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The effect of cigarette burn time on exposure to nicotine and carbon monoxide in adult smokers
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Regulatory Toxicology and Pharmacology 50 (2008) 66–74 www.elsevier.com/locate/yrtph
The effect of cigarette burn time on exposure to nicotine and carbon monoxide in adult smokers Qiwei Liang *, Hans J. Roethig, Peter J. Lipowicz, Yan Jin, Paul E. Mendes Philip Morris USA, Research Center, 4201 Commerce Road, Richmond, VA 23234, USA Received 13 February 2007 Available online 29 August 2007
Abstract Cigarette burn time (CBT), conventionally defined as the time a cigarette burns during smoking, can be affected by cigarette design and smoking behavior. A previous study showed a strong negative correlation between CBT and nicotine yield under machine smoking conditions. This study for the first time examined the relationship of CBT and exposure to nicotine and carbon monoxide in adult smokers in a controlled clinical study. 24 h nicotine equivalents excretion (NE), carboxyhemoglobin (COHb) and CBT were measured in two groups of 20 adults smoking Marlboro Lights and 20 adults smoking Marlboro Ultra on two consecutive days. Approximately 20% of the total variability in CBT was attributed to cigarette brand, 34% to smokers and 1% to study day. The exposure index, defined as the number of cigarettes smoked per day divided by average daily CBT for each smoker, accounted for a large proportion of the total variability in NE (R2 = 0.79–0.91) and COHb (R2 = 0.85–0.90). We conclude that CBT has an important influence on levels of NE and COHb in adult smokers. CBT, along with the number of cigarettes smoked per day, can be used to estimate adult smokers’ exposure to nicotine and carbon monoxide. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Cigarette burn time; Exposure; Nicotine equivalents; Carboxyhemoglobin; Statistical; Relationship; Exposure index; Estimate
1. Introduction Exposure to cigarette smoke, a major health concern, varies considerably from smoker to smoker (Benowitz et al., 1997, 2006; Feng et al., 2006, 2007; Hatsukami et al., 2006; Mendes et al., 2007). The variability in daily exposure is due to not only the number of cigarettes smoked per day, but also, to a large extent, the difference in cigarette puffing behavior. Cigarette puffing behavior can be measured with a special topography device (Bridges et al., 1990; Hammond et al., 2005; Lee at al., 2003; Roethig et al., 2007). However, the everyday puffing behavior of a smoker may be impacted by the measuring procedure because a smoker has to smoke through the device. Cigarette burn time (CBT) is conventionally defined as the time a cigarette burns during smoking. It is influenced *
Corresponding author. Fax: +1 804 2742891. E-mail address: [email protected] (Q. Liang).
0273-2300/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.yrtph.2007.08.010
by cigarette design (Browne, 1990; Robertson, 2000) and the puffing behavior of the individual smoker. Compared to cigarette puffing topography, CBT can relatively easily be measured without special devices and it is not expected to impact the everyday puffing behavior of a smoker. Under machine smoking conditions with a FTC puffing regimen (Federal Register, 1967, 1980), CBT is typically about 7 min (Lipowicz and Dwyer, 2001). Lipowicz and Dwyer (2001) conducted a study under 27 different machine puffing regiments to examine CBT in different test cigarettes. They found that CBT is influenced by puff volume, inter puff interval and ventilation hole blocking. They also found a strong negative correlation between CBT and nicotine yield, meaning that the longer the CBT (i.e. the less intensive the smoking), the lower the nicotine yield. The findings established a quantitative relationship between CBT and nicotine yield and identified a potentially important source of variability in nicotine yield. However, smoking machine conditions do not reflect the full
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complexity of human smoking behavior. So the question arises whether CBT determined during human smoking would reflect human smoking behavior and might, in part, explain the large variability in exposure observed in adult smokers (Benowitz et al., 1997, 2006; Roethig et al., 2005, 2007; Feng et al., 2006, 2007; Mendes et al., 2007). Nicotine and carbon monoxide are frequently measured constituents of cigarette smoke, which is a complex chemical mixture with about 4800 chemical constituents identified in mainstream smoke (Green and Rodgman, 1996). Nicotine equivalents excretion in urine and carboxyhemoglobin (COHb) in blood are validated biomarkers of exposure for these two smoke constituents (Stratton et al., 2001). Substantial inter-subject variability of these two biomarkers has been found in smokers (Roethig et al., 2005, 2007). Knowledge of CBT and its relation to smoker’s levels of nicotine equivalents and COHb might be important for understanding some aspects of smoking behavior and therefore some sources of variability in nicotine equivalents and COHb in smokers. The purposes of this study were to: (1) characterize the variability of CBT between cigarette brands, study days and smokers; (2) examine the quantitative relationships between average daily CBT and adult smokers’ exposure to nicotine and carbon monoxide as determined from biomarkers of exposure and (3) develop a statistical model to predict nicotine equivalents and COHb based on an index of daily cigarette consumption and average daily CBT. 2. Clinical study design This study was a sub-study within a larger, randomized, controlled short-term exposure study to evaluate different cigarette products. The results of the main study have been published elsewhere and details can be found there (Roethig et al., 2005). The study was performed at the clinical study site of MDS Pharma Services at Lincoln, Nebraska and was approved by the local institutional review board (IRB). All participating subjects had given written informed consent before participating in the study and were free to withdraw from the study at any time for any reason. 100 adult, male and female smokers of an 11 mg tar (FTC) cigarette (Marlboro LIGHTS) were confined to the clinical unit for 10 days. After taking baseline measurements, the adult smokers were randomized to 5 different groups: group A continued smoking Marlboro Lights; groups B was switched to Marlboro Ultra (Philip Morris International, 3 mg FTC tar); C and D were switched to two different types of Electrically Heated Cigarette Smoking Systems (3 mg tar FTC) and group E stopped smoking for 8 days. Controlled smoking was used as a measure to keep the numbers of cigarettes smoked per day as constant as possible. Adult smokers could smoke up to a maximum number of cigarettes per day determined as the number they would smoke regularly per day. Smoking was only allowed from 7:00 A.M. to 11:00 P.M. with smoking opportunities every 32 min.
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CBT was measured on two consecutive study days during this 8 day period in 20 adult male and female smokers of Marlboro Lights King Size (Philip Morris USA brand, 11 mg tar, 0.8 mg nicotine, 12 mg CO, FTC delivery) and in another 20 adult male and female smokers of Marlboro Ultra (Philip Morris International brand, 3 mg tar, 0.3 mg nicotine, 4 mg CO, FTC delivery). All cigarettes smoked during these two days had been marked with a line 3 mm from the filter wrapper. One day before measuring the CBT, all smokers were trained on how to monitor and record their CBT. During the two study days, smokers documented CBT for all cigarettes smoked during that day. The start of CBT coincided with the lighting of the cigarette and the end of CBT was when the lit part of the cigarette reached 3 mm from the filter wrapper (this was marked with a pencil line). The smokers were asked to smoke in their usual way but had to stop puffing when the burning zone of the cigarette reached the 3 mm line. They could stop puffing whenever they wanted to, but had to let the cigarette burn until the burning zone reached the 3 mm line. All measurements were supervised by the study staff to ensure accuracy and compliance with the study protocol. Urine nicotine and five of its metabolites (nicotine-Nglucuronide, cotinine, cotinine-N-glucuronide, trans-3 0 hydroxycotinine, and trans-3 0 -hydroxycotinine-O-glucuronide) were measured by a liquid chromatography/tandem mass spectrometry (LC-MS/MS) method (Roethig et al., 2005) in 24 h urine collections on the first and second days. Blood samples for COHb were taken at 7:00 P.M. on the first day and were assayed spectrophotometrically with a CO oximeter (IL Multi-4, Instrumentation Laborartory, Lexington, Mass).
3. Statistical methods Descriptive statistics including mean, range, standard deviation and coefficient of variation were used to characterize the CBT data by smoker, cigarette brand and study day. In order to characterize the sequential change of CBT from one cigarette to the next, the CBT from all smokers were averaged sequentially from the first cigarette smoked in a day to the last cigarette. Box-and-whisker plots were used to characterize the average CBT series. The runs test, a non-parametric statistical method (Farnum and Stanton, 1989), was applied to examine whether the series of average CBTs was random, meaning no trend and independence between two adjacent cigarettes. The runs test is based on the premise that any observational series with randomness is equally likely to be above or below the median of the series. In the test, a run is defined as a series of increasing burn time values or a series of decreasing values. The number of increasing, or decreasing, value is the length of the run. The null hypothesis is that the CBT series is a random series and the test statistic is the number of runs above and below the median. Auto-regression method was also used to describe the autocorrelation between two adjacent cigarettes, which is called the first order autocorrelation in statistics. Autocorrelation of higher orders was not explored due to the relatively small sample size of the study. T test was used to test the statistical significance of difference in CBT between the first cigarette and the subsequent cigarettes (pooled) by cigarette brand and study day. Variance component analysis (Hicks, 1982) was used to examine the effect of cigarette brand, smokers and study day on CBT.
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The model included terms for cigarette brand, smokers and study day. The variance component for each factor was presented as the percentage of the total variability. Nicotine equivalents were calculated as the molar sum of total nicotine, total cotinine, and total trans-3 0 -hydroxycotinine excreted in the 24 h urine (Roethig et al., 2005). Linear regression analysis was used to examine the relationship between average daily CBT and daily nicotine equivalents excretion and COHb. An exposure index was defined and calculated as the number of cigarettes smoked per day divided by average daily CBT. It was used to predict nicotine equivalent excretion and COHb. In order to examine the relationships between CBT and cigarette puffing variables, we requested the raw dataset from Lipowicz and Dwyer (2001) and analyzed it with multiple linear regression. In the model, the response variable was CBT and the independent variables were puff volume, inter-puff interval and vent hole blocking. All statistical analyses were run with SAS Version 9.1 (SAS Institute, 2004).
4. Results 4.1. Demographics The age of the smokers for the cigarette burn time study ranged from 21 to 65 with an average age of 30.7 in the Marlboro Lights group and 30.8 in the Marlboro Ultra group. Male and female smokers were distributed almost
Table 1 Demographics Characteristic
Marlboro Lights group
Marlboro Ultra group
Age*
30.7 (10.5) 21–56 10/10 16/4 23.79 (2.75) 21–30
30.8 (10.6) 21–56 9/11 19/1 24.47 (3.49) 20–32
Gender** Race*** BMI* * ** ***
Mean (SD), range. Female/male. Caucasian/others.
equally between the two groups. The average body mass index was 23.8 in the Marlboro Lights group and 24.5 in the Marlboro Ultra group (Table 1). The smokers had smoked between 5 and 25 cigarettes per day for at least 5 years. 4.2. Statistical characteristics of cigarette burn time Mean CBT was 6 min and 45 s in the Marlboro Lights group and about 1 min shorter in the Marlboro Ultra group. Mean CBT was statistically significantly longer (p < 0.05) and more variable in the Marlboro Lights group than in the Marlboro Ultra group (Table 2). In the Marlboro Lights group, mean CBT was significantly different between the two study days (p < 0.05). In the Marlboro Ultra group, in contrast, mean CBT was not statistically different between the two study days (Table 3). By smoker, the coefficient of variation (CV) of mean CBT ranged from 9% to 33% with an average of about 17%. CBT also showed fluctuation from cigarette to cigarette for the two cigarette brands and during the two study days (Fig. 1). The run test and auto-regression analysis did not show a consistent trend in the fluctuations of average CBT from one cigarette to another. In both the Marlboro Lights group and the Marlboro Ultra group, the fluctuation was random on Day 1 (p > 0.10), but not on Day 2 (p < 0.05). In the Marlboro Lights group, there was no autocorrelation in average CBT between two adjacent cigarettes on Day 1 (p > 0.05), but there was a positive autocorrelation on Day 2 (p < 0.05), meaning that two adjacent cigarettes’ CBT tended to change in the same direction. For the Marlboro Ultra group, no autocorrelation between adjacent cigarettes was found on Day 1 (p > 0.10), but there was a negative autocorrelation on Day 2 (p < 0.05), meaning that two adjacent cigarettes’ CBT tended to change in opposite direction.
Table 2 Cigarette burn time (seconds) by cigarette brand Cigarette brand
N
Mean
Max
Min
SD
CV (%)
Marlboro Lights group Marlboro Ultra group
619 545
387.1* 324.5
919.0 692.0
204.0 162.0
93.7 66.5
24.20 20.48
*
Statistically significant from the Marlboro Ultra group (p < 0.05).
Table 3 Cigarette burn time (seconds) by cigarette brand and study day Cigarette brand and study day b
ML on day 1 ML on day 2 MULc on day 1 MUL on day 2 a b c * **
Na
Mean
Max
Min
SD
CV (%)
295 324 260 285
397.8* 377.4 324.7** 324.4
919.0 686.0 692.0 505.0
204.0 205.0 179.0 162.0
105.3 80.7 73.8 59.1
26.46 21.38 22.73 18.21
Total number of cigarettes smoked. Marlboro Lights Group. Marlboro Ultra Group. Statistically significant (p < 0.05) from ML on day 2. Not statistically significant (p > 0.05) from MUL on day 2.
1000
1000
900
900
Cigarette Burn Time (seconds)
Cigarette Burn Time (seconds)
Q. Liang et al. / Regulatory Toxicology and Pharmacology 50 (2008) 66–74
800 700 600 500 400 300 200
800 700 600 500 400 300 200 100
100 1
2
3 4
5
6
7
8 9 10 11 12 13 14 15 16 17 18 19 20 Cigarette Sequence
1
2 3
4 5 6
1
2
4 5
7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Cigarette Sequence
1000 Cigarette Burn Time (seconds)
1000 Cigarette Burn Time (seconds)
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900 800 700 600 500 400 300 200 100
900 800 700 600 500 400 300 200 100
1
2
3 4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20
Cigarette Sequence
3
6
7 8
9 10 11 12 13 14 15 16 17 18 19 20 21 Cigarette Sequence
Fig. 1. Box and whisper plot of cigarette burn time by each cigarette for adult smokers in: (a) the Marlboro Lights group on Day 1; (b) the Marlboro Lights group on Day 2; (c) the Marlboro Ultra group on Day 1 and (d) the Marlboro Ultra group on Day 2. The top and the bottom edges of the box represent the 75% and 25 percentiles, respectively. The middle line in the box represents the median and the plus symbol is for the mean. The upper and the lower ends of the whisper represent the maximum and the minimum values.
About 20% of the total variability in CBT was attributed to difference between cigarette brands, 34% to difference between smokers and 1% to difference between study days. The remaining 45% variability was due to other unknown factors (Table 4). Both nicotine equivalents (R2 = 0.71–0.80) and COHb (R2 = 0.69–0.74) showed a good correlation with the number of cigarettes smoked per day (Tables 5 and 6). 4.3. Relationship between cigarette burn time and nicotine equivalents There was a linear relationship between average daily CBT and 24-h nicotine equivalents excretion in urine.
The relationship was closer in the Marlboro Lights group than in the Marlboro Ultra group (Table 5). In the Marlboro Lights group, mean daily CBT accounted for an average of about 40% (R2 = 0.31 on Day 1 and R2 = 0.50 on Day 2) of the total variability in nicotine equivalent excretion. In the Marlboro Ultra group, it accounted for an average of about 36% (R2 = 0.42 on Day 1 and R2 = 0.29 on Day 2) of the variability in nicotine equivalent excretion. The exposure index, calculated as the number of cigarettes smoked per day divided by the average daily CBT, explained an average of 85% of the total variability in nicotine equivalents in both the Marlboro Lights (R2 = 0.79 on Day 1 and 0.91 on Day 2) and the Marlboro Ultra
Table 4 Variance component analysis of cigarette burn time Variation source*
Df
MS
EMS**
%***
Brand Smoker (brand) Day (smoker (brand)) Error
1 38 94 1048
1135870 89220 5123 3903
rerror + 17.6rday (smoker (brand)) + 34.8rsmoker (brand) + 579.7rbrand rerror + 14.6rday (smoker (brand)) + 28.79rsmoker (brand) rerror + 574.74rday (smoker (brand)) rerror
20 34 1 45
Total
1163
* ** ***
Smoker is considered to be nested within cigarette brand and study day is nested within smoker and cigarette brand. Based on Type 1 method. Percent variability attributable from the source.
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Table 5 Regression analysis of nicotine equivalents (mg/24 h) against cigarette burn time (seconds), number of cigarettes smoked per day and exposure index (number of cigarettes smoked per day divided by average cigarette burn time) Cigarette brand a
ML ML ML ML ML ML ML ML ML MULb MUL MUL MUL MUL MUL MUL MUL MUL a b c d e
Study day 1 1 1 2 2 2 Pooled Pooled Pooled 1 1 1 2 2 2 Pooled Pooled Pooled
Model c
NE = 40.32 0.0679 burn-time NE = 3.80 + 1.13 cigarettesd NE = 0.83 + 357.72 exposure indexe NE = 52.36 0.1033 burn-time NE = 2.36 + 0.93 cigarettes NE = 0.63 + 302.07 exposure index NE = 43.75 0.0786 burn-time NE = 2.66 + 0.9942 cigarettes NE = 0.33 + 317.95 exposure index NE = 37.83 0.0783 burn-time NE = 1.12 + 0.9662 cigarettes NE = 0.48 + 291.13 exposure index NE = 35.54 0.0760 burn-time NE = 1.42 + 0.81 cigarettes NE = 1.23 + 253.57 exposure index NE = 36.50 0.0766 burn-time NE = 0.8967 + 0.8545 cigarettes NE = 0.61 + 265.55 exposure index
R2
SE of Slope
0.31 0.71 0.79 0.50 0.75 0.91 0.37 0.71 0.82 0.42 0.73 0.83 0.29 0.80 0.87 0.35 0.73 0.82
0.0240 0.1708 43.87 0.0242 0.1278 22.00 6.6940 1.73 23.95 0.0219 0.1382 31.23 0.0279 0.0962 23.08 5.78 1.29 20.43
Marlboro Lights group. Marlboro Ultra group. Average cigarette burn time per subject. Number of cigarettes smoked per day. Number of cigarettes smoked per day/average cigarette burn time.
Table 6 Regression analysis of carboxyhemoglobin (% sat) against cigarette burn time (seconds), number of cigarettes smoked per day and exposure index (number of cigarettes smoked per day divided by average cigarette burn time) Cigarette brand a
ML ML ML MULb MUL MUL a b c d e
Study day 1 1 1 1 1 1
Model c
COHb = 13.58 0.022 burn-time COHb = 0.05 + 0.30 cigarettes d COHb = 0.65 + 99.04 exposure indexe COHb = 10.33 0.02 burn-time COHb = 0.51 + 0.21 cigarettes COHb = 0.55 + 65.94 exposure index
R2
SE of Slope
0.51 0.74 0.90 0.60 0.69 0.85
0.0052 0.0418 7.5891 0.0041 0.0330 6.5618
Marlboro Lights group. Marlboro Ultra group. Average cigarette burn time per subject. Number of cigarettes smoked per day. Number of cigarettes smoked per day/mean cigarette burn time.
groups (R2 = 0.83 on Day 1 and 0.87 on Day 2) (Table 6, Figs. 2–5). When the two study days were pooled, the exposure index accounted for 82% of the total variability in nicotine equivalents for both the Marlboro Lights and the Marlboro Ultra groups (Table 5). 4.4. Relationship between cigarette burn time and carboxyhemoglobin COHb was measured on Day 1 only. Average daily CBT alone accounted for more than 50% (R2 = 0.51 and 0.60, respectively) of the total variability of COHb in both the Marloboro LIGHTs and the Marlboro Ultra groups, which was slightly higher than that for nicotine equivalents. When the exposure index was used as the predictor, the regression R2 value increased to 0.90 in Marlboro
Lights and 0.85 in Marlboro Ultra, meaning that the exposure index accounted for 90% and 85% of the total variability in COHb (Table 6, Figs. 6 and 7). 4.5. Relationship between cigarette burn time and puffing variables in smoking machines In the analysis of the smoking machine data from Lipowicz and Dwyer (2001), CBT showed a very strong linear relationship with puff volume, inter-puff interval and vent hole blocking. In the multiple regression model, the three cigarette puffing variables together accounted for 97% of the variability in the response variable CBT (R2 = 0.97). Inter-puff interval was the most important factor for CBT and it accounted for 79% of the total variability in CBT. Puff volume and vent hole blocking accounted for
Q. Liang et al. / Regulatory Toxicology and Pharmacology 50 (2008) 66–74
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Y= - 0.83 + 357.72 X (R-square=0.79)
Nicotine Equivalents (mg/24h)
30
25
20
15
10
5
0 0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
Exposure Index (number of cigarettes smoked per day / cigarette burn time (sec.))
Fig. 2. Regression of the exposure index (number of cigarette smoked per day divided by average cigarette burn time) against nicotine equivalents for adult smokers of Marlboro Lights on Day 1. The band represents 95% prediction interval.
Y=-0.63 + 302.07 X (R-square=0.91)
Nicotine Equivalents (mg/24h)
30
25
20
15
10
5
0 0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.10
0.11
Exposure Index (number of cigarettes smoked per day / cigarette burn time(sec.))
Fig. 3. Regression of the exposure index (number of cigarette smoked per day divided by average cigarette burn time) against nicotine equivalents for adult smokers of Marlboro Lights on Day 2. The band represents 95% prediction interval.
13% and 5% of the total variability in CBT, respectively. The model also showed that CBT would become longer with longer inter-puff interval, but shorter with larger puff volume or more vent hole blocking. 5. Discussion This was the first study to examine CBT and its relationships with nicotine and carbon monoxide exposure in adult smokers in a controlled clinical study. A controlled study has the limitation that it might not fully reflect smoking behavior in the everyday life settings. But since the main objective of our study was to examine the relationship between cigarette burn time and exposure to nicotine and CO, a controlled study has the advantage of controlling
confounding factors and unknown sources of variability. We have previously showed that smokers’ exposure to NE and COHb in controlled smoking studies was comparable to that in the normal life-settings (Sarkar et al., 2004). The total variability in CBT was largely attributed to the sources from cigarette brand (20%) and smokers (34%). The day-to-day difference appeared to be small (1%). About 45% of the total variability was due to other unknown factors. The cigarette-to-cigarette difference in CBT was obvious for both the Marlboro Lights and the Marlboro Ultra groups. Average CBT was longer for the first cigarette in the morning than the subsequent cigarettes smoked during a day in the Marlboro Lights group, suggesting that the first cigarette in the morning was actually smoked less intensely than other cigarettes during the
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Q. Liang et al. / Regulatory Toxicology and Pharmacology 50 (2008) 66–74 Y =-0.48 + 291.13 X (R-square=0.83)
Nicoitne Equivalents (mg/24h)
30
25
20
15
10
5
0 0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
Exposure Index (number of cigarettes smoked per day / cigarette burn time(sec.))
Fig. 4. Regression of the exposure index (number of cigarette smoked per day divided by average cigarette burn time) against nicotine equivalents for adult smokers of Marlboro Ultra on Day 1. The band represents 95% prediction interval. Y=-1.23 + 253.57 X (R-square=0.87)
Nicotine Equvalents (mg/24h)
30
25
20
15
10
5
0 0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.10
0.11
Exposure Index (number of cigarettes smoked per day / cigarette burn time (sec.))
Fig. 5. Regression of the exposure index (number of cigarette smoked per day divided by average cigarette burn time) against nicotine equivalents for adult smokers of Marlboro Ultra on Day 2. The band represents 95% prediction interval. Y=0.65+ 99.04 X (R-square=0.90)
Carboxyhemoglobin (% Sat.)
10
8
6
4
2
0 0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
Exposure Index (number of cigarettes smoked per day / cigarette burn time (sec.))
Fig. 6. Regression of the exposure index (number of cigarette smoked per day divided by average cigarette burn time) against COHb for adult smokers of Marlboro Lights on Day 1. The band represents 95% prediction interval.
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Y=0.55+ 65.94 X (R-square=0.85)
Carboxyhemoglobin (% Sat.)
10
8
6
4
2
0 0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
Exposure Index (number of cigarettes smoked per day / cigarette burn time (sec.))
Fig. 7. Regression of the exposure index (number of cigarette smoked per day divided by average cigarette burn time) against COHb for adult smokers of Marlboro Ultra on Day 1. The band represents 95% prediction interval.
day. This is in contrast to the general belief that smokers smoke their first cigarette more intensively in the morning. We see this result as an indication of the complexity of smoking behavior and further investigation may be needed. The several differences in the statistical characteristics of mean CBT between the Marlboro Lights group and the Marlboro Ultra group is expected. Unlike the Marlboro Lights smokers who continued to smoke the same kind of cigarette, smokers in the Marlboro Ultra group were switched from an 11 mg tar (FTC) cigarette to the 3 mg tar cigarette. There was a limited flexibility to smoke such a low tar cigarette. CBT showed a negative correlation with nicotine equivalents and COHb, meaning that a longer burn time would result in lower exposure to nicotine and carbon monoxide. In smoking machines, cigarette burn time alone accounted for more than 92% of the nicotine yield in all test cigarettes and under all smoking conditions (Lipowicz and Dwyer, 2001). But in smokers, the highest value was only 50%. One major diference between machine smoking and human smoking is the number of cigarettes smoked. The number does not change in machine smoking whereas it varies between adult smokers and smoking days. We thus introduced the exposure index which takes the number of cigarette smoker per day into consideration. The exposure index explained a large proportion of the total variability in nicotine equivalents excretion and COHb. In the Marlboro Lights group the exposure index accounted for about 85% of the total variability in nicotine equivalents excretions and 90% of the total variability in COHb. In the Marlboro Ultra group, similar results were obtained. A regression model using the exposure index as the estimator variable appeared to estimate smokers’ exposure to nicotine and carbon monoxide well. It has to be pointed out that the adult smokers in this study smoked cigarettes under controlled smoking conditions and the
linear relationships between the number of cigarettes smoked per day and nicotine equivalents and COHb (R2 > 0.7) were unusually strong. When all smokers in the clinical study (include those not involved in the CBT investigation) were considered, the R2 value was 0.55 for nicotine equivalents against the number of cigarette smoked per day and 0.66 for COHb against the number of cigarette smoked per day. In other clinical studies with similar controlled smoking conditions, the R2 value for each of the two biomarkers against the number of cigarettes smoked per day was in the range of 0.2–0.4 (Mendes et al., 2007; Roethig et al., 2007). It is obvious that the usefulness of the exposure index will be dependent on a reasonable large R2 value. Since the results of this study were based on 40 subjects and over two study days only, further studies are warranted to confirm the results in larger populations and under more circumstances. The results of our study were in line with the results of a recently published study (Benowitz et al., 2006) in which, ‘‘time to smoke cigarette’’, a similar concept as CBT, was used to characterize smokers’ puffing behavior. Same as our results, the study showed that cigarettes with a higher tar level were smoked significantly longer than those with a lower tar level. CBT is relatively easy to measure and seems to capture aspects of puffing behavior relevant for exposure to nicotine and CO. The exposure index, with cigarette daily consumption as the numerator and CBT as the denominator, reflects the positive relationship of exposure with cigarettes per day and the negative relationship with CBT. Acknowledgements We would like to thank Jane Lewis, Mary Ellen Counts, and Bill Dwyer for reviewing the manuscript and their helpful comments.
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References Benowitz, N.L.O., Zevin, S., Jacob, P., 1997. Sources of variability in nicotine and cotinine levels with use of nicotine nasal spray, transdermal nicotine, and cigarette smoking. British Journal of Clinical Pharmocology 43 (3), 259–267. Benowitz, N.L., Jacob, P., Herrera, B., 2006. Nicotine intake and dose response when smoking reduced—nicotine content cigarettes. Clinical Pharmacology & Therapeutics 80 (6), 703–714. Bridges, R.B., Combs, J.G., Humble, J.W., Turbek, J.A., Rehm, S.R., Haley, N.J., 1990. Puffing topography as a determinant of smoke exposure. Pharmacology, Biochemistry, and Behavior 37 (1), 29. Browne, C.L., 1990. Design of Cigarettes. third ed. Hoechst Celanese. Charlotte, N.C. Federal Register, August 1, 1967. Cigarettes: Testing for Tar and Nicotine Content. 32(147), 11178. Federal Register, July 10, 1980. Cigarettes and related matters: carbon monoxide, tar and nicotine contents of the cigarette smoke. Description of New Machine and Methods to be Used in Testing. 45(134), 46483–46487. Farnum, N.R., Stanton, L.W., 1989. Quantitative Forecasting Methods. PWS-Kent Publishing Company, Boston, pp. 57–63. Feng, S., Roethig, H., Liang, Q., Kinser, R., Jin, Y., Scherer, G., Urban, M., Engl, J., Riedel, K., 2006. Evaluation of urinary 1-hydroxypyrene, S-phenylmercapturic acid, trans,trans-muconic acid, 3-methyladenine, 3-ethyladenine, 8-hydroxy-2 0 -deoxyguanosine, and thioethers as biomarkers of exposure to cigarette smoke. Biomarkers 11 (1), 28–52. Feng, S., Plunkett, S.E., Lam, K., Kapur, S., Muhammad, R., Jin, Y., Zimmermann, M., Mendes, P., Kinser, R., Roethig, H.J., 2007. A new method for estimating the retention of selected smoke constituents in the respiratory tract of smokers during cigarette smoking. Inhalation Toxicology 19 (1), 169–179. Green, C., Rodgman, A., 1996. The Tobacco Chemists’ ResearchConference: a half century forum for advances in analytical methodology oftobacco and its products. Recent Advances in Tobacco Science 22 (1), 131–306. Hammond, D., Fong, G.T., Cummings, K.M., Hyland, A., 2005. Smoking topography, brand switching, and nicotine delivery: results from an in vivo study. Cancer Epidemiology Biomarkers & Prevention 14 (6), 1370–1375.
Hatsukami, D.K., Benowitz, N.L., Rennard, S.I., Oncken, C., Hecht, S.S., 2006. Biomarkers to assess the utility of potential reduced tobacco products. Nicotine & Tobacco Research 8 (2), 169–191. Hicks, C.R., 1982. Fundamental Concepts in the Design of Experiments, Third ed. CBS College Publishing, pp. 210–224. Lee, E.M., Malson, J.L., Waters, A.J., Moolchan, E.T., Pickworth, W.B., 2003. Smoking topography: reliability and validity in dependent smokers. Nicotine & Tobacco Research 5 (5), 673–679. Lipowicz, P.J., Dwyer, R.W., 2001. Correlation of Nicotine Yield with Decreased Cigarette Burn Time for 27 Machine Puff Regimens Suggests that Burn Time may be a Useful Estimator of Nicotine Intake. Society for Nicotine and Tobacco Research Annual Meeting Seattle, March 23–25, 2001. Mendes, P., Kapur, S., Wang, J., Feng, S., and Roethig, H., 2007. A randomized, controlled exposure study in adult smokers of full flavor cigarettes switching to Lights or Ultra Lights cigarettes. Poster Presentation. 108th Annual Meeting of the American Society for Clinical Pharmacology and Therapeutics (ASCPT). Anaheim, California, March 21–24; Clinical Pharmacology and Therapeutics 81, S45. Robertson, C., 2000. The design and engineering of a cigarette. WHO Conference: Advancing Knowledge on Regulating Tobacco Products. Oslo, Norway. February, 2000. Roethig, H.J., Kinser, R.D., Lau, R.W., Walk, R.A., Wang, N., 2005. Short-term exposure evaluation of adult smokers switching from conventional to first generation electrically heated cigarettes during controlled smoking. Journal of Clinical Pharmacology 45 (1), 133–145. Roethig, H.J., Zedler, B.K., Kinser, R.B., Feng, S., Nelson, B.L., Liang, Q., 2007. Short-term clinical exposure evaluation of a second-generation electrically heated cigarette smoking system. Journal of Clinical Pharmacology 47 (4), 518–530. Sarkar, M.; Liang, Q.; Feng, S.; Kinser, R.; Roethig, H.-J.; 2004. Evaluation of a clinical model to determine exposure from conventional cigarettes in adult smokers. Journal of Clinical Pharmacology 44, 10, 1213; 33rd Annual Meeting of the American College of Clinical Pharmacology, Litchfield Park, Phoenix, Az, October 3–5, 2004. SAS Institute. 2004. SAS/Stat. Version 9.1. Stratton, K., Shetty, P., Wallace, R., et al. (Eds.), 2001. Clearing the Smoke: Assessing the Science Base for Tobacco Harm Reduction. Institute of Medicine. National Academy Press, Washington, D.C.
Regulatory Toxicology and Pharmacology 50 (2008) 66–74 www.elsevier.com/locate/yrtph
The effect of cigarette burn time on exposure to nicotine and carbon monoxide in adult smokers Qiwei Liang *, Hans J. Roethig, Peter J. Lipowicz, Yan Jin, Paul E. Mendes Philip Morris USA, Research Center, 4201 Commerce Road, Richmond, VA 23234, USA Received 13 February 2007 Available online 29 August 2007
Abstract Cigarette burn time (CBT), conventionally defined as the time a cigarette burns during smoking, can be affected by cigarette design and smoking behavior. A previous study showed a strong negative correlation between CBT and nicotine yield under machine smoking conditions. This study for the first time examined the relationship of CBT and exposure to nicotine and carbon monoxide in adult smokers in a controlled clinical study. 24 h nicotine equivalents excretion (NE), carboxyhemoglobin (COHb) and CBT were measured in two groups of 20 adults smoking Marlboro Lights and 20 adults smoking Marlboro Ultra on two consecutive days. Approximately 20% of the total variability in CBT was attributed to cigarette brand, 34% to smokers and 1% to study day. The exposure index, defined as the number of cigarettes smoked per day divided by average daily CBT for each smoker, accounted for a large proportion of the total variability in NE (R2 = 0.79–0.91) and COHb (R2 = 0.85–0.90). We conclude that CBT has an important influence on levels of NE and COHb in adult smokers. CBT, along with the number of cigarettes smoked per day, can be used to estimate adult smokers’ exposure to nicotine and carbon monoxide. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Cigarette burn time; Exposure; Nicotine equivalents; Carboxyhemoglobin; Statistical; Relationship; Exposure index; Estimate
1. Introduction Exposure to cigarette smoke, a major health concern, varies considerably from smoker to smoker (Benowitz et al., 1997, 2006; Feng et al., 2006, 2007; Hatsukami et al., 2006; Mendes et al., 2007). The variability in daily exposure is due to not only the number of cigarettes smoked per day, but also, to a large extent, the difference in cigarette puffing behavior. Cigarette puffing behavior can be measured with a special topography device (Bridges et al., 1990; Hammond et al., 2005; Lee at al., 2003; Roethig et al., 2007). However, the everyday puffing behavior of a smoker may be impacted by the measuring procedure because a smoker has to smoke through the device. Cigarette burn time (CBT) is conventionally defined as the time a cigarette burns during smoking. It is influenced *
Corresponding author. Fax: +1 804 2742891. E-mail address: [email protected] (Q. Liang).
0273-2300/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.yrtph.2007.08.010
by cigarette design (Browne, 1990; Robertson, 2000) and the puffing behavior of the individual smoker. Compared to cigarette puffing topography, CBT can relatively easily be measured without special devices and it is not expected to impact the everyday puffing behavior of a smoker. Under machine smoking conditions with a FTC puffing regimen (Federal Register, 1967, 1980), CBT is typically about 7 min (Lipowicz and Dwyer, 2001). Lipowicz and Dwyer (2001) conducted a study under 27 different machine puffing regiments to examine CBT in different test cigarettes. They found that CBT is influenced by puff volume, inter puff interval and ventilation hole blocking. They also found a strong negative correlation between CBT and nicotine yield, meaning that the longer the CBT (i.e. the less intensive the smoking), the lower the nicotine yield. The findings established a quantitative relationship between CBT and nicotine yield and identified a potentially important source of variability in nicotine yield. However, smoking machine conditions do not reflect the full
Q. Liang et al. / Regulatory Toxicology and Pharmacology 50 (2008) 66–74
complexity of human smoking behavior. So the question arises whether CBT determined during human smoking would reflect human smoking behavior and might, in part, explain the large variability in exposure observed in adult smokers (Benowitz et al., 1997, 2006; Roethig et al., 2005, 2007; Feng et al., 2006, 2007; Mendes et al., 2007). Nicotine and carbon monoxide are frequently measured constituents of cigarette smoke, which is a complex chemical mixture with about 4800 chemical constituents identified in mainstream smoke (Green and Rodgman, 1996). Nicotine equivalents excretion in urine and carboxyhemoglobin (COHb) in blood are validated biomarkers of exposure for these two smoke constituents (Stratton et al., 2001). Substantial inter-subject variability of these two biomarkers has been found in smokers (Roethig et al., 2005, 2007). Knowledge of CBT and its relation to smoker’s levels of nicotine equivalents and COHb might be important for understanding some aspects of smoking behavior and therefore some sources of variability in nicotine equivalents and COHb in smokers. The purposes of this study were to: (1) characterize the variability of CBT between cigarette brands, study days and smokers; (2) examine the quantitative relationships between average daily CBT and adult smokers’ exposure to nicotine and carbon monoxide as determined from biomarkers of exposure and (3) develop a statistical model to predict nicotine equivalents and COHb based on an index of daily cigarette consumption and average daily CBT. 2. Clinical study design This study was a sub-study within a larger, randomized, controlled short-term exposure study to evaluate different cigarette products. The results of the main study have been published elsewhere and details can be found there (Roethig et al., 2005). The study was performed at the clinical study site of MDS Pharma Services at Lincoln, Nebraska and was approved by the local institutional review board (IRB). All participating subjects had given written informed consent before participating in the study and were free to withdraw from the study at any time for any reason. 100 adult, male and female smokers of an 11 mg tar (FTC) cigarette (Marlboro LIGHTS) were confined to the clinical unit for 10 days. After taking baseline measurements, the adult smokers were randomized to 5 different groups: group A continued smoking Marlboro Lights; groups B was switched to Marlboro Ultra (Philip Morris International, 3 mg FTC tar); C and D were switched to two different types of Electrically Heated Cigarette Smoking Systems (3 mg tar FTC) and group E stopped smoking for 8 days. Controlled smoking was used as a measure to keep the numbers of cigarettes smoked per day as constant as possible. Adult smokers could smoke up to a maximum number of cigarettes per day determined as the number they would smoke regularly per day. Smoking was only allowed from 7:00 A.M. to 11:00 P.M. with smoking opportunities every 32 min.
67
CBT was measured on two consecutive study days during this 8 day period in 20 adult male and female smokers of Marlboro Lights King Size (Philip Morris USA brand, 11 mg tar, 0.8 mg nicotine, 12 mg CO, FTC delivery) and in another 20 adult male and female smokers of Marlboro Ultra (Philip Morris International brand, 3 mg tar, 0.3 mg nicotine, 4 mg CO, FTC delivery). All cigarettes smoked during these two days had been marked with a line 3 mm from the filter wrapper. One day before measuring the CBT, all smokers were trained on how to monitor and record their CBT. During the two study days, smokers documented CBT for all cigarettes smoked during that day. The start of CBT coincided with the lighting of the cigarette and the end of CBT was when the lit part of the cigarette reached 3 mm from the filter wrapper (this was marked with a pencil line). The smokers were asked to smoke in their usual way but had to stop puffing when the burning zone of the cigarette reached the 3 mm line. They could stop puffing whenever they wanted to, but had to let the cigarette burn until the burning zone reached the 3 mm line. All measurements were supervised by the study staff to ensure accuracy and compliance with the study protocol. Urine nicotine and five of its metabolites (nicotine-Nglucuronide, cotinine, cotinine-N-glucuronide, trans-3 0 hydroxycotinine, and trans-3 0 -hydroxycotinine-O-glucuronide) were measured by a liquid chromatography/tandem mass spectrometry (LC-MS/MS) method (Roethig et al., 2005) in 24 h urine collections on the first and second days. Blood samples for COHb were taken at 7:00 P.M. on the first day and were assayed spectrophotometrically with a CO oximeter (IL Multi-4, Instrumentation Laborartory, Lexington, Mass).
3. Statistical methods Descriptive statistics including mean, range, standard deviation and coefficient of variation were used to characterize the CBT data by smoker, cigarette brand and study day. In order to characterize the sequential change of CBT from one cigarette to the next, the CBT from all smokers were averaged sequentially from the first cigarette smoked in a day to the last cigarette. Box-and-whisker plots were used to characterize the average CBT series. The runs test, a non-parametric statistical method (Farnum and Stanton, 1989), was applied to examine whether the series of average CBTs was random, meaning no trend and independence between two adjacent cigarettes. The runs test is based on the premise that any observational series with randomness is equally likely to be above or below the median of the series. In the test, a run is defined as a series of increasing burn time values or a series of decreasing values. The number of increasing, or decreasing, value is the length of the run. The null hypothesis is that the CBT series is a random series and the test statistic is the number of runs above and below the median. Auto-regression method was also used to describe the autocorrelation between two adjacent cigarettes, which is called the first order autocorrelation in statistics. Autocorrelation of higher orders was not explored due to the relatively small sample size of the study. T test was used to test the statistical significance of difference in CBT between the first cigarette and the subsequent cigarettes (pooled) by cigarette brand and study day. Variance component analysis (Hicks, 1982) was used to examine the effect of cigarette brand, smokers and study day on CBT.
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The model included terms for cigarette brand, smokers and study day. The variance component for each factor was presented as the percentage of the total variability. Nicotine equivalents were calculated as the molar sum of total nicotine, total cotinine, and total trans-3 0 -hydroxycotinine excreted in the 24 h urine (Roethig et al., 2005). Linear regression analysis was used to examine the relationship between average daily CBT and daily nicotine equivalents excretion and COHb. An exposure index was defined and calculated as the number of cigarettes smoked per day divided by average daily CBT. It was used to predict nicotine equivalent excretion and COHb. In order to examine the relationships between CBT and cigarette puffing variables, we requested the raw dataset from Lipowicz and Dwyer (2001) and analyzed it with multiple linear regression. In the model, the response variable was CBT and the independent variables were puff volume, inter-puff interval and vent hole blocking. All statistical analyses were run with SAS Version 9.1 (SAS Institute, 2004).
4. Results 4.1. Demographics The age of the smokers for the cigarette burn time study ranged from 21 to 65 with an average age of 30.7 in the Marlboro Lights group and 30.8 in the Marlboro Ultra group. Male and female smokers were distributed almost
Table 1 Demographics Characteristic
Marlboro Lights group
Marlboro Ultra group
Age*
30.7 (10.5) 21–56 10/10 16/4 23.79 (2.75) 21–30
30.8 (10.6) 21–56 9/11 19/1 24.47 (3.49) 20–32
Gender** Race*** BMI* * ** ***
Mean (SD), range. Female/male. Caucasian/others.
equally between the two groups. The average body mass index was 23.8 in the Marlboro Lights group and 24.5 in the Marlboro Ultra group (Table 1). The smokers had smoked between 5 and 25 cigarettes per day for at least 5 years. 4.2. Statistical characteristics of cigarette burn time Mean CBT was 6 min and 45 s in the Marlboro Lights group and about 1 min shorter in the Marlboro Ultra group. Mean CBT was statistically significantly longer (p < 0.05) and more variable in the Marlboro Lights group than in the Marlboro Ultra group (Table 2). In the Marlboro Lights group, mean CBT was significantly different between the two study days (p < 0.05). In the Marlboro Ultra group, in contrast, mean CBT was not statistically different between the two study days (Table 3). By smoker, the coefficient of variation (CV) of mean CBT ranged from 9% to 33% with an average of about 17%. CBT also showed fluctuation from cigarette to cigarette for the two cigarette brands and during the two study days (Fig. 1). The run test and auto-regression analysis did not show a consistent trend in the fluctuations of average CBT from one cigarette to another. In both the Marlboro Lights group and the Marlboro Ultra group, the fluctuation was random on Day 1 (p > 0.10), but not on Day 2 (p < 0.05). In the Marlboro Lights group, there was no autocorrelation in average CBT between two adjacent cigarettes on Day 1 (p > 0.05), but there was a positive autocorrelation on Day 2 (p < 0.05), meaning that two adjacent cigarettes’ CBT tended to change in the same direction. For the Marlboro Ultra group, no autocorrelation between adjacent cigarettes was found on Day 1 (p > 0.10), but there was a negative autocorrelation on Day 2 (p < 0.05), meaning that two adjacent cigarettes’ CBT tended to change in opposite direction.
Table 2 Cigarette burn time (seconds) by cigarette brand Cigarette brand
N
Mean
Max
Min
SD
CV (%)
Marlboro Lights group Marlboro Ultra group
619 545
387.1* 324.5
919.0 692.0
204.0 162.0
93.7 66.5
24.20 20.48
*
Statistically significant from the Marlboro Ultra group (p < 0.05).
Table 3 Cigarette burn time (seconds) by cigarette brand and study day Cigarette brand and study day b
ML on day 1 ML on day 2 MULc on day 1 MUL on day 2 a b c * **
Na
Mean
Max
Min
SD
CV (%)
295 324 260 285
397.8* 377.4 324.7** 324.4
919.0 686.0 692.0 505.0
204.0 205.0 179.0 162.0
105.3 80.7 73.8 59.1
26.46 21.38 22.73 18.21
Total number of cigarettes smoked. Marlboro Lights Group. Marlboro Ultra Group. Statistically significant (p < 0.05) from ML on day 2. Not statistically significant (p > 0.05) from MUL on day 2.
1000
1000
900
900
Cigarette Burn Time (seconds)
Cigarette Burn Time (seconds)
Q. Liang et al. / Regulatory Toxicology and Pharmacology 50 (2008) 66–74
800 700 600 500 400 300 200
800 700 600 500 400 300 200 100
100 1
2
3 4
5
6
7
8 9 10 11 12 13 14 15 16 17 18 19 20 Cigarette Sequence
1
2 3
4 5 6
1
2
4 5
7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Cigarette Sequence
1000 Cigarette Burn Time (seconds)
1000 Cigarette Burn Time (seconds)
69
900 800 700 600 500 400 300 200 100
900 800 700 600 500 400 300 200 100
1
2
3 4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20
Cigarette Sequence
3
6
7 8
9 10 11 12 13 14 15 16 17 18 19 20 21 Cigarette Sequence
Fig. 1. Box and whisper plot of cigarette burn time by each cigarette for adult smokers in: (a) the Marlboro Lights group on Day 1; (b) the Marlboro Lights group on Day 2; (c) the Marlboro Ultra group on Day 1 and (d) the Marlboro Ultra group on Day 2. The top and the bottom edges of the box represent the 75% and 25 percentiles, respectively. The middle line in the box represents the median and the plus symbol is for the mean. The upper and the lower ends of the whisper represent the maximum and the minimum values.
About 20% of the total variability in CBT was attributed to difference between cigarette brands, 34% to difference between smokers and 1% to difference between study days. The remaining 45% variability was due to other unknown factors (Table 4). Both nicotine equivalents (R2 = 0.71–0.80) and COHb (R2 = 0.69–0.74) showed a good correlation with the number of cigarettes smoked per day (Tables 5 and 6). 4.3. Relationship between cigarette burn time and nicotine equivalents There was a linear relationship between average daily CBT and 24-h nicotine equivalents excretion in urine.
The relationship was closer in the Marlboro Lights group than in the Marlboro Ultra group (Table 5). In the Marlboro Lights group, mean daily CBT accounted for an average of about 40% (R2 = 0.31 on Day 1 and R2 = 0.50 on Day 2) of the total variability in nicotine equivalent excretion. In the Marlboro Ultra group, it accounted for an average of about 36% (R2 = 0.42 on Day 1 and R2 = 0.29 on Day 2) of the variability in nicotine equivalent excretion. The exposure index, calculated as the number of cigarettes smoked per day divided by the average daily CBT, explained an average of 85% of the total variability in nicotine equivalents in both the Marlboro Lights (R2 = 0.79 on Day 1 and 0.91 on Day 2) and the Marlboro Ultra
Table 4 Variance component analysis of cigarette burn time Variation source*
Df
MS
EMS**
%***
Brand Smoker (brand) Day (smoker (brand)) Error
1 38 94 1048
1135870 89220 5123 3903
rerror + 17.6rday (smoker (brand)) + 34.8rsmoker (brand) + 579.7rbrand rerror + 14.6rday (smoker (brand)) + 28.79rsmoker (brand) rerror + 574.74rday (smoker (brand)) rerror
20 34 1 45
Total
1163
* ** ***
Smoker is considered to be nested within cigarette brand and study day is nested within smoker and cigarette brand. Based on Type 1 method. Percent variability attributable from the source.
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Table 5 Regression analysis of nicotine equivalents (mg/24 h) against cigarette burn time (seconds), number of cigarettes smoked per day and exposure index (number of cigarettes smoked per day divided by average cigarette burn time) Cigarette brand a
ML ML ML ML ML ML ML ML ML MULb MUL MUL MUL MUL MUL MUL MUL MUL a b c d e
Study day 1 1 1 2 2 2 Pooled Pooled Pooled 1 1 1 2 2 2 Pooled Pooled Pooled
Model c
NE = 40.32 0.0679 burn-time NE = 3.80 + 1.13 cigarettesd NE = 0.83 + 357.72 exposure indexe NE = 52.36 0.1033 burn-time NE = 2.36 + 0.93 cigarettes NE = 0.63 + 302.07 exposure index NE = 43.75 0.0786 burn-time NE = 2.66 + 0.9942 cigarettes NE = 0.33 + 317.95 exposure index NE = 37.83 0.0783 burn-time NE = 1.12 + 0.9662 cigarettes NE = 0.48 + 291.13 exposure index NE = 35.54 0.0760 burn-time NE = 1.42 + 0.81 cigarettes NE = 1.23 + 253.57 exposure index NE = 36.50 0.0766 burn-time NE = 0.8967 + 0.8545 cigarettes NE = 0.61 + 265.55 exposure index
R2
SE of Slope
0.31 0.71 0.79 0.50 0.75 0.91 0.37 0.71 0.82 0.42 0.73 0.83 0.29 0.80 0.87 0.35 0.73 0.82
0.0240 0.1708 43.87 0.0242 0.1278 22.00 6.6940 1.73 23.95 0.0219 0.1382 31.23 0.0279 0.0962 23.08 5.78 1.29 20.43
Marlboro Lights group. Marlboro Ultra group. Average cigarette burn time per subject. Number of cigarettes smoked per day. Number of cigarettes smoked per day/average cigarette burn time.
Table 6 Regression analysis of carboxyhemoglobin (% sat) against cigarette burn time (seconds), number of cigarettes smoked per day and exposure index (number of cigarettes smoked per day divided by average cigarette burn time) Cigarette brand a
ML ML ML MULb MUL MUL a b c d e
Study day 1 1 1 1 1 1
Model c
COHb = 13.58 0.022 burn-time COHb = 0.05 + 0.30 cigarettes d COHb = 0.65 + 99.04 exposure indexe COHb = 10.33 0.02 burn-time COHb = 0.51 + 0.21 cigarettes COHb = 0.55 + 65.94 exposure index
R2
SE of Slope
0.51 0.74 0.90 0.60 0.69 0.85
0.0052 0.0418 7.5891 0.0041 0.0330 6.5618
Marlboro Lights group. Marlboro Ultra group. Average cigarette burn time per subject. Number of cigarettes smoked per day. Number of cigarettes smoked per day/mean cigarette burn time.
groups (R2 = 0.83 on Day 1 and 0.87 on Day 2) (Table 6, Figs. 2–5). When the two study days were pooled, the exposure index accounted for 82% of the total variability in nicotine equivalents for both the Marlboro Lights and the Marlboro Ultra groups (Table 5). 4.4. Relationship between cigarette burn time and carboxyhemoglobin COHb was measured on Day 1 only. Average daily CBT alone accounted for more than 50% (R2 = 0.51 and 0.60, respectively) of the total variability of COHb in both the Marloboro LIGHTs and the Marlboro Ultra groups, which was slightly higher than that for nicotine equivalents. When the exposure index was used as the predictor, the regression R2 value increased to 0.90 in Marlboro
Lights and 0.85 in Marlboro Ultra, meaning that the exposure index accounted for 90% and 85% of the total variability in COHb (Table 6, Figs. 6 and 7). 4.5. Relationship between cigarette burn time and puffing variables in smoking machines In the analysis of the smoking machine data from Lipowicz and Dwyer (2001), CBT showed a very strong linear relationship with puff volume, inter-puff interval and vent hole blocking. In the multiple regression model, the three cigarette puffing variables together accounted for 97% of the variability in the response variable CBT (R2 = 0.97). Inter-puff interval was the most important factor for CBT and it accounted for 79% of the total variability in CBT. Puff volume and vent hole blocking accounted for
Q. Liang et al. / Regulatory Toxicology and Pharmacology 50 (2008) 66–74
71
Y= - 0.83 + 357.72 X (R-square=0.79)
Nicotine Equivalents (mg/24h)
30
25
20
15
10
5
0 0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
Exposure Index (number of cigarettes smoked per day / cigarette burn time (sec.))
Fig. 2. Regression of the exposure index (number of cigarette smoked per day divided by average cigarette burn time) against nicotine equivalents for adult smokers of Marlboro Lights on Day 1. The band represents 95% prediction interval.
Y=-0.63 + 302.07 X (R-square=0.91)
Nicotine Equivalents (mg/24h)
30
25
20
15
10
5
0 0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.10
0.11
Exposure Index (number of cigarettes smoked per day / cigarette burn time(sec.))
Fig. 3. Regression of the exposure index (number of cigarette smoked per day divided by average cigarette burn time) against nicotine equivalents for adult smokers of Marlboro Lights on Day 2. The band represents 95% prediction interval.
13% and 5% of the total variability in CBT, respectively. The model also showed that CBT would become longer with longer inter-puff interval, but shorter with larger puff volume or more vent hole blocking. 5. Discussion This was the first study to examine CBT and its relationships with nicotine and carbon monoxide exposure in adult smokers in a controlled clinical study. A controlled study has the limitation that it might not fully reflect smoking behavior in the everyday life settings. But since the main objective of our study was to examine the relationship between cigarette burn time and exposure to nicotine and CO, a controlled study has the advantage of controlling
confounding factors and unknown sources of variability. We have previously showed that smokers’ exposure to NE and COHb in controlled smoking studies was comparable to that in the normal life-settings (Sarkar et al., 2004). The total variability in CBT was largely attributed to the sources from cigarette brand (20%) and smokers (34%). The day-to-day difference appeared to be small (1%). About 45% of the total variability was due to other unknown factors. The cigarette-to-cigarette difference in CBT was obvious for both the Marlboro Lights and the Marlboro Ultra groups. Average CBT was longer for the first cigarette in the morning than the subsequent cigarettes smoked during a day in the Marlboro Lights group, suggesting that the first cigarette in the morning was actually smoked less intensely than other cigarettes during the
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Q. Liang et al. / Regulatory Toxicology and Pharmacology 50 (2008) 66–74 Y =-0.48 + 291.13 X (R-square=0.83)
Nicoitne Equivalents (mg/24h)
30
25
20
15
10
5
0 0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
Exposure Index (number of cigarettes smoked per day / cigarette burn time(sec.))
Fig. 4. Regression of the exposure index (number of cigarette smoked per day divided by average cigarette burn time) against nicotine equivalents for adult smokers of Marlboro Ultra on Day 1. The band represents 95% prediction interval. Y=-1.23 + 253.57 X (R-square=0.87)
Nicotine Equvalents (mg/24h)
30
25
20
15
10
5
0 0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.10
0.11
Exposure Index (number of cigarettes smoked per day / cigarette burn time (sec.))
Fig. 5. Regression of the exposure index (number of cigarette smoked per day divided by average cigarette burn time) against nicotine equivalents for adult smokers of Marlboro Ultra on Day 2. The band represents 95% prediction interval. Y=0.65+ 99.04 X (R-square=0.90)
Carboxyhemoglobin (% Sat.)
10
8
6
4
2
0 0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
Exposure Index (number of cigarettes smoked per day / cigarette burn time (sec.))
Fig. 6. Regression of the exposure index (number of cigarette smoked per day divided by average cigarette burn time) against COHb for adult smokers of Marlboro Lights on Day 1. The band represents 95% prediction interval.
Q. Liang et al. / Regulatory Toxicology and Pharmacology 50 (2008) 66–74
73
Y=0.55+ 65.94 X (R-square=0.85)
Carboxyhemoglobin (% Sat.)
10
8
6
4
2
0 0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
Exposure Index (number of cigarettes smoked per day / cigarette burn time (sec.))
Fig. 7. Regression of the exposure index (number of cigarette smoked per day divided by average cigarette burn time) against COHb for adult smokers of Marlboro Ultra on Day 1. The band represents 95% prediction interval.
day. This is in contrast to the general belief that smokers smoke their first cigarette more intensively in the morning. We see this result as an indication of the complexity of smoking behavior and further investigation may be needed. The several differences in the statistical characteristics of mean CBT between the Marlboro Lights group and the Marlboro Ultra group is expected. Unlike the Marlboro Lights smokers who continued to smoke the same kind of cigarette, smokers in the Marlboro Ultra group were switched from an 11 mg tar (FTC) cigarette to the 3 mg tar cigarette. There was a limited flexibility to smoke such a low tar cigarette. CBT showed a negative correlation with nicotine equivalents and COHb, meaning that a longer burn time would result in lower exposure to nicotine and carbon monoxide. In smoking machines, cigarette burn time alone accounted for more than 92% of the nicotine yield in all test cigarettes and under all smoking conditions (Lipowicz and Dwyer, 2001). But in smokers, the highest value was only 50%. One major diference between machine smoking and human smoking is the number of cigarettes smoked. The number does not change in machine smoking whereas it varies between adult smokers and smoking days. We thus introduced the exposure index which takes the number of cigarette smoker per day into consideration. The exposure index explained a large proportion of the total variability in nicotine equivalents excretion and COHb. In the Marlboro Lights group the exposure index accounted for about 85% of the total variability in nicotine equivalents excretions and 90% of the total variability in COHb. In the Marlboro Ultra group, similar results were obtained. A regression model using the exposure index as the estimator variable appeared to estimate smokers’ exposure to nicotine and carbon monoxide well. It has to be pointed out that the adult smokers in this study smoked cigarettes under controlled smoking conditions and the
linear relationships between the number of cigarettes smoked per day and nicotine equivalents and COHb (R2 > 0.7) were unusually strong. When all smokers in the clinical study (include those not involved in the CBT investigation) were considered, the R2 value was 0.55 for nicotine equivalents against the number of cigarette smoked per day and 0.66 for COHb against the number of cigarette smoked per day. In other clinical studies with similar controlled smoking conditions, the R2 value for each of the two biomarkers against the number of cigarettes smoked per day was in the range of 0.2–0.4 (Mendes et al., 2007; Roethig et al., 2007). It is obvious that the usefulness of the exposure index will be dependent on a reasonable large R2 value. Since the results of this study were based on 40 subjects and over two study days only, further studies are warranted to confirm the results in larger populations and under more circumstances. The results of our study were in line with the results of a recently published study (Benowitz et al., 2006) in which, ‘‘time to smoke cigarette’’, a similar concept as CBT, was used to characterize smokers’ puffing behavior. Same as our results, the study showed that cigarettes with a higher tar level were smoked significantly longer than those with a lower tar level. CBT is relatively easy to measure and seems to capture aspects of puffing behavior relevant for exposure to nicotine and CO. The exposure index, with cigarette daily consumption as the numerator and CBT as the denominator, reflects the positive relationship of exposure with cigarettes per day and the negative relationship with CBT. Acknowledgements We would like to thank Jane Lewis, Mary Ellen Counts, and Bill Dwyer for reviewing the manuscript and their helpful comments.
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