Free Radical and Nicotine Yields in Mainstream Smoke of Chinese Marketed Cigarettes: Variation with Smoking Regimens and Cigarette Brands

Abstract

Free radicals and nicotine are components of cigarette smoke that are thought to contribute to the development of smoking-induced diseases. China has the largest number of smokers in the world, yet little is known about the yields of tobacco smoke constituents in different Chinese brand of cigarettes. In this study, gas-phase and particulate-phase free radicals, as well as nicotine yields, were quantified in mainstream cigarette smoke from five popular Chinese brands and two research cigarettes (3R4F and 1R6F). Mainstream smoke was generated under International Organization of Standardization (ISO) and Canadian Intense (CI) smoking regimens using a linear smoking machine. Levels of free radicals and nicotine were measured by electron paramagnetic resonance spectroscopy (EPR) and gas-chromatography with flame-ionization detection, respectively. Under the ISO puffing regimen, Chinese brand cigarettes produced an average of 3.0 ± 1.2 nmol/cig gas-phase radicals, 118 ± 44.7 pmol/cig particulate-phase radicals, and 0.6 ± 0.2 mg/cig nicotine. Under the CI puffing regimen, Chinese brand cigarettes produced an average of 5.6 ± 1.2 nmol/cig gas-phase radicals, 282 ± 92.1 pmol/cig particulate-phase radicals, and 2.1 ± 0.4 mg/cig nicotine. Overall, both gas and particulate-phase free radicals were substantially lower compared to the research cigarettes under both regimens, whereas no significant differences were observed for nicotine levels. When Chinese brands were compared, the highest free radical and nicotine yields were found in “LL” and “BS” brands, while lowest levels were found in “YY”. These results suggested that the lower radical delivery by Chinese cigarettes compared to US reference cigarettes may be associated with reductions in oxidant-related harm.

Graphical Abstract

INTRODUCTION

It is estimated that tobacco kills more than seven million people each year around the world ¹. More than 7000 chemicals have been identified in tobacco with at least 150 of which are known to be harmful to active and passive smokers ². In 2010, the United States (U.S.) Surgeon General clearly delineated tobacco free radicals as important factors in the etiology of tobacco-related diseases ³, such as respiratory and cardiovascular diseases and cancer ^4–6. In the 1980s, Pryor and colleagues characterized cigarette smoke radicals into two types, reactive gas-phase radicals and more stable particulate-phase radicals ⁷. Gas-phase radicals have been shown to cause oxidative stress in the lung and upper digestive tract, while particulate-phase radicals can penetrate deep into the body and result in systemic consequences ⁸. Nicotine is another harmful constituent in mainstream smoke and is responsible for the addictiveness of cigarettes ⁹. High dependence on nicotine is an independent risk factor for several major causes of death from tobacco-related diseases, such as pulmonary impairment, atherosclerosis, and chronic obstructive pulmonary disease ¹⁰. Therefore, it is important to quantitatively evaluate a smoker’s exposure to free radicals and nicotine from mainstream cigarette smoke to better inform public health and potential regulation of tobacco smoke constituents.

In order to compare different tobacco products in the laboratory, standardized machine puffing protocols are used ¹¹. The World Health Organization (WHO) Study Group on Tobacco Product Regulation recommends two common protocols, the International Organization of Standardization (ISO) and the Canadian Intense (CI) smoking regimes, to measure toxicant deliveries. The ISO is a less-intensive method than CI which has a greater puff frequency and puff volume ^{12, 13}. Two different methods are recommended to capture the variation in individual smoking behavior, which can significantly impact toxicant levels generated from cigarettes ^14–16.

Tobacco use is still a serious public health challenge in China due to high rates of smoking prevalence, low cigarette price, and heavy economic burden of tobacco-related diseases ¹⁷. The Chinese Adults Tobacco Survey Report found that the number of adult smokers in China has reached 27.7% (316 million) in 2015 ¹⁸. In 2009, the WHO reported that China consumed 38% (2.2 trillion) of the world’s cigarettes, which is more than the other top four tobacco consuming countries combined ¹⁹. As the largest tobacco producer and consumer, China plays a critical role in the WHO Framework Convention on Tobacco Control ²⁰.

Like other countries, China produces its own domestic brands of cigarettes. Previous studies of U.S. manufactured cigarettes indicated that levels of mainstream smoke toxicants (e.g. volatile organic compounds, nicotine, carbonyls, free radicals) differed by brand with potential variations in tobacco type, ventilation, additives and paper porosity ^21–23. For example, the level of gas-phase radicals varied widely (12-fold) across the 27 popular U.S. brand cigarettes, ranging from 1.2 to 14 nmol per cigarette ²⁴. Although data on free radical and nicotine delivery from cigarette brands from western countries cigarette brands are available, there are limited data on cigarettes from other regions including China.

The United States and the WHO are developing regulatory strategies to limit harmful constituents in tobacco smoke. Quantifying these exposures are necessary first steps to inform regulatory policies. The main aim of this study was to systematically characterize and compare the free radical and nicotine production in mainstream smoke from five popular Chinese domestic brand cigarettes machine-smoked using ISO and CI protocols.

MATERIALS AND METHODS

Cigarette selection.

The 3R4F Kentucky reference cigarette has been widely used as a comparator cigarette for the analysis of mainstream smoke constituents ²⁵. 1R6F, a replacement cigarette for 3R4F, has also been used as a reference in a limited number of studies ^{26, 27}. 3R4F and 1R6F were obtained from the University of Kentucky (Lexington, Kentucky, USA). Five Chinese commercial brands (“BS”, “SX”, “YY”, “LL” and “ZH”) were selected based on their popularity and combined share of the Chinese cigarette market (Figure 1 and Table 1). Among all the brands, “LL” and “SX” occupied the top spots of tobacco consumption in the Taiwan and China mainland markets, respectively. Cartons of each of the five brands were purchased at Pudong International Airport, Shanghai, China in 2017. The cigarettes with the original packaging were shipped to the Tobacco Center of Regulatory Science, Pennsylvania State University College of Medicine, where they were put in airtight plastic bags and coldly stored at −20°C until testing. All selected Chinese cigarettes are king size (84 mm in length) and are not marketed based upon any particular flavor (like menthol).

Figure 1.

Photograph of the Chinese cigarettes used in the study: (1) SX, (2) LL, (3) BS, (4) ZH, (5) YY.

Table 1.

Characteristics of the Chinese cigarette brands tested in this study.

Brand	UPC	Pack type	Filter length (mm)	Tobacco type	Tar content (mg/cig)	Nicotine content (mg/cig)
SX	6 901028 076586	Red hard pack	20	flue-cured	8	0.7
ZH	6 901028 076883	Red/gold hard pack	27	flue-cured	11	1.0
BS	6 901028 191029	White hard pack	20	flue-cured	10	1.0
YY	6 901028 311007	Black/red hard pack	30	flue-cured	11	1.1
LL	4 711588 110901	Yellow hard pack	20	blended	10	0.8

Note: Tobacco type, tar and nicotine content as listed by manufacturer. Filter length was determined in the laboratory.

Materials.

Analytical grade of nitrone spin trap phenyl-N-tert-butylnitrone (PBN), 2,2,6,6-tetramethyl-1-piperidinyloxyl (TEMPO), tert-butylbenzen, heptadecane and 4-hydroxy-2,2,6,6 tetramethylpiperidine 1-oxyl (TEMPOL) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Methanol was obtained from Fisher Scientific (Pittsburgh, PA, USA). Suprasil electron paramagnetic resonance (EPR) tubes (4 mm o.d; Wilmad-Labglass, Vineland, NJ, USA), Schlenk line (Chemglass Life Sciences, Vineland, NJ, USA) and Cambridge filters pads (CFP, Performance Systematix Inc., Grand Rapids, MI, USA) were used as supplied.

Mainstream smoke generation.

Before smoking, the cigarettes were conditioned in a constant relative humidity (60±3%) and temperature (22±2°C) chamber for at least 48 h. Mainstream smoke was generated by a single-port liner smoking machine (Human Puff Profile Cigarette Smoking Machine (CSM-HPP), CH Technologies, NJ, USA). Each research or commercial cigarette was smoked to a length 3 mm prior to the marked filter overwrap (tipping) under both ISO (ISO 3308: 2012: 35 mL puff volume, 60-s puff interval, 2-s duration, and no filter vents blocked) and CI (CI T-115:1999: 55 mL puff volume, 2-s duration, 30-s puff interval and filter vents blocked with clear tape) standard smoking regimens.

Analysis of free radicals and nicotine.

Mainstream smoke was separated into particulate-phase and gas-phase by passing through a 47 mm CFP, which was placed upstream of the smoke machine pump to trap particulate-phase smoke for the free radical and nicotine analysis. An impinger containing 4 mL ice-cold tert-butylbenzene and 0.05 mol/L PBN was located downstream of the pump to trap gas-phase smoke for free radical analysis. After smoking, CFPs were put in an airtight plastic bag, and aliquots of the PBN solution were immediately frozen in liquid nitrogen. They were stored at −80 °C until further analysis.

Both types of radicals were analyzed on a Bruker eScan R spectrometer (Bruker-Biospin, Billerica, MA, USA) operating in X-band as we have done previously ⁸. In brief, particulate-phase radicals trapped on the CFP were analyzed by inserting the CFP directly into the cavity of a spectrometer. The EPR parameters were as follows: microwave frequency, 9.7 GHz; modulation frequency, 86.0 kHz; microwave power, 6.00 mW; scan range, 50 G; modulation amplitude, 1.10 G; sweep time, 5.243 s; time constant, 10.240 ms; and conversion time, 10.240 ms. All measurements were carried out at room temperature (22 ± 1 °C). Spin concentrations were determined by integration of the area under the curve of the EPR signal using WinEPR software (version 0.98, National Institute of Environmental Health Sciences, National Institutes of Health, USA). Standardized concentrations of TEMPOL in methanol or a blank methanol solution pipetted onto CFP were used to quantify the spin concentrations of cigarette particulate-phase radicals. Gas-phase radicals were also analyzed as described previously ⁸. In short, 400 μL aliquots were deoxygenated using a Schlenk line technique of three froze–pump–thaw cycles with argon ²⁸, and then followed by EPR analysis. The EPR parameters for gas phase radicals: microwave frequency, 9.7 GHz; modulation frequency, 86.0 kHz; microwave power, 6.00 mW; scan range, 60 G; modulation amplitude, 1.10 G; sweep time, 41.94 s; time constant, 81.92 ms; and conversion time, 81.92 ms. All measurements were carried out at room temperature (21.5 ± 0.5 °C). Spin quantification of the radical signals obtained was performed in MatLab. Each spectrum was processed automatically to produce a double integral. In the process, point-based-spline baseline correction was applied to the absorption data (first integral) prior calculation of the second integral. Conversion factors from double integral values to spin concentrations we obtained from the known concentrations of a stable radical standard, TEMPO ²⁹.

Following ISO and CI smoking, nicotine was extracted from the CFP using 20 mL of methanol and heptadecane was added as an internal standard. Nicotine was quantitatively analyzed by Gas Chromatography with Flame Ionization Detection (GC-FID) (HP 5890) as described previously ³⁰.

Data analysis.

All experiments were repeated at least in triplicate. The results are presented as mean ± standard deviations (SD) in two different ways (per puff and per cigarette) to provide a full interpretation of the findings. All pairwise comparisons were evaluated by conducting one-way ANOVA with Tukey’s multiple comparison test via GraphPad prism 5.0 software (MacKiev GraphPad Software, Inc., San Diego, CA, 1994–2005), and the level of statistical significance was set to P < 0.05.

RESULTS

Physical characteristics.

Characteristics of the Chinese cigarette brands tested in this study are shown in Table 1. A cross-sectional study conducted in 21 cities in China found that 40.1 % of smokers chose cigarettes at a price of 5 to 10 Yuan per pack (20 manufactured cigarettes) ³¹. The price of cigarettes tested in the current study ranged from 6 to 10 Yuan per pack, except for one brand (30 Yuan per pack) which is considered a high-grade cigarette and occupied more than 60% of the Chinese high-grade cigarette market share in 2010 ³². The filter lengths for each brand ranged from 20 to 30 mm. According to the self-reported information from manufacturers, tar and nicotine content of different brands ranged from 8 to 11 mg /cig, and from 0.7 to 1.1 mg/cig, respectively.

Free radical and nicotine yields under ISO and CI regimens.

As expected, the deliveries of free radicals and nicotine from Chinese cigarette brands were greater when smoke was generated using the more intensive CI protocol compared to the ISO protocol (Figures 2–4). Across all brands measured in this study (i.e. five Chinese and two reference cigarettes), the CI puffing regimen produced a significant 3.6 fold increase (95% CI: 3.0–4.1) in gas-phase radicals, 2.0-fold increase (95% CI: 1.5–2.5) in particulate-phase radicals and 3.3-fold increase (95% CI: 2.9–3.8) in nicotine yields compared to the ISO regimen.

Figure 2.

Comparison of gas-phase free radical yields of cigarettes under ISO and CI standardized smoking regimens. * indicates p < 0.05 compared to the ISO method for the same brand.

Figure 4.

Comparison of nicotine yields per cigarette of cigarettes under ISO and CI standardized smoking regimens. * indicates p < 0.05 compared to the ISO method for the same brand.

Free radical and nicotine yields among different brand cigarettes.

The differences in free radical and nicotine yields generated from Chinese commercial and both research cigarettes were analyzed. As shown in Table 2, gas-phase radical yields from U.S. reference cigarettes (1R6F and 3R4F) were 3.70- to 4.40-fold higher than Chinese commercial brands using the CI protocol (p<0.001) and 1.60- to 1.77-fold higher (p<0.001) using the ISO protocol (p<0.001). For particulate-phase radicals, U.S. reference cigarettes yields were ~1.96-fold higher (p<0.001) than Chinese commercial brands using the CI protocol and 2.19- to 2.76-fold higher using the ISO protocol (p<0.001). However, there was no significant difference in nicotine production between the Chinese commercial brands and 1R6F or 3R4F, under either the ISO (p=0.097 for 1R6F and p=0.134 for 3R4F) or CI methods (p=0.413 for 1R6F and p=0.284 for 3R4F).

Table 2.

Comparison of free radicals and nicotine yields in mainstream smoke of Chinese brand and the reference cigarettes.

	ISO regimen			CI regimen
	Chinese cigarettes	3R4F	1R6F	Chinese cigarettes	3R4F	1R6F
Gas-phase radicals, nmol/cig	3.0 ± 1.2	4.8 ± 0.7	5.3 ± 0.6	5.6 ± 1.2	24.6 ± 5.7	20.7 ± 4.3
Particulate-phase radicals, pmol/cig	118 ± 44.7	326 ± 32.9	259 ± 54.1	282 ± 92.1	551 ± 55.9	553 ± 63.9
Nicotine, mg/cig	0.6 ± 0.2	0.7 ± 0.0	0.7 ± 0.1	2.1 ± 0.4	2.3 ± 0.1	2.1 ± 0.2

In addition, we compared the free radical and nicotine yields from mainstream smoke of five commercial cigarette brands (Table 3 and Figure 2–4). Overall, results of gas-phase radicals across five Chinese cigarettes varied 3.5-fold for ISO and 2.0-fold for the CI regimen. Similar variations were also found in particulate-phase radicals (4.7-fold (ISO) and 2.0-fold (CI)) and nicotine (3.5-fold (ISO) and 2.0-fold (CI)). Specifically, “YY” brand generated the lowest of both gas- and particulate-phase radicals and nicotine under ISO, and the lowest particulate-phase radicals under CI, while “ZH” and “SX” brands produced the lowest gas-phase radical and nicotine delivery under CI. “LL” generated the highest gas- and particulate-phase radicals under ISO, and the highest gas-phase radical (6.8 ± 0.0 nmol/cig) under the CI method. “BS” brand produced the highest nicotine under both smoking regimens. The highest particulate-phase radical production under CI was found in “SX” brand. Significant differences of the one-way ANOVA analysis are listed in Table 4.

Table 3.

Comparison of free radicals and nicotine yields in mainstream smoke among different Chinese brand cigarettes.

	Gas-phase radicals, nmol/cig		Particulate-phase radicals, pmol/cig		Nicotine, mg/cig
	ISO regimen	CI regimen	ISO regimen	CI regimen	ISO regimen	CI regimen
SX	3.6 ± 0.6	6.3 ± 1.1	130.7 ± 54.3	406.3 ± 77.6	0.5 ± 0.2	1.5 ± 0.2
ZH	2.2 ± 1.2	4.5 ± 1.0	92.6 ± 7.4	295.8 ± 70.4	0.6 ± 0.1	1.9 ± 0.2
LL	4.3 ± 1.1	6.8 ± 0.0	156.7 ± 58.1	282.3 ± 32.6	0.6 ± 0.1	2.5 ± 0.2
YY	1.7 ± 0.2	4.6 ± 0.8	81.9 ± 29.8	163.7 ± 33.4	0.3 ± 0.1	2.1 ± 0.4
BS	3.2 ± 0.4	5.9 ± 0.9	128.3 ± 34.1	260.8 ± 33.4	0.8 ± 0.1	2.5 ± 0.2

Table 4.

Statistical significant difference based on the ANOVA for the comparison of free radicals and nicotine yields among different brands Chinese cigarettes.

	Brand	p-value (ISO regimen)				p-value (CI regimen)
		SX	ZH	LL	YY	SX	ZH	LL	YY
Gas-phase radicals	ZH	0.032*				0.511
	LL	0.266	0.003*			0.868	0.412
	YY	0.006*	0.444	0.000*		0.551	0.951	0.447
	BS	0.530	0.169	0.113	0.047*	0.888	0.604	0.759	0.647
Particulate-phase radicals	ZH	0.296				0.019*
	LL	0.472	0.087			0.010*	0.758
	YY	0.185	0.766	0.049*		0.000*	0.006*	0.013*
	BS	0.946	0.326	0.433	0.207	0.003*	0.428	0.624	0.037*
Nicotine	ZH	0.038*				0.058*
	LL	0.035*	0.967			0.000*	0.017*
	YY	0.131	0.001*	0.001*		0.013*	0.460	0.074*
	BS	0.000*	0.023*	0.025*	0.000*	0.000*	0.008*	0.722	0.037*

^*p<0.05

Furthermore, considering the difference in the number of puffs (5–10 puffs for ISO and 7–11 puffs for CI), we also calculated free radical and nicotine yields per puff from mainstream smoke of all cigarettes, the results were shown in Table S1 and Figure S1–S3. Gas-phase radical yield per puff was higher in 1R6F and 3R4F than in Chinese commercial brands cigarettes. Similar associations for particulate-phase radicals were found between the research cigarettes and Chinese commercial brand cigarettes. It should be noted that nicotine yield was same in both the research cigarettes and Chinese commercial brand cigarettes, with the exception of “LL” under CI.

Associations of free radicals and nicotine.

Free radical levels were reported to be associated with cigarette ventilation ¹⁵, which also is an important determinant of nicotine yield. To determine if free radicals are associated with nicotine yield, correlational analyses were performed to examine the potential associations among nicotine, gas-phase radical and particulate-phase radical delivery in Chinese brand cigarettes. Free radical levels in the gas-phase was moderately correlated with those in the particulate-phase (r = 0.39 for ISO method, r= 0.36 for CI method). No strong correlations were found for nicotine yield with gas-phase free radicals (r = 0.20 for ISO method, r = 0.01 for CI method) or particulate-phase free radicals (r = 0.25 for ISO method, r = 0.01 for CI method).

DISCUSSION

Free radical delivery from cigarette smoking may inflict oxidative stress and result in oxidative DNA, protein and lipid damage, which are commonly accepted as a critical cause of respiratory, cardiovascular and other diseases ^33–35. The amount of nicotine delivered from cigarette smoking is also critical in causing diseases, as it can be directly associated with smoking dependence and cigarette usage ³⁶. However, there is little information on the levels of free radicals and nicotine generated from Chinese cigarettes. The present study selected five popular commercial brands from the Chinese cigarette market, and conducted a comparative examination of the effect of cigarette brand on free radical and nicotine yields when smoked under both less intensive (ISO) or more intensive (CI) conditions. As expected, higher levels of nicotine and free radicals were observed for the CI protocol compared to the ISO protocol. Generally, among the five Chinese brand cigarettes, free radical yield was lower in “YY” brand and higher in “LL” brand, and nicotine yield was higher in “BS” brand. Lower gas- and particulate-phase radical yields were found in all Chinese cigarettes than both of the research cigarettes (1R6F and 3R4F); however, nicotine yields were similar between Chinese brands and the reference cigarettes.

When cigarettes were smoked using the CI protocol, there was a significant (p<0.05), 2- to 4-fold higher yields for gas- and particulate-phase radicals as well as nicotine over the ISO protocol. These results corroborated the findings of a great deal of the previous work on cigarettes in other countries ^{11, 15, 27, 37}. For example, a study on Korean cigarettes were conducted to test 25 compounds containing nicotine and other toxic constituents from five domestic brands, and consistently found that most of the constituents showed a tendency to be detected at levels 2 to 4 times higher under the CI regime than under the ISO regime ³⁷. The higher free radical and nicotine yields under the CI regimen may be attributed to its machine-smoking protocol including larger total puff volume, puff frequency, and blocked filter vents compared to ISO ^{16, 38, 39}.

Here, we reported for the first time that the levels of free radical and nicotine yields in mainstream smoke of Chinese cigarettes. Both gas- and particulate-phase radical yields of 3R4F and 1R6F reference cigarettes in this study were similar to those measured in our previous study under both ISO and CI methods ¹⁵. We also found that Chinese commercial cigarettes produced lower levels of free radicals as compared to U.S. commercial cigarettes ¹⁵. Nicotine yields of 3R4F and 1R6F reference cigarettes in this study were similar to those measured in a recent study ²⁵. The mean nicotine yields of the reference cigarettes were not significantly different with that of Chinese brand cigarettes (0.6 ± 0.2 mg/cig for ISO and 2.1 ± 0.4 mg/cig for CI regime). Under either smoking protocol, the mean nicotine yields of the US reference cigarettes were not significantly different with that of Chinese brand cigarettes and were consistent with previous results from US cigarette brands ^{22, 40}, but higher than that from Korean cigarettes ³⁷. These results suggested that differences in free radical and nicotine delivery from different country’s cigarettes should be considered in comparative disease epidemiology studies ⁴¹.

Furthermore, we found that the levels of free radical and nicotine in the mainstream smoke varied widely by Chinese cigarette brands. The variations of cigarette composition levels by brand may result from each cigarette’s design features (e.g. filter length, filter ventilation and tobacco blend) ^{42, 43}. For example, a previous study documented that the length of the cigarette filter had a significant effect on the labsorption of the hazardous chemical components produced by cigarettes ⁴⁴. Lower free radical and nicotine levels of “YY” brand may be attributed to its longer filter (30 mm). In addition, previous studies have shown that different types of tobacco (e.g. reconstituted, burley, oriental, bright tobacco) varied widely free radical delivery and produced different levels of cytotoxicity ^{15, 45}. U.S. brand cigarettes and the reference cigarettes (3R4F and 1R6F) are typically made from a mixture of different tobacco types ⁴⁶, while, most of the Chinese brand cigarettes tested were made with flue-cured tobacco, with the exception of “LL” (Table 1). Tobacco blend variety is an important modifiable design of cigarettes that could be manipulated to minimize toxicant exposure.

Our study has some limitations. Firstly, we were unable to assess more brands in the Chinese cigarette market. Secondly, in the present study, we focused primarily on the determination of total nicotine yields without specifically looking at differences based on protonation states of nicotine. Finally, we do not clearly understand the factors contributing to brand-specific differences in free radical and nicotine generation. The potential impacts of both physical and design characteristics (e.g. filter ventilation, flavor additives, paper porosity, tobacco blend and packing density) of Chinese brand cigarettes on tobacco carcinogen delivery warrant further exploration.

In summary, the present study provided new information on the free radical and nicotine levels generated by commercial Chinese cigarettes. Smoking regimens and brands are key factors in determining free radical and nicotine production in Chinese cigarette. Strikingly different production of free radicals and nicotine levels under different smoking-regimens suggest that variation in human smoking behaviors may be associated with greater levels of oxidative stress and nicotine addiction in smokers. Differing levels of free radical and nicotine delivery may be helpful in better understanding international differences in smoking-related disease epidemiology. For example, the risk of lung cancer associated with cigarette smoking is lower in China compared to the U.S. 47. Further investigation into the wide brand-to-brand variation in free radicals and nicotine production may help identify ways to reduce smoking exposure and to guide potential future regulatory strategies.

Figure 3.

Comparison of particulate-phase free radical yields of cigarettes under ISO and CI standardized smoking regimens. * indicates p < 0.05 compared to the ISO method for the same brand.

ABBREVIATIONS

CFP

Cambridge filters pads

CI

Canadian Intense

EPR

electron paramagnetic resonance

ISO

International Organization of Standardization

PBN

phenyl-N-tert-butylnitrone

TEMPO

2,2,6,6-tetramethyl-1-piperidinyloxyl

WHO

World Health Organization