photon.

$$\text{\textbullet \text{\textbullet l}} \text{\textbullet \text{\textbullet l}} \text{\textbullet \text{\textbullet l}} \text{\textbullet \text{\textbullet l}} \text{\textbullet \text{\textbullet l}} \text{\textbullet \text{\textbullet l}} \text{\textbullet \text{\textbullet l}} \text{\textbullet \text{\textbullet l}} \text{\textbullet \text{\textbullet l}} \text{\textbullet \text{\textbullet l}} \text{\textbullet \text{\textbullet l}} \text{\textbullet \textbullet \textbullet \textbullet l}$$

Lucigenin-luminescence is a reductive dioxygenation.

$$\text{\textbullet \text{\textquotedblleft}lucigennin}\text{\textquotedblright}\text{H}\_2\text{O}\_2 \rightarrow \text{\textquotedblleft N-methylacrodone}\text{\textquotedblright}\text{\textquotedblleft}\text{Photon}\tag{16}$$

As per Eq. (16), lucigenin-luminescence requires the spin-allowed reactive addition of molecular oxygen plus 2RE, that is, <sup>1</sup> H2O2. The product of this reductive dioxygenation is a dioxetane intermediate that breaks down to one ground state 1 N-methylacridone and one **nπ\***electronically excited <sup>1</sup> N-methylacridone\*. Relaxation of the <sup>1</sup> N-methylacridone\* yields a photon.

Luminol dioxygenation is not reductive. The net dioxygenation incorporates molecular oxygen to produce an endoperoxide intermediate with the breakdown release of 1 N2 and formation of a **nπ\***electronically excited aminophthalate. As indicated by Eq. (14), 1 luminol does not react with ground state oxygen. Spin conservation and frontier orbital overlap problems restrict such direct reaction. As illustrated in **Figure 1**, the frontier orbitals of 3 O2 are its two degenerates **π\*** SOMOs. Hund's maximum multiplicity rule is satisfied when the electrons of each SOMO have the same spin. Each of the two **π\*** orbitals of <sup>3</sup> O2 have fermionic character that restricts overlap with the bosonic frontier orbitals of luminol. By contrast, the frontier **π\*** orbitals of 1 O2\* are bosonic and include one LUMO **π\*** orbital and one HOMO **π\*** orbital. Overlap of the LUMO of <sup>1</sup> O2\* with the HOMO of <sup>1</sup> luminol satisfies the symmetry requirements for reaction.

There are three mechanistic possibilities for 1 luminol reactions yielding luminescence. The fermionic (doublet multiplicity/radical) pathway requires two steps as illustrated by Eqs. (17) and (18).

$$\text{\textbullet \text{\textbullet l}}\text{\textbullet \text{\textbullet l}}\text{\textbullet \text{\textbullet l}}\text{\textbullet \text{\textbullet l}}\text{\textbullet \text{\textbullet l}}\text{\textbullet \text{\textbullet l}}\text{\textbullet \text{\textbullet l}}\text{\textbullet \text{\textbullet l}}\text{\textbullet \text{\textbullet l}}\text{\textbullet \text{\textbullet l}}\text{\textbullet \textbullet \textbullet \textbullet l}\text{\textbullet \textbullet l}$$

The radical 2 luminol-1RE can participate in SOMO-SOMO reaction with superoxide (2 O2 <sup>−</sup>) yielding singlet multiplicity electronically excited aminophthalate ( 1 aminophthalate\*) that relaxes with photon emission.

$$\text{\textbullet}^{2}\text{luminol}\_{\text{-}1\text{RE}}\text{+}^{2}\text{O}\_{2}\text{\textbullet}^{1}\text{\textbullet}^{1}\text{\textbullet}^{1}\text{\textbullet}^{1}\text{\textbullet}^{1}\text{N}\_{2}\text{+}^{1}\text{Photon}\tag{18}$$

The bosonic (singlet multiplicity/nonradical) pathway can occur by a single reaction as illustrated by Eq. (19),

$$\text{\textbullet \text{\textquotedblleft}l\text{\textquotedblright}}\text{\textquotedblright}\text{\textquotedblleft O}\_2\text{\textquotedblright}\rightarrow\text{\textquotedblleft \textquotedblright}\text{\textquotedblright}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblright}\text{\textquotedblleft}\text{\textquotedblright}\text{\textquotedblright}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblright}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblright}\text{\textquotedblright}\text{\textquotedblright}\text{\textquotedblright}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblright}\text{\textquotedblright}\text{\textquotedblright}\text{\textquotedblright}\text{\textquotedblright}\end{\text{\textquotedblleft}}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textqu0}\text{\textqu$$

The bosonic (singlet multiplicity/nonradical) pathway can also occur by a twostep reaction as illustrated by Eqs. (20) and (21).

 <sup>1</sup> luminol + <sup>1</sup> OCl<sup>−</sup> → <sup>1</sup> luminol−2RE + <sup>1</sup> Cl− (20)

 <sup>1</sup> luminol−2RE + <sup>1</sup> H2O2 → <sup>1</sup> aminophthalate + <sup>1</sup> N2 + Photon (21)

Although luminol is versatile with regard to reactive mechanism, dioxygenation is ultimately required for chemiluminescence. In an alkaline milieu, classical peroxidase or hemoglobin can catalyze <sup>1</sup> H2O2-dependent luminol-luminescence. The peroxidase-catalyzed mechanism of luminol-luminescence described by Dure and Cormier illustrates the kinetics of the fermionic pathway [70]. For such reaction, a classical peroxidase is first oxidized by 1 H2O2, that is, 2RE are transferred to <sup>1</sup> H2O2 producing two 1 H2O as described in Eq. (22).

$$\text{peroxide} \star \, ^1\text{H}\_2\text{O}\_2 \rightarrow \text{Cpx} \, \text{1}\_{\text{-2RE}} + 2^1\text{H}\_2\text{O} \tag{22}$$

**77**

*Essence of Reducing Equivalent Transfer Powering Neutrophil Oxidative Microbicidal Action…*

limiting with regard to luminescence, but this reaction is necessary for regeneration

allowed SOMO-SOMO reaction, that is, a doublet-doublet annihilation, yielding

luminol + Cpx 1−2RE → Cpx 2−RE + <sup>2</sup> luminol−1RE (23)

luminol + Cpx 2−RE → peroxidase + <sup>2</sup> luminol−1RE (24)

luminol (starting reactant) and nonradical 2RE-oxidized luminol

H2O2-dependent oxidation of peroxidase to Cpx 1-2RE allows it to cata-

luminol + <sup>1</sup>

Metalloenzymes and cytochromes are suited to 1RE transfers and under proper

lyze the initial fermionic 1RE oxidation of luminol in an alkaline milieu. Hemoglobin has peroxidase activity under alkaline conditions, thus explaining the sensitivity of luminol-luminescence for detecting the presence of blood erythrocytes by alkaline peroxide methods. Luminol-luminescence by the classical plant peroxidase-catalyzed reactions of Eqs. (22)–(25) is sensitive to pH, decreasing with increasing acidity. Acidification of the reaction milieu to a pH of about 5 ± 1 effectively eliminates classical peroxidase-catalyzed luminol luminescence. This is quantitatively demonstrated in the Michaelis-Menten enzyme kinetic analyses of luminol-luminescence

Alkaline pH favors the fermionic luminol-luminescence reactions catalyzed by

ricyanide-catalyzed luminol luminescence reaction is most efficient in the pH range from 10.4 to 10.8 [72]. In **Table 2**, note that no significant luminescence is observed from HRP-catalyzed luminol reaction at pH 4.9. The maximum luminescence velocity (*V*max) values are low and standard errors (SE) are high. However, a relatively weak but significant luminescence is observed at pH 7.0, that is, Michaelis-Menten

for myeloperoxidase and horse radish peroxidase presented in **Table 2** [71].

analysis of the HRP luminescence shows a low *V*max, but an acceptable SE. Of special note, Michaelis-Menten kinetic analysis indicates that the HRPcatalyzed luminol-luminescence velocity is first order with respect to H2O2 concentration, but second order with respect to luminol concentration, that is, the luminescence velocity is directly proportional to the square of the luminol concentration. These results are consistent with those reported by Dure and Cormier [70], and with the fermionic radical reactive pathway described in Eqs. (22)–(25) and Eq. (21). Although luminol solubility becomes a problem at low pH, acidity favors the bosonic haloperoxidase luminol-luminescence catalyzed by MPO. Note that bosonic, haloperoxidase-catalyzed luminol luminescence is first order with respect to luminol, chloride, or bromide, but second order with respect to H2O2, that is, luminescence activity is proportional to the square of the H2O2 concentration. The MPO-catalyzed luminol-luminescence kinetic finding is the opposite of those observed for HRP-catalyzed luminol-luminescence, and are consistent with

plant peroxidase, hemoglobin, and heavy metals. The pKa of 1

the bosonic reactive pathway for luminol-luminescence via <sup>1</sup>

luminol-1RE can proceed as a spin

luminol-2RE with 1

aminophthalate\*) that relaxes by photon

luminol is slow and rate

luminol−2RE (25)

substrate producing 2

H2O2 yields

H2O2 is 11.75. The fer-

O2\* reaction described

sub-

*DOI: http://dx.doi.org/10.5772/intechopen.81543*

of the starting peroxidase, as per Eq. (24).

Disproportionation of the two radical <sup>2</sup>

2<sup>2</sup> luminol−1RE → <sup>1</sup>

electronically excited aminophthalate (1

As per Eq. (21), the spin-allowed reaction of 1

reaction conditions can catalyze the 1RE oxidation of a 1

The reaction of complex 2 (Cpx 2-RE) with another 1

<sup>1</sup>

<sup>1</sup>

the nonradical <sup>1</sup>

luminol-2RE).

emission.

strate-1RE. The 1

( 1

This 2RE oxidized peroxidase, referred to as complex 1 (Cpx 1), can now readily oxidize <sup>1</sup> luminol by removing 1RE producing <sup>2</sup> luminol-1RE, as per Eq. (23).

*Essence of Reducing Equivalent Transfer Powering Neutrophil Oxidative Microbicidal Action… DOI: http://dx.doi.org/10.5772/intechopen.81543*

$$\text{ ${}^1$ }\text{luminol} \text{ + } \text{Cpx } \text{ ${}^1$ }\_{-2\text{RE}} \rightarrow \text{ ${}^1$ }\text{px } \text{ ${}^2$ }\_{-\text{RE}} \text{ + } \text{ ${}^2$ }\text{luminol}\_{-1\text{RE}}\tag{23}$$

The reaction of complex 2 (Cpx 2-RE) with another 1 luminol is slow and rate limiting with regard to luminescence, but this reaction is necessary for regeneration of the starting peroxidase, as per Eq. (24).

$$\text{\textquotedblleft lumimod + Cpx \,\Omega\text{\textquotedblright}\text{\textquotedblleft}\text{\textquotedblright}\text{\textquotedblleft}\text{\textquotedblright}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblright}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblright}\text{\textquotedblright}\text{\textquotedblright}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblright}\text{\textquotedblright}\text{\textquotedblright}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblright}}\text{\textquotedbl$$

Disproportionation of the two radical <sup>2</sup> luminol-1RE can proceed as a spin allowed SOMO-SOMO reaction, that is, a doublet-doublet annihilation, yielding the nonradical <sup>1</sup> luminol (starting reactant) and nonradical 2RE-oxidized luminol ( 1 luminol-2RE).

$$2^2 \text{luminol}\_{\text{-2RE}} \twoheadrightarrow {}^1 \text{luminol} \text{+} \text{ }^1 \text{luminol}\_{\text{-2RE}} \tag{25}$$

As per Eq. (21), the spin-allowed reaction of 1 luminol-2RE with 1 H2O2 yields electronically excited aminophthalate (1 aminophthalate\*) that relaxes by photon emission.

Metalloenzymes and cytochromes are suited to 1RE transfers and under proper reaction conditions can catalyze the 1RE oxidation of a 1 substrate producing 2 substrate-1RE. The 1 H2O2-dependent oxidation of peroxidase to Cpx 1-2RE allows it to catalyze the initial fermionic 1RE oxidation of luminol in an alkaline milieu. Hemoglobin has peroxidase activity under alkaline conditions, thus explaining the sensitivity of luminol-luminescence for detecting the presence of blood erythrocytes by alkaline peroxide methods. Luminol-luminescence by the classical plant peroxidase-catalyzed reactions of Eqs. (22)–(25) is sensitive to pH, decreasing with increasing acidity. Acidification of the reaction milieu to a pH of about 5 ± 1 effectively eliminates classical peroxidase-catalyzed luminol luminescence. This is quantitatively demonstrated in the Michaelis-Menten enzyme kinetic analyses of luminol-luminescence for myeloperoxidase and horse radish peroxidase presented in **Table 2** [71].

Alkaline pH favors the fermionic luminol-luminescence reactions catalyzed by plant peroxidase, hemoglobin, and heavy metals. The pKa of 1 H2O2 is 11.75. The ferricyanide-catalyzed luminol luminescence reaction is most efficient in the pH range from 10.4 to 10.8 [72]. In **Table 2**, note that no significant luminescence is observed from HRP-catalyzed luminol reaction at pH 4.9. The maximum luminescence velocity (*V*max) values are low and standard errors (SE) are high. However, a relatively weak but significant luminescence is observed at pH 7.0, that is, Michaelis-Menten analysis of the HRP luminescence shows a low *V*max, but an acceptable SE.

Of special note, Michaelis-Menten kinetic analysis indicates that the HRPcatalyzed luminol-luminescence velocity is first order with respect to H2O2 concentration, but second order with respect to luminol concentration, that is, the luminescence velocity is directly proportional to the square of the luminol concentration. These results are consistent with those reported by Dure and Cormier [70], and with the fermionic radical reactive pathway described in Eqs. (22)–(25) and Eq. (21).

Although luminol solubility becomes a problem at low pH, acidity favors the bosonic haloperoxidase luminol-luminescence catalyzed by MPO. Note that bosonic, haloperoxidase-catalyzed luminol luminescence is first order with respect to luminol, chloride, or bromide, but second order with respect to H2O2, that is, luminescence activity is proportional to the square of the H2O2 concentration.

The MPO-catalyzed luminol-luminescence kinetic finding is the opposite of those observed for HRP-catalyzed luminol-luminescence, and are consistent with the bosonic reactive pathway for luminol-luminescence via <sup>1</sup> O2\* reaction described

*Neutrophils*

release of 1

Relaxation of the <sup>1</sup>

indicated by Eq. (14), 1

frontier **π\*** orbitals of 1

<sup>1</sup>

The radical 2

<sup>1</sup>

<sup>1</sup>

producing two 1

<sup>1</sup>

O2

oxide (2

( 1

illustrated by Eqs. (17) and (18).

1

dioxygenation is a dioxetane intermediate that breaks down to one ground state

Luminol dioxygenation is not reductive. The net dioxygenation incorporates molecular oxygen to produce an endoperoxide intermediate with the breakdown

conservation and frontier orbital overlap problems restrict such direct reaction. As

Hund's maximum multiplicity rule is satisfied when the electrons of each SOMO

that restricts overlap with the bosonic frontier orbitals of luminol. By contrast, the

cence. The fermionic (doublet multiplicity/radical) pathway requires two steps as

H2O2—peroxidase → <sup>2</sup> luminol−1RE +<sup>1</sup>

<sup>−</sup>) yielding singlet multiplicity electronically excited aminophthalate

The bosonic (singlet multiplicity/nonradical) pathway can occur by a single

The bosonic (singlet multiplicity/nonradical) pathway can also occur by a two-

→ <sup>1</sup>

Although luminol is versatile with regard to reactive mechanism, dioxygenation is ultimately required for chemiluminescence. In an alkaline milieu, classical per-

H2O2 → Cpx 1−2RE + 2<sup>1</sup>

This 2RE oxidized peroxidase, referred to as complex 1 (Cpx 1), can now readily

peroxidase-catalyzed mechanism of luminol-luminescence described by Dure and Cormier illustrates the kinetics of the fermionic pathway [70]. For such reaction, a

OCl<sup>−</sup>

H2O2 → <sup>1</sup>

luminol-1RE can participate in SOMO-SOMO reaction with super-

aminophthalate + <sup>1</sup>

aminophthalate<sup>−</sup> + <sup>1</sup>

luminol−2RE + <sup>1</sup>

aminophthalate + <sup>1</sup>

Cl−

H2O2-dependent luminol-luminescence. The

H2O2, that is, 2RE are transferred to 1

luminol-1RE, as per Eq. (23).

N2 and formation of a **nπ\***electronically excited aminophthalate. As

luminol does not react with ground state oxygen. Spin

O2\* are bosonic and include one LUMO **π\*** orbital and one

O2\* with the HOMO of <sup>1</sup>

N-methylacridone\* yields a photon.

N-methylacridone\*.

O2 are its two degenerates **π\*** SOMOs.

O2 have fermionic character

luminol reactions yielding lumines-

luminol satis-

H2O (17)

N2 + Photon (18)

N2 + Photon (19)

N2 + Photon (21)

H2O (22)

(20)

H2O2

N-methylacridone and one **nπ\***electronically excited 1

illustrated in **Figure 1**, the frontier orbitals of 3

HOMO **π\*** orbital. Overlap of the LUMO of <sup>1</sup>

fies the symmetry requirements for reaction. There are three mechanistic possibilities for 1

luminol + <sup>1</sup>

<sup>2</sup> luminol−1RE + 2O2

reaction as illustrated by Eq. (19),

luminol + <sup>1</sup>

luminol−2RE + <sup>1</sup>

oxidase or hemoglobin can catalyze <sup>1</sup>

classical peroxidase is first oxidized by 1

peroxidase + <sup>1</sup>

step reaction as illustrated by Eqs. (20) and (21).

aminophthalate\*) that relaxes with photon emission.

O2 <sup>∗</sup> → <sup>1</sup>

luminol + <sup>1</sup>

H2O as described in Eq. (22).

luminol by removing 1RE producing <sup>2</sup>

− →<sup>1</sup>

have the same spin. Each of the two **π\*** orbitals of <sup>3</sup>

**76**

oxidize <sup>1</sup>


*Michaelis-Menten enzyme kinetic analyses of classical peroxidase (horse radish peroxidase) and haloperoxidase (myeloperoxidase) activities with regard to H*

*and pH.*

**79**

*Essence of Reducing Equivalent Transfer Powering Neutrophil Oxidative Microbicidal Action…*

in Eq. (19) or the sequential bosonic pathway described in Eqs. (20), (21). By either

Under alkaline conditions, luminol-luminescence provides high sensitivity for

Under acid conditions, the luminol-luminescence provides a method for specific quantification of haloperoxidase-dependent dioxygenation activity. In **Table 2**,

requirement is first order with respect to halide, and that the Michaelis constant (

activity is exclusively bosonic. Reactants are all singlet multiplicity, involving

used for measurement of phagocyte oxygenation activities. Its original applica

tion was an attempt to amplify the relatively weak native luminescence signal from stimulated macrophages. Comparing the luminol-luminescence responses of neutrophils with those of macrophages illustrates that the MPO-rich neutrophils responses are several magnitudes greater than the luminol-luminescence responses

of phagocytes [58, 65]. The luminol-dependent activities of MPO-positive

activity is not inhibited by the MPO inhibitor azide (N3

oxidase-dependent reactions described in reactions Eqs. (17)–(18).

**7. Circulating neutrophils reflect the state of inflammation**

1 H 2 O

Luminol was the first, and remains the most common, chemiluminigenic probe

Comparing MPO-rich human neutrophils with the MPO-deficient heterophile leukocytes of chickens further illustrates how chemiluminigenic probing can be used as a sensitive method for quantifying and differentiating the oxygenating activities

human neutrophil leukocytes are a hundredfold higher than those of MPO-negative chicken heterophil leukocytes. Despite the diminution in luminol-luminescence, dioxygenation activity is still quantifiable from MPO-negative phagocytes. Such

haloperoxidase, luminol-luminescence most probably reflects the type of fermionic

Under normal conditions, large numbers of neutrophils are produced by the hematopoietic marrow and released into the circulating blood each day, highlight

ing the importance of neutrophils for innate host protection against infection. To accomplish its microbicidal role, neutrophils undergo specific degranulation and mobilization of appropriate membrane receptors in response to a constellation of microbial peptides, complement activation products, cytokines, interleukins, and lipid activators. Such activities prepare neutrophils for phagocytosis, but do not directly trigger respiratory burst activity [73]. Priming actuates neutrophil locomo

tion and increases neutrophil recognition of and phagocytic response to opsonin-

Activation of systemic inflammation in response to infection directly affects circulating blood neutrophils. The chemical signals of inflammation alter the state of neutrophil alert. As such, the state of neutrophil priming reflects the state of host immune activation [76]. Selective *in vitro* measurement of unprimed and maxi

mally-primed circulating blood neutrophil activities by sensitive chemiluminigenic probing allows rapid multi-metric analysis using less than a half drop of anticoagu

lated whole blood. Analysis of such blood neutrophil luminescence metrics using classification statistical approaches, especially discriminant function analysis, allows assessment of the *in vivo* state of immune activation. The state of neutrophil

priming gauges the state of host systemic inflammation [77, 78].

<sup>−</sup> is required for MPO-catalyzed luminol-luminescence, that the

<sup>−</sup> is expectedly greater that for Br

1 H 2 O

2, but relatively low specificity.

2 are required for

<sup>−</sup>. Haloperoxidase

<sup>−</sup>) [66]. In the absence of

*K*M)






*DOI: http://dx.doi.org/10.5772/intechopen.81543*

detection of classical peroxidase catalysts or

HOMO-LUMO frontier orbital interaction.

from MPO-deficient macrophage [57].

labeled microbes [56, 74, 75].

luminol dioxygenation.

<sup>−</sup> or Br

for the more electronegative Cl

note that Cl

pathway, and consistent with the second order findings, two

 *O2*

*2, halide (Cl- or Br−), luminol,* 

#### *Neutrophils*

*Essence of Reducing Equivalent Transfer Powering Neutrophil Oxidative Microbicidal Action… DOI: http://dx.doi.org/10.5772/intechopen.81543*

in Eq. (19) or the sequential bosonic pathway described in Eqs. (20), (21). By either pathway, and consistent with the second order findings, two <sup>1</sup> H2O2 are required for luminol dioxygenation.

Under alkaline conditions, luminol-luminescence provides high sensitivity for detection of classical peroxidase catalysts or1 H2O2, but relatively low specificity. Under acid conditions, the luminol-luminescence provides a method for specific quantification of haloperoxidase-dependent dioxygenation activity. In **Table 2**, note that Cl<sup>−</sup> or Br<sup>−</sup> is required for MPO-catalyzed luminol-luminescence, that the requirement is first order with respect to halide, and that the Michaelis constant (*K*M) for the more electronegative Cl<sup>−</sup> is expectedly greater that for Br<sup>−</sup>. Haloperoxidase activity is exclusively bosonic. Reactants are all singlet multiplicity, involving HOMO-LUMO frontier orbital interaction.

Luminol was the first, and remains the most common, chemiluminigenic probe used for measurement of phagocyte oxygenation activities. Its original application was an attempt to amplify the relatively weak native luminescence signal from stimulated macrophages. Comparing the luminol-luminescence responses of neutrophils with those of macrophages illustrates that the MPO-rich neutrophils responses are several magnitudes greater than the luminol-luminescence responses from MPO-deficient macrophage [57].

Comparing MPO-rich human neutrophils with the MPO-deficient heterophile leukocytes of chickens further illustrates how chemiluminigenic probing can be used as a sensitive method for quantifying and differentiating the oxygenating activities of phagocytes [58, 65]. The luminol-dependent activities of MPO-positive human neutrophil leukocytes are a hundredfold higher than those of MPO-negative chicken heterophil leukocytes. Despite the diminution in luminol-luminescence, dioxygenation activity is still quantifiable from MPO-negative phagocytes. Such activity is not inhibited by the MPO inhibitor azide (N3 <sup>−</sup>) [66]. In the absence of haloperoxidase, luminol-luminescence most probably reflects the type of fermionic oxidase-dependent reactions described in reactions Eqs. (17)–(18).

## **7. Circulating neutrophils reflect the state of inflammation**

Under normal conditions, large numbers of neutrophils are produced by the hematopoietic marrow and released into the circulating blood each day, highlighting the importance of neutrophils for innate host protection against infection. To accomplish its microbicidal role, neutrophils undergo specific degranulation and mobilization of appropriate membrane receptors in response to a constellation of microbial peptides, complement activation products, cytokines, interleukins, and lipid activators. Such activities prepare neutrophils for phagocytosis, but do not directly trigger respiratory burst activity [73]. Priming actuates neutrophil locomotion and increases neutrophil recognition of and phagocytic response to opsoninlabeled microbes [56, 74, 75].

Activation of systemic inflammation in response to infection directly affects circulating blood neutrophils. The chemical signals of inflammation alter the state of neutrophil alert. As such, the state of neutrophil priming reflects the state of host immune activation [76]. Selective *in vitro* measurement of unprimed and maximally-primed circulating blood neutrophil activities by sensitive chemiluminigenic probing allows rapid multi-metric analysis using less than a half drop of anticoagulated whole blood. Analysis of such blood neutrophil luminescence metrics using classification statistical approaches, especially discriminant function analysis, allows assessment of the *in vivo* state of immune activation. The state of neutrophil priming gauges the state of host systemic inflammation [77, 78].

*Neutrophils*

**Michaelis-Menten kinetics**

**78**

**Substrate [S], variable** 

**pH** **H**

**O2 2, mM**

**Cl−, mEq/L**

**Br−,** 

**Luminol,** 

**M-M equation**

**Km ± SE**

**Vmax ± SE**

**mEq/L**

**μM**

**Substrates, constant**

**(conc. range)**

Haloperoxidase:

H

H

O2

2 (0.01–1.4 mM)

Cl− (0.2–7.7 mEq/L)

Br− (14–882 μEq/L)

Luminol (0.0018–15 μM)

Luminol (0.0018–0.47

7.0

2.27

90

0

variable

μM)

Classical

H

H

O2

2 (0.01–1.4 mM)

Cl− (0.2–900 mEq/L)

Br− (14–882 μEq/L

Luminol (0.0147–30 μM)

Luminol (0.0018–7.5

7.0

2.27 *Reaction milieu was 50 mM acetate buffer (pH 5.0, 4.9) or phosphate buffer (pH 7.0) in a 0.3 mL volume. The indicated conc. of Cl− or Br− was added in a 0.3 mL volume.*

*The enzymes, 78 pmol MPO and 10 pmol HRP as indicated, were added in a 0.1 mL volume. The final concentration was 78 nM for MPO and 10 nM for HRP.*

*O2* *Chemiluminescence velocity (v) and Vmax are expressed as peak kilocounts of relative light units (RLU × 10–3) per sec measured during the initial 20 sec post H*

*Michaelis-Menten enzyme kinetic analyses of classical peroxidase (horse radish peroxidase) and haloperoxidase (myeloperoxidase) activities with regard to H*

*2 in a 0.3 mL volume. The final volume was 1.0 mL.*

*O2*

*2 injection.*

*O2*

*2, halide (Cl- or Br−), luminol,* 

*The luminescence reaction was initiated by injecting the indicated concentration of H*

**Table 2.**

*and pH.*

0

0

variable

v = Vmax[S]2/(Km + [S])2

0.90 ± 0.17

189 ± 2

μM)

4.9

2.27

0

0

variable

5.0

2.27

0

variable

45

v = Vmax[S]/Km + [S]

v = Vmax[S]2/(Km + [S])2

0.0 ± 0.0

0 ± 0

6.23 ± 2.68

3 ± 0

5.0

2.27

variable

0

45

v = Vmax[S]/Km + [S]

0.0 ± 0.7

0 ± 0

5.0

variable

0

4.5

77

v = Vmax[S]/Km + [S]

17.49 ± 3.84

156 ± 34

O2

2 (0.01–1.4 mM)

5.0

variable

90

0

77

v = Vmax[S]/Km + [S]

31.02 ± 0.05

280 ± 55

Peroxidase: Horse

Radish Peroxidase

4.9

2.27

90

0

variable

5.0

2.27

0

variable

45

v = Vmax[S]/Km + [S]

v = Vmax[S]/Km + [S]

v = Vmax[S]/Km + [S]

0.10 ± 0.02

3252 ± 219

8.80 ± 0.77

1490 ± 70

0.68 ± 0.05

2280 ± 96

5.0

2.27

variable

0

45

v = Vmax[S]/Km + [S]

7.60 ± 2.60

1105 ± 253

5.0

variable

0

4.5

77

O2

2 (0.01–1.4 mM)

5.0

variable

90

0

77

v = Vmax[S]2/(Km + [S])2

v = Vmax[S]2/(Km + [S])2

0.58 ± 0.03

2932 ± 2

2.82 ± 0.05

3900 ± 1

Myeloperoxidase
