Sodium azide as a specific quencher of singlet oxygen during chemiluminescent detection by luminol and Cypridina luciferin analogues

Reactive oxygen species (ROS) are presently thought to play important role in an increasing number of the physiological and pathological processes in living organisms. Various chemiluminescent (CL) compounds have been studied in order to find suitable and specific probes for the detection of particular ROS species. The CL of luminol is known to be non‐specific and can be induced by various oxidants. Two Cypridina luciferin analogues, CLA and MCLA, have been used for the detection of ROS in vivo. CLAs are thought to emit light only when reacting with superoxide and singlet oxygen. It is possible to distinguish the particular ROS by using a specific quencher or scavenger, e.g. superoxide dismutase (SOD) or sodium azide (NaN₃). The CL reactions of luminol (3‐aminophthalhydrazide), CLA [2‐methyl‐6‐phenyl‐3,7‐dihydroimidazo(1,2α) pyrazin‐3‐one] and MCLA [2‐methyl‐6‐(p‐methoxyphenyl)‐3,7‐dihydroimidazo(1,2α) pyrazin‐3‐one] were studied in three hydrogen peroxide decomposition systems (H₂O₂–HRP; H₂O₂–CuSO₄; and H₂O₂–NaOCl). The measurements were carried out in phosphate buffer, pH 7.4, at 25 °C, using a luminometer (Fluoroskan Ascent FL and Sirius C). NaN₃ was used as the specific quencher of singlet oxygen. The results demonstrate that the proclaimed specifity of the CL of Cypridina luciferin analogues towards singlet oxygen has to be discussed. Copyright © 2011 John Wiley & Sons, Ltd.     

Study on the reaction mechanism and the static injection chemiluminescence method for detection of acetaminophen

Acetaminophen, also called paracetamol, is found in Tylenol, Excedrin and other products as over–the‐counter medicines. In this study, acetaminophen as a luminol signal enhancer was used in the chemiluminescence (CL) substrate solution of horseradish peroxidase (HRP) for the first time. The use of acetaminophen in the luminol–HRP–H₂O₂ system affected not only the intensity of the obtained signal, but also its kinetics. It was shown that acetaminophen was to be a potent enhancer of the luminol–HRP–H₂O₂ system. A putative enhancement mechanism for the luminol–H₂O₂–HRP–acetaminophen system is presented. The resonance of the nucleophilic amide group and the benzene ring of acetaminophen structure have a great effect on O‐H bond dissociation energy of the phenol group and therefore on phenoxyl radical stabilization. These radicals act as mediators between HRP and luminol in an electron transfer reaction that generates luminol radicals and subsequently light emission, in which the intensity of CL is enhanced in the presence of acetaminophen. In addition, a simple method was developed to detect acetaminophen by static injection CL based on the enhanced CL system of luminol–H₂O₂–HRP by acetaminophen. Experimental conditions, such as pH and concentrations of substrates, have been examined and optimized. The proposed method exhibited good performance, the linear range was from 0.30 to 7.5 mM, the relative standard deviation was 1.86% (n = 10), limit of detection was 0.16 mM and recovery was 99 ± 4%. Copyright © 2013 John Wiley & Sons, Ltd.     

Characterization of peroxidases in luminol chemiluminescence coupled with copper-catalysed oxidation of cysteamine

Hydrogen peroxide formed during the course of the copper(II)-catalysed oxidation of cysteamine with oxygen was continuously determined by a peroxidase (POD)-catalysed luminol chemiluminescence (CL) method. Horseradish peroxidase (HRP), lactoperoxidase (LPO) and Arthromyces ramosus peroxidase (ARP) were used as a CL catalyst. The respective PODs gave specific CL intensity-time profiles. HRP caused a CL delay, and ARP gave a time-response curve which followed the production rate of H₂O₂. LPO gave only a weak CL flash which decayed promptly. These differences of CL response curves could be explained in terms of the different reactivities of PODs for superoxide anion and the different formation rate of luminol radicals in the peroxidation of luminol catalysed by POD.     

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.     

Long‐term chemiluminescence signal is produced in the course of luminol oxidation catalyzed by enhancer‐independent peroxidase purified from Jatropha curcas leaves

Isoenzyme c of horseradish peroxidase (HRP‐C) is widely used in enzyme immunoassay combined with chemiluminescence (CL) detection. For this application, HRP‐C activity measurement is usually based on luminol oxidation in the presence of hydrogen peroxide (H₂O₂). However, this catalysis reaction was enhancer dependent. In this study, we demonstrated that Jatropha curcas peroxidase (JcGP1) showed high efficiency in catalyzing luminol oxidation in the presence of H₂O₂. Compared with HRP‐C, the JcGP1‐induced reaction was enhancer independent, which made the enzyme‐linked immunosorbent assay (ELISA) simpler. In addition, the JcGP1 catalyzed reaction showed a long‐term stable CL signal. We optimized the conditions for JcGP1 catalysis and determined the favorable conditions as follows: 50 mM Tris buffer (pH 8.2) containing 10 mM H₂O₂, 14 mM luminol and 0.75 M NaCl. The optimum catalysis temperature was 30°C. The detection limit of JcGP1 under optimum condition was 0.2 pM. Long‐term stable CL signal combined with enhancer‐independent property indicated that JcGP1 might be a valuable candidate peroxidase for clinical diagnosis and enzyme immunoassay with CL detection. Copyright © 2014 John Wiley & Sons, Ltd.     

Determination of horserdish peroxidase concentration using the chemiluminescence of Cypridina luciferin analogue, 2 methyl-6-(p-methyoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-one

The chemiluminescence of the Cypridina luciferin analogue, 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (MCLA) was observed at 462nm in the presence of horseradish peroxidase (HRP) and the total spectrum of light emitted was found to depend linearly on HRP concentration. Methods for the determination of HRP concentration using the chemiluminescence was investigated. HRP could be detected in the range from 100 pmol/L to 100nmol/L under the optimum condition, H₂O₂ (10mmol/L) and MCLA (10μmol/L) at pH 5.8.     

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.     

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.     

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.     

Improvement of mimetic peroxidase activity of gold nanoclusters on the luminol chemiluminescence reaction by surface modification with ethanediamine

Peroxidase is a commonly used catalyst in luminol–H₂O₂ chemiluminescence (CL) reactions. Natural peroxidase has a sophisticated separation process, short shelf life and unstable activity, therefore it is important to develop peroxidases that have both high catalytic activity and good stability as alternatives to the natural enzyme. Gold nanoclusters (Au NCs) are an alternative peroxidase with catalytic activity in the luminol–H₂O₂ CL reaction. In the present study, ethanediamine was modified on the surface of Au NCs forming cationic Au NCs. The zeta potential of the cationic Au NCs maintained its positive charge when the pH of the solution was between 4 and 9. The cationic Au NCs showed higher catalytic activity in the luminol–H₂O₂ CL reaction than did unmodified Au NCs. A mechanism study showed that the better performance of cationic Au NCs may be attributed to the generation of ¹O₂ on the surface of cationic Au NCs and a positive surface charge, for better affinity to luminol. Cationic Au NC, acting as a peroxidase mimic, has much better stability than horseradish peroxidase over a wide range of temperatures. We believe that cationic Au NCs may be useful as an artificial peroxidase for a wide range of potential applications in CL and bioanalysis.     

Scavenging of reactive oxygen species by N‐substituted indole‐2 and 3‐carboxamides

Indole‐2 and 3‐carboxamides (IDs) are proposed to be selective cyclooxygenase inhibitors. Since cyclooxygenase‐1 may be involved in reactive oxygen species (ROS) production, we hypothesize that these indole derivatives have antioxidative properties. We have employed chemiluminescence (CL) and electron spin resonance (ESR) spin trapping to examine this hypothesis. We report here the results of a study of reactivity of 10 selected indole derivatives towards ROS. The following generators of ROS were applied: potassium superoxide (KO₂) as a source of superoxide radicals (O₂^·?), the Fenton reaction (Co‐EDTA/H₂O₂) for hydroxyl radicals (HO^·), and a mixture of alkaline aqueous H₂O₂ and acetonitrile for singlet oxygen (¹O₂). Hydroxyl radicals were detected as 5,5‐dimethyl‐1‐pyrroline‐N‐oxide (DMPO) spin adduct, whereas 2,2,6,6‐tetramethyl‐piperidine (TEMP) was used as a detector of ¹O₂. Using the Fenton reaction, 0.5 mmol/L IDs were found to inhibit DMPO‐?H radical formation in the range 7–37%. Furthermore the tested compounds containing the thiazolyl group also inhibited the ¹O₂‐dependent TEMPO radical, generated in the acetonitrile + H₂O₂ system. About 20% inhibition was obtained in the presence of 0.5 mmol/L IDs. 1 mmol/L IDs caused an approximately 13–70% decrease in the CL sum from the O₂^·? generating system (1 mmol/L). The aim of this paper is to evaluate these indole derivatives as antioxidants and their abilities to scavenge ROS. Copyright © 2004 John Wiley & Sons, Ltd.     

Reactivation of horseradish peroxidase with imidazole for continuous determination of hydrogen peroxide using a microflow injection–chemiluminescence detection system

A method for reactivation of inactivated horseradish peroxidase (HRP) was studied and exploited in an assay for hydrogen peroxide (H₂O₂). Addition of imidazole into a mobile phase made continuous determination of hydrogen peroxide (H₂O₂) possible by micro?ow injection based on horseradish‐catalysed luminol chemiluminescence. For reproducible determination of H₂O₂ with HRP, the inactivation of HRP via protonation of the active sites of HRP caused by reaction with H₂O₂ must be avoided. We successfully reactivated protonated HRP (inactive HRP) with exogenous imidazole in the mobile phase of the micro?ow injection system. The imidazole successfully removed the attached proton from the inactive sites of the HRP. This assay was reproducible (within‐run reproducibility, CV = 4.0%) and the detection limit for H₂O₂ was 5 pmol. Copyright © 2003 John Wiley & Sons, Ltd.     

Comparison of oxidative stress induced by ciprofloxacin and pyoverdin in bacteria and in leukocytes to evaluate toxicity

Oxidative stress induced by ciprofloxacin and pyoverdin, a leukotoxic pigment, was studied by comparing their effect in bacteria and leukocytes. Chemiluminescence (CL) assays with lucigenin or luminol were adapted to measure the stimuli of superoxide anion (O₂^?) and other reactive species of oxygen (ROS) in bacteria. Ciprofloxacin principally induced the production of O₂^? in the three species studied: Staphylococcus aureus, Enterococcus faecalis and Escherichia coli. Lucigenin CL assay showed high oxidative stress in S. aureus due to its low superoxide dismutase (SOD) activity, whereas E. coli exhibited important SOD activity, responsible for little production of O₂^? in absence or presence of ciprofloxacin. Reduction of nitroblue of tetrazolium (NBT) was applied. This assay indicated that there was higher oxidative stress in S. aureus and E. faecalis than in E. coli. The comparison of oxidative stress generated in bacteria and leukocytes was used to check the selective toxicity of ciprofloxacin in comparison with pyoverdin. Ciprofloxacin did not generate significant stimuli of O₂^? in neutrophils, while pyoverdin duplicated the production of O₂^?. CL and NBT were useful to study the leukotoxicity of ciprofloxacin. Oxidative stress caused by the antibiotic and the leukotoxic pigment was similar in bacteria. Copyright © 2003 John Wiley & Sons, Ltd.     

Chemiluminescence of off‐line and on‐line gold nanoparticle‐catalyzed luminol system in the presence of flavonoid

It was found that flavonoids could remarkably inhibit the chemiluminescence (CL) intensity of an off‐line gold nanoparticle (AuNP)‐catalyzed luminol–H₂O₂ CL system. By contrast, flavonoids enhanced the CL intensity of an on‐line AuNP‐catalyzed luminol–H₂O₂ CL system. In the off‐line system, the AuNPs were prepared beforehand, whereas in the on‐line system, AuNPs were produced by on‐line mixing of luminol prepared in a buffer solution of NaHCO₃ ? Na₂CO₃ and HAuCl₄ with no need for the preliminary preparation of AuNPs. The on‐line system had prominent advantages over the off‐line system, namely a lowering of the background noise and improvements in the stability of the CL system. The results show that differences in the signal suppression effect of flavonoids on the off‐line AuNP‐catalyzed CL system are influenced by the combined action of a free radical scavenging effect and occupy‐sites function; the latter was proved to be predominant using controlled experiments. Enhancement of the on‐line system was ascribed to the presence of flavonoids promoting the on‐line formation of AuNPs, which better catalyzed the luminol–H₂O₂ CL reaction, and the enhancement activity of the six flavonoids increased with the increase in reducibility. This work broadens the scope of practical applications of an AuNP‐catalyzed CL system.     

Application of optimized chemiluminescence assay for determination of the antioxidant capacity of herbal extracts

A chemiluminescence (CL) assay for the determination of antioxidant capacity (AOC) has been optimized and applied to analyses of herbal extracts in the present study. The optimal concentrations of reagents (luminol, H₂O₂, horseradish peroxidase) have been determined, as well as the optimal reaction conditions (wavelength, pH, temperature, sample volume). All of the measurements were performed at the emission maximum of the oxidized form of luminol (425 nm). The optimal concentrations of the reagents were determined as follows: 1.6 mmol/L luminol, 7.5 mmol/L H₂O₂ and 0.14 U/mL horseradish peroxidase activity in the reaction mixture. Analyses were carried out in phosphate buffer, pH 7.4, at room temperature. With the optimized CL assay, the AOCs of various water and methanol herbal extracts were determined (dog rose hips, plantain leaves and coltsfoot and thyme flowers) and the results were compared to those obtained by other classical methods for the evaluation of antioxidants. Strong correlations (r > 0.9) with the Folin–Ciocalteau assay and the 2,2‐diphenyl‐1‐picrylhydrazyl radical (DPPH)^● assay are confirmed, although there is no correlation between AOC and the concentration of ascorbic acid in the samples analysed. This optimized CL assay is simple, rapid and reliable, and it represents a good alternative to classical methods (Folin–Ciocalteau, DPPH^●) for the determination of AOC of herbal extracts and other food samples. Copyright © 2012 John Wiley & Sons, Ltd.     

Enhanced chemiluminescence in the oxidation of luminol and an isoluminol cortisol conjugate by hydrogen peroxide in reversed micelles

The chemiluminescent oxidation of luminol and an isoluminol cortisol conjugate (ABICOR) by hydrogen peroxide has been studied in cetyltrimethylammonium bromide (CTAB) reversed micelles in octane-chloroform (1 : 1). The maximum chemiluminescence intensity of both compounds is dependent on the initial concentrations of the H₂O₂ and substrates, the pH value of the micelle polar phase and the H₂O/CTAB ratio. The optimum pH ranged from 8.5 to 9.5. Under comparable conditions, the chemiluminescence intensity for luminol was 15-fold higher than for the ABI-COR conjugate. A mechanism of oxidation of the substrates in reversed micelles is proposed and the possible mechanisms of inhibition by the substrate and oxidant is discussed.     

Pyrophosphate as substrate for alkaline phosphatase activity: A convenient flow‐injection chemiluminescence assay

A sensitive and convenient flow‐injection chemiluminescence (FI‐CL) turn‐on assay for alkaline phosphatase (ALP) activity without any label and synthesis is developed. Cu²⁺ can catalyze the luminol–H₂O₂ CL reaction. Pyrophosphate (PPi) can chelate Cu²⁺ and therefore the Cu²⁺‐mediated luminol‐H₂O₂ CL reaction is inhibited. The addition of ALP can catalyze the hydrolysis of PPi into phosphate ions, Cu²⁺ is released and the chemiluminescence recovers. A detection limit of 1 mU/mL ALP is obtained.     

Nitric oxide synthase inhibitors decrease human polymorphonuclear leukocyte luminol-dependent chemiluminescence

Nitric oxide synthase (NOS) inhibitors have been reported to modulate luminol-dependent chemiluminescence (CL) in rat macrophages, whereas the potent oxidant peroxynitrite (ONOO^-) was shown to react with luminol to yield CL in a cellfree system. We evaluated the role of the -arginine/NOS pathway in luminol CL by phorbol ester-activated human polymorpho-nuclear (PMN) leukocytes using the NOS inhibitors N^G-monomethyl- -arginine ( -NMMA) and N-iminoethyl- -omithine ( -NIO). Nitric oxide (·NO) release was determined by oxidation of oxymyoglobin. In addition, the effect of NOS inhibitors on superoxide anion O₂^-) production was measured. Luminol CL was notably diminished by -NMMA in a dose-dependent manner. Superoxide dismutase (SOD) also decreased luminol CL and -NMMA potentiated light emission decrease produced by SOD. Nitric oxide and O₂^·- production was significantly decreased by -NMMA; moreover, luminol-dependent CL but not O₂^·- production was attenuated by -NIO. These data suggest that products of catalytic activity of both ·NO synthase and NADPH oxidase are required to elicit maximal luminol CL in this system. These studies demonstrate that the NOS synthase pathway is involved in luminol CL by human PMN, and they suggest that ONOO would be an unrecognized mediator in this phenomenon.     

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.     

Studies of visible oscillating chemiluminescence in luminol–H2O2–KSCN–CuSO4 system using (2‐hydroxyethyl) trimethylammonium hydroxide

Visible oscillating chemiluminescence (CL) of luminol–H₂O₂–KSCN–CuSO₄ was studied using the organic base (2‐hydroxyethyl)trimethylammonium hydroxide. The effect of concentrations of luminol, H₂O₂, KSCN, CuSO₄ and the base were investigated in a batch reactor. This report shows how the concentration of components involved in the oscillating CL system influenced the oscillation period, light amplitude and total time of light emission. The oscillating CL with different bases was also investigated. Results indicated that using 2‐HETMAOH causes regular oscillating CL with nearly the same oscillating period. Copyright © 2008 John Wiley & Sons, Ltd.