Near-real-time determination of hydrogen peroxide generated from cigarette smoke

Abstract

The ability to monitor hydrogen peroxide (H₂O₂) in aqueous smoke extracts will advance our understanding of the relationship between cigarette smoke-induced oxidative stress, inflammation, and disease and help elucidate the pathways by which the various smoke constituents exert their pathogenic effects. We have demonstrated, for the first time, the measurement of H₂O₂ production from cigarette smoke without prior separation of the sample. Cigarettes were tested on a commercial smoking machine, such that the whole smoke or gas vapor phase was bubbled through phosphate buffered saline solution at pH 7.4. Aliquots of these solutions were analyzed using an Amplex Red/horseradish peroxidase fluorimetric assay that required only a 2 minute incubation time, facilitating the rapid, facile collection of data. Catalase was used to demonstrate the selectivity and specificity of the assay for H₂O₂ in the complex smoke matrix. We measured ∼7–8 μM H₂O₂ from two reference cigarettes (i.e., 1R4F and 2R4F). We also observed 9× more H₂O₂ from whole smoke bubbled samples compared to the gas vapor phase, indicating that the major constituent(s) responsible for H₂O₂ formation reside in the particulate phase of cigarette smoke. Aqueous solutions of hydroquinone and catechol, both of which are particulate phase constituents of cigarette smoke, generated no H₂O₂ even though they are free radical precursors involved in the production of reactive oxygen species in the smoke matrix.

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Introduction

Hydrogen peroxide, being the precursor to other reactive oxygen species (ROS),¹ occupies a central role in oxygen metabolism. Hydrogen peroxide is produced by various reactions in several subcellular compartments. In addition to its role in cellular toxicity, H₂O₂ has recently gained much attention as a signalling molecule involved in transduction pathways that induce cellular damage and apoptosis.^2–4 Increased intracellular levels of ROS such as superoxide radical anion, hydroxyl radical, or hydrogen peroxide are referred to as oxidative stress, an imbalance between oxidants and antioxidants in favor of the former. A variety of environmental insults may produce oxidative stress in individuals exposed to such insults. Cigarette smoking can cause an acute inflammatory reaction in the lung, producing ROS and reactive nitrogen species (RNS). For a number of years, there has been speculation that various ROS species are involved in the well-known deleterious biological effects of cigarette smoke.^5–14 Electron paramagnetic resonance (EPR) evidence suggests that semiquinone free radical species are involved in causing DNA damage that could lead to cancer. It was hypothesized that H₂O₂ is also involved.¹⁰ As shown in the reaction below, hydroquinones (H₂Q) present in smoke can reduce dioxygen (O₂) to produce superoxide (O₂⁻˙) and semiquinone (Q⁻˙) radical anions.⁵ The superoxide radical anion is thought to be the immediate precursor to H₂O₂, which can then be reduced to the hydroxyl radical by metals such as iron. The resulting HO˙ radical can then damage DNA.

H₂Q + O₂ → O₂⁻˙ + Q⁻˙ + 2H⁺

Cigarette smoke is among the most extensively studied complex matrices. Various investigators have reported the detection of superoxide radical, H₂O₂, and hydroxyl radical in aqueous smoke solutions. The radicals can be detected using an EPR spin-trap method,¹¹ although other techniques such as chemiluminescence¹² and colorimetric methods have been used.⁶ The use of spin trap methods requires expensive EPR instrumentation and does not provide detailed structural information without prior fractionation of the sample. Therefore there is a need to develop simple, rapid analytical techniques to measure ROS species generated by whole, unseparated cigarette smoke.

Spectroscopic techniques can offer advantages as analytical tools. Fluorescence spectroscopy can provide very sensitive analyte assays. A variety of fluorescence assays for the detection of H₂O₂ have been devised, employing a number of substrates. Using horseradish peroxidase (HRP) as a catalyst, H₂O₂ is detected by measuring either an increase or a decrease in the substrate’s fluorescence after oxidation.^15–21

Many constituents of cigarette smoke, such as carbon monoxide, carbon dioxide, ammonia, methane etc. can be directly monitored using a variety of commercial instruments. However, the ability to detect other potentially more injurious constituents (e.g. ROS) in near real-time scenarios has not been demonstrated. Furthermore, most of the methods developed to detect H₂O₂ in its pure form or in simple matrices are not suitable for cigarette smoke-related studies, especially in situ, as cigarette smoke is an exceedingly complex matrix consisting of several thousand constituents.^22,23 A variety of chemical, electrochemical, spectroscopic, and enzymatic techniques have been used to detect H₂O₂ in fractionated cigarette smoke or its various extracts. For example, Nakayama et al. assayed the H₂O₂ concentration in whole smoke-bubbled buffer solution using a colorimetric procedure involving HRP and 2,2′-azinodi (3-ethylbenzothiazoline-6-sulfonate) (ABTS).⁶ This colorimetric technique required isolation of the organic soluble fraction with ethyl acetate and ether, followed by bubbling with nitrogen gas to remove the residual organic solvent before addition of the colorimetric substrate. They subsequently reported that this colorimetric method was not suitable for analysis of the total particulate matter (TPM) trapped on the Cambridge filter pad (CFP), because of rapid reduction of the oxidized form of the colorimetric substrate by redox-active TPM constituents.⁷ Alternatively they used differential pulse polarography to assay H₂O₂ concentration in the aqueous CFP extracts, and were able to develop a sensitive (less than 10 μM H₂O₂) and reproducible (standard deviation less than 2%) assay. However, the pre-treatment of the sample before polarography involved separation through a reverse-phase column, as well as treatment of the extract by bubbling with air for 1 minute and by aging the extract for 4 hours before the assay.

Several groups have used a nonfluorescent, fluorogenic derivative (Amplex Red = 10-acetyl-3,7-dihydroxxyphenoxazine) of resorufin (7-hydroxy-3H-phenoxazine-3-one) as the basis for a sensitive assay for H₂O₂, employing HRP as a catalyst. ^24–27 Mohanty et al. applied this methodology to the detection of H₂O₂ release from activated human leukocytes,²⁷ while Zhou et al. used the same assay system to detect the activity of phagocyte NADPH oxidase.²⁴ More recent investigations have studied the reactions involved in this assay from the standpoint of kinetic analysis. ^25,26 This assay is becoming increasingly popular as a method to analyze H₂O₂, both because of its sensitivity and simplicity. We have developed a rapid and sensitive method employing 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red) and HRP for the near-real-time determination of H₂O₂ produced when cigarette smoke is bubbled through phosphate buffered saline solutions. To the best of our knowledge, we demonstrate, for the first time, the feasibility of measuring H₂O₂ production by cigarette smoke without prior separation of smoke components.

Experimental procedures

Cigarettes and chemicals

Reference cigarettes (1R4F and 2R4F) were obtained from the Kentucky Tobacco Research and Development Center.^28,29 The 1R4F Kentucky reference cigarette was produced in 1983. Due to depletion of the supply, a subsequent identical reference cigarette, 2R4F, was made in 2003. Descriptive data for each of the cigarettes is shown in Table 1. Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine, Catalog No. A-6550) was purchased from Molecular Probes, Inc. (Eugene, OR). Hydrogen peroxide (stabilized with 200 ppm acetanilide), dimethylsulfoxide (DMSO), horseradish peroxidase (HRP, catalog No. P-2088), and catalase (from bovine liver, Catalog No. C-100) were purchased from Sigma Chemical Co. (St. Louis, MO) and used without further purification. The H₂O₂ reagent was stored in the refrigerator at 4 °C wrapped in aluminium foil. The stock solution of Amplex Red was 10 mM in analytically pure DMSO (60 μg in 60 μL) and stored at −20 °C before use. The stock solution of HRP was 10 U mL⁻¹ in sodium phosphate buffer. The buffer solution was prepared from Dulbecco’s phosphate buffered saline (PBS) powder in 1 L increments with 50 mM strength at pH 7.4 (Cat. No. 21600-010, Invitrogen Gibco Cell Culture Products, Carlsbad, CA). Water used to prepare aqueous solutions was from a Barnstead Mega-Pure Deionized Water System (Model: A 440267, Barnstead/Thermdyne, Dubuque, IA). The working solution was prepared fresh each day using 50 μL of 10 mM Amplex Red stock, 100 μL of 10 U mL⁻¹ HRP stock and 4.85 mL of 50 mM PBS. This working solution was sufficient for 10 experiments using 0.5 mL aliquots. Hydrogen peroxide calibration standards of 0.2 μM, 1 μM, 2.5 μM, 5 μM, and 10 μM were made in sodium phosphate buffer.

Table 1 Descriptive data for cigarettes under study

Properties	1R4F	2R4F
Filter length/mm	27.2	27.0
Cig. length/mm	84	84
Cig. weight/mg	1085	1060
Filter ventilation (%)	28.4	28.0
Total particulate matter/mg	14.6	13.6

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Detection of hydrogen peroxide using the Amplex Red assay

Cuvettes containing 0.5 mL of the working solution and either 0.5 mL of the various H₂O₂ calibration standards or 0.5 mL aliquots of the smoke bubbled PBS were incubated for either 30 minutes at room temperature or 2 minutes at 37 °C. Fluorescence was then measured with a spectrofluorimeter (FluoroMax-3, Jobin Yvon, NJ) using excitation at 530 ± 2 nm and fluorescence detection at 585 ± 2 nm. Background fluorescence, determined for a control reaction lacking H₂O₂, was subtracted from each value. For statistical purposes, triplicate measurements were obtained for each data point. The buffer solution was not oxygen-free since we wanted to approximate physiological conditions.

Smoking protocol

Cigarettes (unconditioned) were smoked on a five-port smoking machine (KC Automation, Inc. Richmond, VA) in single port mode. The puff parameters were: sine wave flow profile, 35 mL per puff for a 2-second duration every 60 s. All smoke collection was performed in a laboratory fume hood, where the flow rate across the cigarette coal was not controlled, and at ambient temperature and relative humidity. A total of 5 puffs were collected for each cigarette tested. This condition was chosen to have enough smoke sample to collect a reasonable amount of aqueous smoke-bubbled extract and to allow standardization for the various cigarettes tested. Generally, a maximum of 7–10 puffs can be collected from a burning cigarette.

Preparation of aqueous cigarette smoke samples

The first five puffs of smoke were bubbled through the PBS contained in a gas washing bottle or impinger with a 25 mm diameter fritted disc (Catalogue #7163, Aceglass, NJ). In order to ensure all, or at least a very high percentage, of the smoke was successfully trapped, a minimum volume of 50 mL PBS was used in the gas washing bottle. Whole smoke bubbled extract refers to smoke collected directly without using a CFP, while smoke passed first through the 44 mm diameter CFP before being collected in PBS, is called gas vapor phase bubbled extract. TPM is the particulate phase that is trapped on the CFP, and is the sum of nicotine, water, and “tar”. All smoke samples were prepared immediately before experiments unless otherwise indicated. Samples were maintained at room temperature (∼24 °C) under ambient light during the experiment, and 0.5 mL aliquots from the PBS fractions were used in all measurements. The gas washing bottles were soaked in acetone and a mixture of strong acids overnight in order to remove materials trapped in the glass frit.

Results and discussion

H₂O₂ calibration curves

The enzymatic determination of H₂O₂ can be accomplished with high sensitivity and specificity using Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine), a highly sensitive and chemically stable fluorogenic probe.²⁴ Amplex Red is a colorless and non-fluorescent molecule which produces the highly fluorescent product, resorufin (7-hydroxy-3H-phenoxazine-3-one) following HRP-catalyzed H₂O₂ oxidation (see Scheme 1). In the presence of peroxidase, the Amplex Red reagent reacts with H₂O₂ in a 1 ∶ 1 stoichiometry to produce the deacetylated product.²⁴ Mohanty et al. observed that at least a 5-fold excess of Amplex Red over H₂O₂ present in the assay mixture was required to obtain a linear dose–response.²⁷ Zhou et al. report that if equimolar concentrations of Amplex Red and H₂O₂ are incubated in the presence of HRP, there is a quantitative conversion of the substrate to resorufin.²⁴ If the concentration of H₂O₂ is higher than that of Amplex Red, however, then the fluorescence decreases and essentially disappears at ratios of H₂O₂ to Amplex Red of 2 ∶ 1. Zhou et al. believe that this further oxidized derivative of resorufin, produced by the action of excess H₂O₂, is a nonfluorescent complex polymer.²⁴ The relative kinetics of the two oxidations were reported by Zhou et al. as follows.²⁴ The rate of conversion of resorufin to the nonfluorescent derivative was more than 30-fold slower than the rate of conversion of Amplex Red to resorufin. Several conclusions from the foregoing are germane to the present studies. To obtain a reasonable quantitative estimate of H₂O₂ concentrations in cigarette smoke, it was important to maintain a molar excess of Amplex Red relative to H₂O₂. Several reports suggested that H₂O₂ levels in smoke samples were μM or less, ^6,7,9,30 hence the H₂O₂ calibration range was set at 0.2 to 10 μM and the final concentration of Amplex Red reagent was kept at 50 μM for all measurements, to ensure at least a 5 ∶ 1 ratio of Amplex Red to H₂O₂. A second important point to note is that as the molar ratio of H₂O₂ increases toward 1 ∶ 1 with respect to Amplex Red, then some conversion of the resorufin to nonfluorescent products might be expected to occur. In fact Mohanty et al. note this phenomenon.²⁷ For example, when 50 μM Amplex Red is used in the assay, then a linear dose–response is seen for H₂O₂ concentrations in the range of 1–10 μM, 20 μM H₂O₂ falls off slightly from linearity, while 50 μM H₂O₂ shows the same fluorescence as 20 μM. (Incubation times of 15 min at room temperature for all experiments). Mohanty et al. attributed this decreased fluorescence with higher concentrations of H₂O₂ to the further oxidation of the first oxidized product of Amplex Red.²⁷ Finally, since the kinetics of the second oxidation step are quite slow compared to the first oxidation, shorter reaction times will favor the stable production of resorufin, without significant further oxidation of the resorufin to nonfluorescent products (vide infra).

Scheme 1 Reaction for the enzymatic determination of H₂O₂ using the non-fluorescent Amplex Red reagent and horseradish peroxidase to produce the strongly fluorescent Resorufin product.

According to Towne et al.,²⁵ the Amplex Red reagent may be unstable at high pH (>8.3), since de-N-acetylation due to nucleophilic substitution could occur. Furthermore, the absorption and fluorescence of the resorufin reaction product are also somewhat pH-dependent. We chose to use PBS, pH 7.4, throughout our experiments. Fig. 1 shows the H₂O₂ calibration curves for various incubation times at room temperature or at 37 °C, in a water bath. A linear dose–response relationship between fluorescence intensity and H₂O₂ concentration was observed over the range of H₂O₂ concentrations from 0.2–5 μM. There was a small deviation from linearity at a H₂O₂ concentration of 10 μM. The results in this present paper are consistent with the results reported by Zhou et al.,²⁴ in that Zhou et al. also showed a small deviation from linearity at 10 μM H₂O₂, with increasing deviation as the concentration of H₂O₂ increased above 10 μM. Mohanty et al. observed deviations from linearity at 20 μM H₂O₂ and above.²⁷ There was no apparent difference in fluorescence intensity for samples incubated for 2 min at 37 °C, 1 min at 37 °C, or 30 min at 24 °C (recommended in the product literature). As mentioned above, the shorter incubation times are to be preferred, since the competing reaction resulting in a further oxidation of resorufin is thereby minimized. Mohanty et al. incubated Amplex Red with HRP and H₂O₂ for 15 min at room temperature for their dose–response data,²⁷ while Zhou et al. incubated for 5 min at room temperature.²⁴ In the data immediately following, the amount of H₂O₂ production from aqueous cigarette smoke fractions is calculated based upon converting fluorescence intensity readings (as obtained from Amplex Red/HRP assays of replicate aliquots of the smoke-bubbled solution at various time intervals after collection) to H₂O₂ concentrations. The assays were either carried out for 2 min at 37 °C or 30 min at room temperature. Comparisons with the appropriate dose–response curves of Fig. 1 allowed one to calculate the H₂O₂ concentration associated with a fluorescence intensity for a given set of assay conditions.

Fig. 1 Standard calibration curves for H₂O₂ at different temperatures and incubation times. Each data point represents the average of triplicate measurements, with 1σ standard deviation.

The data shown in Fig. 1 were somewhat surprising in that the calibration curve obtained with a 30 min incubation at room temperature was essentially the same as the calibration using an assay time of 2 min at 37 °C. The original literature using the Amplex Red assay for H₂O₂ determination used assay times of 5 or 15 min at room temperature,^24,27 although the product literature from Molecular Probes suggests an assay time of 30 min. Our data seem to indicate that a 30 min assay at room temperature gives equivalent results to a 2 min assay at 37 °C. However, as indicated in the discussion above, a shorter incubation time is to be preferred as competing reactions resulting in degradation of resorufin are thus minimized.

Time profile of H₂O₂ production from whole smoke bubbled PBS samples

Fig. 2 shows the H₂O₂ production vs. time for the 1R4F and 2R4F reference cigarettes under two sets of assay conditions with the whole smoke bubbled PBS stored at room temperature (∼24 °C) under ambient light. The data in Fig. 2A and 2B were obtained using a 30 min incubation time at room temperature, while the data in Fig. 2C and 2D were obtained using a 2 min incubation time at 37 °C. The results in Fig. 2 show that there is little difference in the results from each of the assay conditions. This result is consistent with the data in Fig. 1, which show essentially no difference between the two sets of assay conditions when calibration standards of H₂O₂ were used. The data in Fig. 2 also show that over time, there is initially a rather large increase in H₂O₂ production, followed by a more gradual increase which plateaus by ∼4 h.

Fig. 2 Plots of H₂O₂ concentration (μM) versus time (min) for cigarette smoke bubbled PBS solutions incubated at the times and temperatures indicated. Aliquots from the smoke-bubbled PBS solutions were taken at various times and assayed for H₂O₂ using the Amplex Red assay. The fluorescence intensity produced from the Amplex Red assay was used, in conjunction with the H₂O₂ dose–response data in Fig. 1, to estimate the concentration of H₂O₂ present in the smoke-bubbled aqueous extracts. The estimation of H₂O₂ for the two assay conditions (30 min at room temperature or 2 min at 37 °C) was based upon the corresponding dose–response data from Fig. 1. Each data point represents the average of triplicate measurements from the same smoke bubbled PBS solution (1 cigarette), with 1σ standard deviation. Trend lines were generated in Microsoft® Excel using a polynomial equation which calculates the least squares fit through points by using the following equation: y = b + c1x + c2x2, where b and c are constants.

The observed temporal profiles are not unrealistic since tobacco smoke is formed by a complex series of processes and as it leaves the immediate environment of the cigarette, is not at chemical or physical equilibrium. However, as the smoke is diluted with the surrounding air or absorbing buffer solutions in this case, chemical reactions and physical processes probably slow to the point where, for many constituents, a quasi-equilibrium is attained.³¹ Thus, the concentrations of H₂O₂ shown in Fig. 2 may reflect a continuous depletion/production of H₂O₂ rather than a stable fixed concentration. We are unable to distinguish which is the case in these present experiments. The observed kinetics may be convolved with the effect of limits imposed by one or more of the reactants and/or deactivation of the enzyme rather than the actual H₂O₂ formation. More work is required to directly correlate changes in H₂O₂ evolution with total and constituent cigarette deliveries. We also found that the H₂O₂ concentration did not change much with further aging, from 4 h up to 48 h at room temperature and under ambient light.

Regarding the magnitude of H₂O₂ measured, it is interesting to compare the results presented here with the only other published report of H₂O₂ generated by whole cigarette smoke. Our data show an increase in H₂O₂ with time, reaching an apparent plateau at ∼240 min at ∼7–8 μM H₂O₂ for 5 puffs of smoke. Nakayama et al. also observed an increase in H₂O₂ in cigarette smoke bubbled PBS (pH 6.1), with a plateau at ∼120 min at 4 μg or 0.118 μM H₂O₂ for the entire cigarette.⁶ Unfortunately insufficient experimental details regarding the cigarette such as cigarette identity, puff count, and TPM delivery are provided for a quantitative comparison. In addition Nakayama subsequently noted that this colorimetric assay was not suitable for measuring H₂O₂ from aqueous TPM extracts from the CFP because, as noted previously, of the rapid reduction of the oxidized form of the colorimetric substrate by redox-active TPM constituents.⁷ Since TPM is a part of whole smoke, this causes us to question his H₂O₂ value.

Time profile of H₂O₂ production from gas vapor phase bubbled PBS sample

Smoke consists of lower molecular weight permanent gases, such as carbon monoxide and carbon dioxide, and semi-volatile constituents that reside predominantly in the vapor phase and higher molecular weight compounds (and some inorganic species), which reside predominantly in the particulate phase.²³ Experimentally, the gas phase is defined as those materials that pass through the CFP. We measured the production of H₂O₂ from the gas vapor phase of the 1R4F reference cigarette using a CFP to trap the particulate matter. Hydrogen peroxide concentration was calculated from fluorescence measurements based upon the standard dose–response data in the same manner as explained for Fig. 2. These data are shown in Fig. 3 with 1R4F whole smoke data at 150 min for comparison. The amount of H₂O₂ from the gas vapor phase bubbled PBS solution did not vary much from blank controls (see inset in Fig. 3). Thus there was little or no H₂O₂ production from the smoke gas vapor phase constituents, suggesting that the major source of H₂O₂ is from constituents present in the particulate phase.

Fig. 3 A comparison between 1R4F-gas/vapor phase bubbled PBS sample (after aging for various times up to 150 min) and 1R4F-whole smoke bubbled PBS sample (after aging for 150 min). Inset: expanded time profile of H₂O₂ production for 1R4F-gas/vapor phase bubbled PBS sample at room temperature. Each data point represents the average of triplicate measurements after blank subtraction with 1σ standard deviation, obtained after 30 min incubation with the Amplex Red working solution at room temperature.

Assuming the particulate phase is mostly responsible for H₂O₂ production, we can compare our whole smoke results to the two other reports of H₂O₂ from aqueous cigarette tar extracts. Nakayama et al. reported 37 μg per cig. of H₂O₂ from one 1R4F research cigarette.⁷ From Fig. 2 we measured approximately 8 μM from five puffs of whole smoke. Based upon volumes of buffer used and assuming 8 puffs per cigarette, our yield was ∼22 μg per cig. The agreement is quite good despite the very different methods. Their method involves an initial separation of the TPM and gas phase constituents using a CFP, followed by an aeration step, 4 h of aging, a reverse-phase liquid chromatography separation, and finally differential pulse polarography using a mercury drop electrode. Interestingly, the authors noted that both storage temperature and buffer solution pH affected H₂O₂ formation. Larger amounts of H₂O₂ were formed when the solution was aged over 4 h at elevated temperature. The difference between aging at 4 °C and 37 °C was almost two-fold. As the buffer solution pH was increased from 6.3 to 8.2, the yield of H₂O₂ increased almost two-fold.

The other study of aqueous cigarette tar extracts involved chromatographic separation into seventy-eight 2 mL fractions with H₂O₂ determination of each fraction via oxygen consumption as measured with a Clarke-type oxygen electrode.⁹ The comparison is difficult because the researchers used the 2R1 reference cigarette, smoked in a non-standard way, with no report of puff count or filter pad weight (i.e. TPM). The 2R1 reference cigarette is unfiltered and is thus also non-ventilated. It produces 28.6 mg of TPM per cig. when smoked per the Federal Trade Commission method, 35 mL per puff for a 2 s duration every 60 s.³² In this study the cigarette was sampled taking a 30 mL per puff of unspecified duration every 30 s. In addition 5 cigarettes were smoked on one CFP of unspecified diameter. There is an upper mg weight limit beyond which the pad loses its efficiency to trap particulate matter. There are also many TPM constituents that are not water soluble, despite sonication. Summing over all the fractions, they report 313 μM H₂O₂ from five 2R1 cigarettes. Dividing through by 5 cigarettes and assuming 28.6 mg per cig., we calculate 2.2 μM H₂O₂ mg TPM⁻¹. Converting our value of 8 μM to per cigarette per mg TPM basis, 8 μM × (8 puffs/cig/5 puffs) × 13.6 mg cig⁻¹, the result is 0.9 μM H₂O₂ mg TPM⁻¹. The agreement is reasonable and consistent with the TPM deliveries of the two different cigarettes.

Selectivity study of Amplex Red assay for the determination of H₂O₂

With the exception of some of the major constituents, such as CO and CO₂, many of the volatile organic compounds of cigarette smoke are present at concentrations of 0.1 to 1000 μg per cig.¹³ Many of the trace constituents of the particulate phase may be present at levels one to three orders of magnitude lower, parts per billion or parts per trillion levels. Due to the multitude of components present in cigarette smoke, it is possible that the oxidation of Amplex Red observed when the reagent is reacted with aqueous bubbled cigarette smoke is due to some component other than H₂O₂. To demonstrate the selectivity of the Amplex Red reaction for H₂O₂ present or produced in whole smoke samples, catalase with a final concentration of 13 392 units was added to an aged whole smoke bubbled PBS sample (after 24 h at room temperature and ambient light). Catalase catalyzes the disproportion of H₂O₂ to form O₂ and H₂O. Each molecule of the enzyme is capable of decomposing 40 000 molecules of H₂O₂ per second at 0 °C and producing 10¹² molecules of oxygen per second. This is a standard procedure for selectively removing H₂O₂ (e.g. see Mohanty et al.,²⁷). The addition of 10 μL catalase consumed all of the H₂O₂ formed from the aged cigarette smoke-bubbled PBS solutions. These data are shown in Fig. 4, where H₂O₂ concentration is plotted vs. emission wavelength for 1R4F whole smoke extract, a blank sample with no smoke, and the same 1R4F smoke extract with catalase. This result indicated that the fluorescent product of Amplex Red was induced mainly or exclusively by H₂O₂ produced in smoke bubbled solutions and not from any other constituents in the whole smoke. The selectivity of the Amplex Red reaction for H₂O₂ has also been investigated by Mohanty et al.,²⁷ who found that superoxide anion (O₂⁻), generated by xanthine oxidase and hypoxanthine did not increase the fluorescence of Amplex Red.

Fig. 4 Effect of catalase addition on fluorescence intensity of aged whole smoke bubbled PBS sample. Amplex Red working solution was added to two aliquots of aged (24 h) whole smoke-bubbled PBS samples, one of which had been pre-treated with catalase. After incubation for 2 min at 37 °C, the fluorescence emission spectrum was determined.

H₂O₂ production from hydroquinone and catechol

Since H₂O₂ was generated at physiological pH and room temperature by the interaction of cigarette smoke with buffered saline solution, it is reasonable to assume that the same types of reactions may produce ROS species, including H₂O₂, in the respiratory tract.²⁰ It has been asserted that the dihydroxybenzenes, particularly hydroquinone and catechol, are the free radical precursors in aqueous cigarette smoke responsible for the production of injurious ROS.⁹ Pryor et al. reported that the polyphenolic fraction of aqueous tar extract: (1) caused most of the observed DNA damage in rat thymocytes, (2) generated 90 times more H₂O₂ than the unfractionated aqueous tar extract solution, and (3) contained the stable o- and p-benzosemiquinone radicals.⁹ Using our assay, we studied the production of H₂O₂ from hydroquinone and catechol for up to three hours. Either 0.5 mL of 100 mM hydroquinone or catechol in PBS was added to 0.5 mL of the working solution. No H₂O₂ was observed from either compound, similar to the gas vapor phase bubbled extract data shown in Fig. 3. This is in contrast to Nakayama et al. who reported H₂O₂ generation from catechol, 3-methylcatechol, 4-methylcatechol, and hydroquinone, using 5 μmol in DMSO added to 100 mM phosphate buffer at pH 7.4 and aged for 30 min.⁶ Yang et al. measured semiquinone radicals, the presumed precursor of H₂O₂, from catechol and hydroquinone solutions that were aged for 6 months in 50 mM phosphate buffer at pH 7.4 under air in an open bottle and then investigated in a carbonate buffer at pH 9.0.³³ Pederson showed that semiquinone anion radicals of para- and ortho-hydroquinones exist at physiological pH in buffered solution under continuous access to molecular oxygen in a flowing system.³⁴ These observations suggest that other, perhaps multiple, compounds are necessary to initiate the quinone/semiquinone/hydroquinone catalytic redox cycle to produce H₂O₂. Metal ions, such as Fe²⁺, are most likely required, but an alkaline pH or continuous access to molecular oxygen may also be necessary.

Finally, although we have used the Amplex Red assay to successfully detect H₂O₂ in smoke-bubbled aqueous solutions, and the results have been found to be similar to other analyses by other investigators, there are a number of complications with this assay which are pointed out by Towne et al.²⁵ Among these are the fact that the fluorescent product, resorufin, of the Amplex Red assay is itself a substrate for HRP, that oxidation of resorufin leads to nonfluorescent product(s), variation in fluorescence intensity of resorufin in aqueous solution in the pH range 6.2–7.7, and the fact that inhibition/inactivation of HRP may take place when high levels of H₂O₂ are present. Extensive studies by Towne et al. revealed that both pH and resorufin concentration had significant effects on the kinetics of fluorescence decay of resorufin.²⁵ In practical terms, this means that the pH of the buffer used for the assay must be carefully chosen, and that the buffering capacity must be sufficient for the anticipated use. Towne et al. further observed that below the K_m of H₂O₂ (1.55 μM), the reaction rate is approximately proportional to H₂O₂ concentration at a given Amplex Red concentration, when the HRP concentration was 0.0041 U mL⁻¹.²⁵ As the authors point out, however, much higher amounts of HRP (100–500 fold higher) are used for routine quantitative H₂O₂ assay, as this assures quick and complete turnover of H₂O₂ by excess Amplex Red and HRP, thus minimizing the undesirable conversions of resorufin to other compounds. Towne et al. show an extensive dose–response study for the assay of H₂O₂ by Amplex Red, and found that a reasonable proportionality was found between 0.1 and 100 μM concentrations of H₂O₂ and fluorescence intensity, when the Amplex Red concentration was 160 μM, HRP concentration was 0.41 U mL⁻¹, and incubation times were 2 or 15 min at room temperature.²⁵ Above 100 μM H₂O₂, the fluorescence decreased markedly. The assay conditions in this present paper were within the ranges where proportionality between H₂O₂ concentration and fluorescence intensity would be expected to occur. Towne et al. provide a enzymatic rate of 0.0032 μmol min⁻¹ for the reaction of Amplex Red with H₂O₂, when the HRP concentration is 0.0041 U mL⁻¹.²⁵ Seong et al.,²⁶ using a continuous flow microfluidics system, observed a V_max of 1.90 μmol min⁻¹ when the Amplex Red concentration was 10 μM, and the HRP concentration was 0.01 U mL⁻¹ in pH 7.4 buffer. These kinetic data suggest again that shorter assay times are to be preferred for the H₂O₂ assay. Towne et al., ²⁵ based on their studies, suggest several practical suggestions for using the Amplex Red assay for H₂O₂. Among these are keeping the incubation time as short as possible and lowering the HRP concentration at high H₂O₂ concentrations.

Conclusions

For the first time, we have demonstrated the feasibility of measuring H₂O₂ production from aqueous extracts of cigarette smoke without prior separation of the complex cigarette sample matrix. Concentrations of 3–8 μM H₂O₂ were found in aqueous solutions of whole smoke bubbled samples, while there was negligible H₂O₂ formation from gas vapor phase bubbled samples. The time dependence of H₂O₂ production in cigarette smoke-bubbled PBS as measured by Amplex Red assay shows an increase up to 120 min and reaches a plateau thereafter, suggesting H₂O₂ was formed over this time period by some reaction(s) involving the highly complex mixture of chemical constituents present in cigarette smoke. The data in this work suggest that the major constituents responsible for H₂O₂ formation are in the particulate phase. The ability to monitor H₂O₂in situ and in model systems will advance our understanding of the relationship between cigarette smoke-induced oxidative stress, inflammation, and disease and help elucidate the pathogenic mechanisms and the causative smoke constituents.

Acknowledgements

This research was sponsored by Philip Morris USA. Serene Williams was supported by an appointment to the Department of Energy Science Undergraduate Laboratory Internships (SULI) program. Fei Yan and Ramesh Jagannathan are also supported by an appointment to the Oak Ridge National Laboratory Postdoctoral Research Associates Program. The Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the U.S. Department of Energy under contract DE-AC05-00OR22725.

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