Validation of different chemilumigenic substrates for detecting extracellular generation of reactive oxygen species by phagocytes and endothelial cells

Chemiluminescence is a widely used tool to detect extracellular generation of reactive oxygen species (ROS). In the present study we tested four different chemilumigenic substrates (CLS)—luminol, isoluminol, lucigenin and pholasin—to detect extracellular CL in different cell types: polymorphonuclear leukocytes (PMN); DMSO‐differentiated HL‐60 cells; murine macrophages (RAW 264.7); and TNFα‐stimulated human endothelial cells (HUVEC). Extracellular ROS production was calculated by subtracting intracellular CL response in the presence of superoxide dismutase and catalase from the overall CL response in the absence of enzymes. CL varied considerably in dependence on the CLS and the stimulus used to evoke ROS generation. Luminol (oxidized LDL and zymosan stimulation) and isoluminol (FMLP and PMA stimulation) were the most effective CLS for PMN. Using 5 µmol/L lucigenin as CLS, small but consistent CL responses could be obtained in macrophages stimulated with PMA, zymosan or oxidized LDL. FMLP‐stimulated extracellular CL in H‐60 cells, HUVEC and macrophages was detected with the greatest sensitivity by pholasin. Our results demonstrate that none of the investigated CLS consistently yielded the highest CL quantum, either in different cell types with one stimulating agent or by different stimulating agents in one cell type. To get the highest CL quantum in experimental studies, we recommend optimizing the CLS depending on the cell type and the ROS‐generating stimulus used. Copyright © 2003 John Wiley & Sons, Ltd.     

Reactive oxygen metabolite production is inhibited by histamine and H1-antagonist dithiaden in human PMN leukocytes

The study evaluated the distinction between extracellular and intracellular production of reactive oxygen metabolites (ROM) in isolated polymorphonuclear leukocytes (PMNL) stimulated with opsonised zymosan (OZ) and investigated its modulation by the endogenous mediator histamine (0.1-100 μmol/l) and by the H 1 -antagonist dithiaden (1-100 μmol/l). For this observation, a modified luminol and an isoluminol amplified chemiluminescence (CL) technique were used. Our results showed that PMNL activated with OZ responded with a respiratory burst accompanied by both extra- and intracellular generation of ROM. Histamine and dithiaden significantly decreased both the extra- and intracellular component of chemilumiescence stimulated with OZ. While dithiaden decreased both the extra- and intracellular part of CL with the same potency, histamine decreased preferentially the extracellular part of CL. The fact that histamine as well as the H 1 -antagonist dithiaden decreased the respiratory burst indicates that not only histamine receptors but also non-receptor mechanisms could be involved in the reduction of CL. Interaction with enzymes (NADPH-oxidase, myeloperoxidase, phospholipase A 2 ) or interference with PMNL membrane structure may well result in reduction of the chemiluminescence signal.     

Reactive Oxygen Metabolite Production is Inhibited by Histamine and H 1 -antagonist Dithiaden in Human PMN Leukocytes

The study evaluated the distinction between extracellular and intracellular production of reactive oxygen metabolites (ROM) in isolated polymorphonuclear leukocytes (PMNL) stimulated with opsonised zymosan (OZ) and investigated its modulation by the endogenous mediator histamine (0.1-100 &#119 mol/l) and by the H 1 -antagonist dithiaden (1-100 &#119 mol/l). For this observation, a modified luminol and an isoluminol amplified chemiluminescence (CL) technique were used. Our results showed that PMNL activated with OZ responded with a respiratory burst accompanied by both extra- and intracellular generation of ROM. Histamine and dithiaden significantly decreased both the extra- and intracellular component of chemilumiescence stimulated with OZ. While dithiaden decreased both the extra- and intracellular part of CL with the same potency, histamine decreased preferentially the extracellular part of CL. The fact that histamine as well as the H 1 -antagonist dithiaden decreased the respiratory burst indicates that not only histamine receptors but also non-receptor mechanisms could be involved in the reduction of CL. Interaction with enzymes (NADPH-oxidase, myeloperoxidase, phospholipase A 2 ) or interference with PMNL membrane structure may well result in reduction of the chemiluminescence signal.     

Myeloperoxidase-Based Chemiluminescence of Polymorphonuclear Leukocytes and Monocytes

Luminol and lucigenin chemiluminescence (CL) responses produced by separated human blood polymorphonuclear leukocytes (pmn) and monocytes (mono) have been studied following stimulation with the surface-receptor agonist fMLP (a synthetic chemotactic peptide) and the protein kinase C activator phorbol myristate acetate (PMA). Pmn produced two- to threefold the luminol CL and superoxide anion (O₂⁻) levels of mono; lucigenin CL was similar for both cell-types. The myeloperoxidase (MPO) inhibitor salicylhydroxamic acid (SHA) abrogated luminol but not lucigenin CL in both cell types, but did not further inhibit the already grossly subnormal luminol CL responses seen with MPO-deficient cells which produced normal lucigenin CL. SHA also profoundly inhibited the luminol CL response in a cell-free MPO–H₂O₂ system. Mono lucigenin CL does not appear to specifically measure O₂⁻ production. These data show that luminol CL provides a useful measure of pmn and also mono MPO activity. However, analysis of the effects of various reactive oxygen species (ROS) scavengers, assessed on phagocyte and cell-free CL systems (both MPO–H₂O₂ and superoxide generating) suggest that the luminol CL signal is not entirely dependent on MPO activity.     

Inhibitory effect of stobadine on FMLP-induced chemiluminescence in human whole blood and isolated polymorphonuclear leukocytes.

The chemiluminescence (CL) technique with luminol and isoluminol was used to characterize the effect of stobadine on reactive oxygen metabolites (ROM) generation in human whole blood and in isolated polymorphonuclear leukocytes (PMNL) stimulated with N-formyl-methionyl-leucyl-phenyl-alanine (FMLP). In whole blood and in isolated PMNL, stobadine in the concentrations of 1, 10 and 100 micromol/L significantly inhibited the CL signal after FMLP, which activated predominantly extracellular generation of ROM. The same concentrations of stobadine were effective on CL in a cell-free system. On the other hand, myeloperoxidase (MPO) liberation was decreased by stobadine only in the concentration of 100 micromol/L. The results showed stobadine to act as a potent inhibitor/scavenger of extracellularly produced ROM in human PMNL and indicated interference of stobadine with ROM as well as with signalling events resulting in NADPH-oxidase activation and MPO liberation.     

Evaluation of Opsonophagocytic Dysfunctions in Severely Burned Patients by Luminol-Dependent Chemiluminescence

We compared the luminol-dependent chemiluminescence (CL) response of peripheral blood from severely burned patients with that from normal controls to evaluate the primary defense level against bacterial infection in the patients. The CL was measured upon addition to diluted whole blood of a soluble stimulus, phorbol myristate acetate (PMA) or particulate stimuli such as bacteria or zymosan without special opsonization. In the early post-burn days, the initial rate of whole blood CL induced with the particulate stimulus was much lower than that in the normal controls, whereas the rate was higher when PMA was used as a stimulus. The number of granulocytes in the patients' blood had increased and isolated polymorphonuclear leukocytes (PMNs) from the patients exhibited higher CL responses to the particulate or soluble stimulus as compared with those of normal controls. The results suggest that the PMNs in burn patients were activated and normally mobilized in the early post-burn period but the opsonizing capacity in the blood decreased. In fact, the serum levels of complement, immunoglobulins and fibronectin were found to be lower in the blood from the patients than those from normal controls and a supplement of freshly frozen plasma of human immunoglobulin preparations restored the initial rate of the whole blood CL upon phagocytosis. The prognosis is still poor when severe infection occurs in the patients with decreased CL response of whole blood. Recombinant human granulocyte colony-stimulating factor (rhG-CSF) enhanced the CL response of PMNs from burn patients. The administration of rhG-CSF may be useful for decreasing the morbidity of severe infection following burn injury in the near future.     

A new chemiluminescence paradox: selective inhibition of isoluminol‐amplified activity in phagocytes by peptides from annexin AI

Chemiluminescence systems enhanced by either isoluminol or luminol in combination with a peroxidase are sensitive methods for the detection of reactive oxygen species (ROS) generated by phagocyte NADPH oxidase. The two amplifying substrates are structurally very similar, differing only in the position of the amino group in the aromatic ring of the molecules. This difference renders isoluminol a less lipophilic molecule that is less permeable to biological membranes. The use of isoluminol is consequently restricted to studies dealing with the secretion of oxygen metabolites. In this study we show that synthetic peptides derived from the N‐terminal domain of the calcium‐regulated protein annexin AI interfere with the detection of radicals in an isoluminol‐amplified, but not in a luminol‐amplified, system. The annexin AI‐derived peptides reduce the light output with isoluminol excited by superoxide and horseradish peroxidase (HRP) in formyl‐methionyl‐leucyl‐phenylalanine‐ and phorbol myristate acetate‐stimulated cells, as well as by hydrogen peroxide and HRP. The precise mechanism for the inhibition is not known. The results presented strongly suggest that a reduced cellular response detected with isoluminol‐amplified chemiluminescence should be confirmed with an alternative technique to determine release of superoxide anions and hydrogen peroxide. Copyright © 2008 John Wiley & Sons, Ltd.     

Chemiluminescence of non‐differentiated THP‐1 promonocytes: developing an assay for screening anti‐inflammatory milk proteins and peptides

Human leukemic THP‐1 promonocytes are widely used as a model for peripheral blood monocytes. However, superoxide production during respiratory burst (RB) of non‐differentiated THP‐1 (nd‐THP‐1) cells is very low. Here we present a rapid and low‐cost method for measuring the chemiluminescence (CL) of opsonized zymosan (OZ) induced RB which allows detection of Escherichia coli lipopolysaccharide (LPS) induced priming of nd‐THP‐1 cells on the basis of CL reaction kinetics. Maximum CL intensity obtained was 2.20 ± 0.25 and 1.30 ± 0.11 relative light units, while CL peak time was achieved at 18.1 ± 2.6 and 28.7 ± 1.3 min in primed and non‐primed cells, respectively. The priming of nd‐THP‐1 cells with LPS evoked typical TNF‐α and IL‐6 production. We tested the effects of bovine lactoferrin and protein fractions from Lactobacillus helveticus BGRA43 fermented milk for potential anti‐inflammatory effects on LPS primed nd‐THP‐1 cells. Four fractions were found to inhibit the OZ‐induced CL in a dose‐dependent manner (IC₅₀ 3–30 µg/mL), while lactoferrin inhibited CL to a lesser extent (IC₅₀ 270 µg/mL). These results suggest that measuring CL response of nd‐THP‐1 cells can serve as a method for screening anti‐inflammatory compounds which could be used in reducing the risk of phagocyte‐mediated inflammatory diseases. Copyright © 2010 John Wiley & Sons, Ltd.     

Luminol‐dependent chemiluminescence of human phagocyte cell lines: comparison between DMSO differentiated PLB 985 and HL 60 cells

The human promyelocytic leukemia HL 60 and PLB 985 cell lines can differentiate into terminally mature neutrophil‐like cells via dimethyl sulfoxide (DMSO) induction. In this study the luminol‐dependent chemiluminescence (LCL) of both neutrophil‐like cells was analayzed and compared in response to phorbol myristate acetate (PMA) and opsonized zymosan (OZ) stimulants. It was shown that, like human blood neutrophils, both neutrophil‐like cells expressed high levels of CD11b, but unlike human blood neutrophils these cells almost lack LCL‐detectable intracellular oxidase activity. By studying the pattern of activation to OZ and PMA and priming with GM‐CSF, we concluded that there is no difference between the percentage of differentiation and function of DMSO‐induced HL 60 and PLB 985. However, the LCL capacity (area under the curve) of DMSO induced PLB 985 cells was higher than that of HL 60 cells in response to both PMA and OZ, which implies a higher capacity to generate reactive oxygen species in PLB 985 cells. Copyright © 2009 John Wiley & Sons, Ltd.     

Evaluation of lophine derivatives as L‐012 (luminol analog)‐dependent chemiluminescence enhancers for measuring horseradish peroxidase and H2O2

8‐Amino‐5‐chloro‐7‐phenylpyrido[3,4‐d]pyridazine‐1,4(2H,3H)dione (L‐012) was recently synthesized as a new chemiluminescence (CL) probe; the light intensity and the sensitivity of L‐012 are higher than those of other CL probes such as luminol. Previously, our group developed four lophine‐based CL enhancers of the horseradish peroxidase (HRP)‐catalyzed CL oxidation of luminol, namely 2‐(4‐hydroxyphenyl)‐4,5‐diphenylimidazole (HDI), 2‐(4‐hydroxyphenyl)‐4,5‐di(2‐pyridyl)imidazole (HPI), 4‐(4,5‐diphenyl‐1H‐imidazol‐2‐yl)phenylboronic acid (DPA), and 4‐[4,5‐di(2‐pyridyl)‐1H‐imidazol‐2‐yl]phenylboronic acid (DPPA), and showed that DPPA was suitable for the photographic detection of HRP. In this study, we replaced luminol with L‐012 and evaluated these as L‐012‐dependent CL enhancers. In addition, to detect HRP and/or H₂O₂ with higher sensitivity, each detection condition for the L‐012–HRP–H₂O₂ enhanced CL was optimized. All the derivatives enhanced the L‐012‐dependent CL as well as luminol CL; HPI generated the highest enhanced luminescence. Under optimized conditions for HRP detection, the detection limit of HRP was 0.08 fmol. By contrast, the detection limit of HRP with the enhanced L‐012‐dependent CL using 4‐iodophenol, which is a common enhancer of luminol CL, was 1.1 fmol. With regard to H₂O₂ detection, the detection limits for enhanced CL with HPI and 4‐iodophenol were 0.29 and 1.5 pmol, respectively. Therefore, it is demonstrated that HPI is the most superior L‐012‐dependent CL enhancer. Copyright © 2013 John Wiley & Sons, Ltd. Copyright © 2013 John Wiley & Sons, Ltd.     

Chemiluminescence from luminol solution after illumination of a 355 nm pulse laser

Chemiluminescence (CL) on the time scale of microseconds to milliseconds from luminol solution after illumination of a 355 nm pulse laser is reported. It was found that the CL is the emission from 3‐aminophthalate ion (AP*). In CL decay after the pulse laser illumination, a peak was observed from about 200 to 30 µs depending on the laser power and the luminol concentration. It seemed that there was a fast and slow decay process; their kinetics were greatly dependent on the laser power and the luminol concentration. Dissolved oxygen was involved in the CL and played the same role on the whole time scale of microseconds to milliseconds. Involvement of reactive oxygen species such as H₂O₂, ¹O₂, O₂^?? and OH in the CL was examined by adding their scavengers. Experimental results suggested that the possibility of involvement of H₂O₂ and ¹O₂ in the CL was low. The CL in time periods less than 50 µs might be related to ?OH. The ^?O₂^??‐induced CL increased with time after 50 µs and became dominant on the time scale of milliseconds. The CL was considered to be caused by both the photoionization and type I reaction mechanisms. Copyright © 2009 John Wiley & Sons, Ltd.     

Mild hypothermia protects against postischemic hepatic endothelial injury and decreases the formation of reactive oxygen species

Abstract

The ability of mild hypothermia (MH; 34°C) to protect against postischemic endothelial injury and decrease reactive oxygen species' (ROS) formation was studied using lucigenin and luminol enhanced chemiluminescence (CL). Lucigenin CL is largely specific for superoxide, while luminol reacts with many ROS.

Isolated rat livers perfused under constant flow in a non-recirculating system were exposed to 2.5 h of ischemia after 0.5 h perfusion with Krebs-Henseleit buffer at either normothermia (38°C) or mild hypothermia (34°C) (n = 5, all groups). CL (cps), vascular resistance (Woods units), O₂ consumption, and potassium efflux were measured at the end of perfusion, and at 0 min reperfusion, and every 30 min during reperfusion.

For both the lucigenin and luminol groups, CL and vascular resistance increased significantly (repeat measures ANOVA, P <0.05) for normothermia (NT, 38°C) but not mild hypothermia. Potassium efflux did not change significantly for the mild hypothermia groups. In the luminol enhanced group, oxygen consumption was greater in the mildly hypothermic group at 1 h and 1.5 h of reperfusion.

Mild hypothermia decreased postischemic ROS production. Increased vascular resistance in the normothermia group may indicate an endothelial injury. Mild hypothermia appears to protect against this injury.     

Employment of 4‐(1,2,4‐triazol‐1‐yl)phenol as a signal enhancer of the chemiluminescent luminol–H2O2–horseradish peroxidase reaction for detection of hepatitis C virus in real samples

Highly sensitive detection of hepatitis C virus (HCV) in serum is a key method for diagnosing and classifying the extent of HCV infection. In this study, a p‐phenol derivative, 4‐(1,2,4‐triazol‐1‐yl)phenol (4‐TRP), was employed as an efficient enhancer of the luminol–hydrogen peroxide (H₂O₂)–horseradish peroxidase (HRP) chemiluminescence (CL) system for detection of HCV. Compared with a traditional enhancer, 4‐TRP strongly enhanced CL intensity with the effect of prolonging and stabilizing light emission. The developed CL system was applied to detecting HCV core antigen (HCV‐cAg) using a sandwich structure inside microwells. Our experimental results showed that there was good linear relationship between CL intensity and HCV‐cAg concentration in the 0.6–3.6 pg/mL range (R = 0.99). The intra‐ and inter‐assay coefficients of variation were 4.5–5.8% and 5.0–7.3%, respectively. In addition, sensitive determination of HCV‐cAg in serum samples using the luminol–H₂O₂–HRP–4‐TRP CL system was also feasible in clinical settings. Copyright © 2015 John Wiley & Sons, Ltd.     

Development of chemiluminescence method for determination of 10‐hydroxycamptothecin based on luminol–[Ag(HIO6)2]5− reaction in alkaline solution

A novel chemiluminescence (CL) method was developed for the determination of 10‐hydroxycamptothecin(HCPT) based on the CL reaction between [Ag(HIO₆)₂]^5? and luminol in alkaline solution. CL emission of Ag(III) complex–luminol in alkaline medium was very different from that in acidic medium. A possible mechanism of enhanced CL emission was suggested. The enhanced effect of HCPT on CL emission of the [Ag(HIO₆)₂]^5?–luminol system was found. The enhanced degree of CL emission was proportional to HCPT concentration. The effect of the reaction conditions on CL emission was examined. Under optimal conditions, the limit of detection was 6.5 × 10^?9 g mL^?1. The proposed method was applied for the determination of HCPT in real samples with the recoveries of 93.2–109% with the RSD of 1.7–3.3%. Copyright © 2010 John Wiley & Sons, Ltd.     

Effects of inhibitors on chicken polymorphonuclear leukocyte oxygenation activity measured by use of selective chemiluminigenic substrates

Chicken heterophil polymorphonuclear leukocytes (CPMNLs) have NADPH oxidase activity, but lack myeloperoxidase (MPO). Stimulation of CPMNLs by phorbol 12-myristate 13-acetate or chicken opsonified zymosan results in luminol-dependent chemiluminescence (CL) activity, which is small relative to that of human peroxidase-positive neutrophils (HPMNLs), as well as lucigenin-dependent CL, comparable to HPMNL responses. Inhibitors were used to investigate and characterize the CL activity of CPMNLs. Inhibition constants were calculated, using Dixon inhibition analysis, or were reported as the concentration producing 50% inhibition of the magnitude of CL responses. Azide and cyanide are effective inhibitors of luminol CL in HPMNLs, although these peroxidase inhibitors do not inhibit either luminol or lucigenin CL of CPMNLs. Since these agents also inhibit eosinophil peroxidase, lack of inhibition of CPMNL CL indicates that the small percentages of peroxidase-positive eosinophils in CPMNL preparations are not responsible for the luminol CL observed. Iodoacetate and fluoride, pre-oxidase and pre-peroxidase inhibitors of glycolytic metabolism, effectively inhibit lucigenin and luminol CL activities in CPMNLs. Superoxide dismutase competitively inhibits lucigenin and luminol CL in CPMNLs, but catalase is an ineffective inhibitor. Although luminol is efficiently dioxygenated by a MPO-dependent mechanism in HPMNL, use of peroxidase-deficient CPMNLs indicates that this substrate does not exclusively measure peroxidase activity.     

The influence of age and sex on phagocyte chemiluminescence

The process of ageing is associated with increased susceptibility to infection. Phagocytes form the primary defence mechanism against infecting microorganisms, but the influence of ageing on phagocyte function remains controversial. In this study we have applied a microtitre plate phagocyte chemiluminescence (CL) assay suitable for clinical use to compare phagocyte oxidative metabolism in younger healthy subjects (age 20–60 years) and healthy older (60–70 years) subjects. Polymorphonuclear leukocytes (PMNL) and monocytes were stimulated using phorbol myristate acetate (PMA), serum opsonized zymosan (SOZ), and non-opsonized zymosan (ZYM) in the presence of both lucigenin and luminol. Monocytes showed a higher luminolenhanced CL response to PMA in males compared with females in the younger age group. No PMNL differences were observed between the sexes. Although no difference were found in relation to age when cells were stimulated with PMA and SOZ, significantly lower background (unstimulated) CL was obtained from PMNL with luminol. PMNL luminol-enhanced CL responses were also lower in response to ZYM. The findings suggest a reduced response of PMNL from older subjects to minimal stimulation. This could be related to abnormalities in the triggering of the respiratory burst or myeloperoxidase release due to ageing. The influence of age and sex should be taken into account in clinical studies of phagocyte CL.     

Chemiluminescence determination of moxifloxacin in pharmaceutical and biological samples based on its enhancing effect of the luminol–ferricyanide system using a microfluidic chip

A sensitive determination of a synthetic fluoroquinolone antibacterial agent, moxifloxacin (MOX), by an enhanced chemiluminescence (CL) method using a microfluidic chip is described. The microfluidic chip was fabricated by a soft‐lithographic procedure using polydimethyl siloxane (PDMS). The fabricated PDMS microfluidic chip had three‐inlet microchannels for introducing the sample, chemiluminescent reagent and oxidant, and a 500 µm wide, 250 µm deep and 82 mm long microchannel. An enhanced CL system, luminol–ferricyanide, was adopted to analyze the MOX concentration in a sample solution. CL light was emitted continuously after mixing luminol and ferricyanide in the presence of MOX on the PDMS microfluidic chip. The amount of MOX in the luminol–ferricyanide system influenced the intensity of the CL light. The linear range of MOX concentration was 0.14–55.0 ng/mL with a correlation coefficient of 0.9992. The limit of detection (LOD) and limit of quantification (LOQ) were 0.06 and 0.2 ng/mL respectively. The presented method afforded good reproducibility, with a relative standard deviation (RSD) of 1.05% for 10 ng/mL of MOX, and has been successfully applied for the determination of MOX in pharmaceutical and biological samples. Copyright © 2013 John Wiley & Sons, Ltd.     

The early effect of sublethal X-irradiation of phagocytic cells in mouse blood and the influence of cystamine as measured by chemiluminescence

The metabolic burst accompanying phagocytosis of granulocytes (PMN) leads to the generation of activated oxygen species such as O-2, H2O2, 1O2 and OH; which give rise to chemiluminescence (CL) in the presence of luminol. Reliable CL-measurements of stimulated PMN can be carried out in freshly drawn mouse blood, when photon counts are related to the number of PMN. Effects of low dose total body X-irradiation were studied using C57B1/6 mice. It was found that 24 and 48 hours after irradiation (0.24-0.95 Gy) CL of whole blood was slightly decreased. If however CL-counts were related to the number of PMN, an enhanced CL per single granulocyte was recorded. The administration of cystamine leads to an immune stimulating effect of unirradiated animals. In animals, who received 0.95 Gy a distinct radioprotective effect of cystamine can be observed.     

Microflow‐injection chemiluminescence of luminol and hypochlorite enhanced by phloxine B

We report for the first time that the sensitivity of the luminol–hypochlorite chemiluminescence (CL) reaction was enhanced approximately 10 times by the addition of phloxine B. The maximum wavelength of CL emission shifted from 431 to 595 nm in the absence and presence, respectively, of phloxine B, suggesting that an efficient chemiluminescence resonance energy transfer occurred between a luminol donor and a phloxine B acceptor in the luminol–hypochlorite–phloxine B system. Based on this observation, a simple, rapid and sensitive microflow injection CL method, using a microchip with spiral channel configurations, was developed for the determination of hypochlorite. Under optimized conditions, a linear calibration curve (R² = 0.9944) over the range 0.1–10.0 µmol/L was obtained, with a detection limit of 0.025 µmol/L (S:N = 3). The relative standard deviation (RSD) was found to be 4.2% (n = 10) for 2.5 µmol/L hypochlorite. The sample consumption was only 2 μL, with a sample throughput of 90/h. The method has been used for determining trace amounts of hypochlorite in water samples with satisfactory results. Copyright © 2012 John Wiley & Sons, Ltd.     

Enhancement of chemiluminescence of the KIO4–luminol system by gallic acid,acetaldehyde and Mn2+: application for the determination of catecholamines

Chemiluminescence (CL) from the oxidation of luminol with potassium periodate in strong alkaline solutions was greatly enhanced by the combined effect of gallic acid, acetaldehyde and Mn²⁺. The CL spectra exhibited only one emission band at 425 nm, indicating 3‐aminophthalate as the emitting species. Various scavengers for superoxide anion, hydroxyl radical and singlet oxygen quenched the CL emission very efficiently (74–100%), suggesting the possible involvement of these reactive oxygen species (ROS) in the CL reactions. It is postulated that oxidation of gallic acid and acetaldehyde by periodate catalyzed by Mn²⁺ generates these ROS, which then react with luminol to enhance the CL emission. We also found that the enhanced CL emission was strongly inhibited by catecholamines, probably because of their effective scavenging of ROS. Based on this observation, a simple, rapid and sensitive new CL method was developed for the determination of catecholamines. The detection limits (3σ) for dopamine, l‐ dopa, norepinephrine and epinephrine were 0.63, 1.37, 0.56 and 14.3 nmol/L, respectively. The linear range was 1–10 nmol/L; relative standard deviations were 0.71–1.34% for 0.1 µmol/mL catecholamines. This CL method was applied to the determination of catecholamines in pharmaceutical injections with satisfactory results. Copyright © 2009 John Wiley & Sons, Ltd.