The Effect of Tobacco Blend Additives on the Retention of Nicotine

Mar 16, 2004 - The influence of the tobacco additives diammonium hydrogen ... There were no statistically significant differences between the indices ...
0 downloads 0 Views 85KB Size
Download PDF
Chem. Res. Toxicol. 2004, 17, 537-544

537

The Effect of Tobacco Blend Additives on the Retention of Nicotine and Solanesol in the Human Respiratory Tract and on Subsequent Plasma Nicotine Concentrations during Cigarette Smoking Alan K. Armitage,† Michael Dixon,*,‡ Barrie E. Frost,§ Derek C. Mariner,*,§,| and Neil M. Sinclair§ Sycamore Lodge, Abbey Road, Knaresborough, North Yorkshire, HG5 8X, United Kingdom, British American Tobacco, Globe House, 4 Temple Place, London WC2R 2PG, United Kingdom, and British American Tobacco, R & D Centre, Regents Park Road, Southampton, SO15 8TL, United Kingdom Received April 11, 2003

The influence of the tobacco additives diammonium hydrogen phosphate (DAP) and urea on the delivery and respiratory tract retention of nicotine and solanesol and on the uptake of nicotine into venous blood was investigated in 10 smokers under mouth-hold and 75 and 500 mL inhalation conditions. Three cigarettes with identical physical specifications were produced from a common lamina tobacco blend. The control cigarette contained nonammoniated reconstituted tobacco sheet (RTS), whereas DAP and other ammonia compounds were added to the RTS of the second cigarette. Urea was added to the tobacco of the third cigarette. The presence of DAP or urea in the test cigarettes did not significantly influence solanesol retention within the mouth during the mouth-hold condition. Nicotine retention within the mouth during the mouth-hold condition was, however, significantly higher for the DAP cigarette (64.3 ( 10.5%) than for the urea (53.3 ( 11.3%) or control cigarette (46.3 ( 8.6%), but this did not result in an increase in nicotine uptake into venous blood. Solanesol retentions during the 75 and 500 mL inhalation volume conditions and nicotine retentions during the 75 mL inhalation volume condition were not significantly different for the three cigarette types. Although the nicotine retention approached 100% with each cigarette type during the 500 mL inhalation condition, the nicotine retention for the urea-treated cigarette (99.6 ( 0.2%) was marginally, but statistically, significant, higher than for the control (99.1 ( 0.5%) and DAP-treated cigarettes (98.8 ( 0.6%). There were no statistically significant differences between the indices of nicotine uptake into venous blood for the three cigarette types in any of the inhalation conditions.

Introduction Ammonia-forming compounds occur naturally in tobacco (1) and compounds that might be expected to be a further source of ammonia in smoke are sometimes added as flavors, flavor precursors, and processing agents during the manufacture of some types of cigarettes (2). Recently, it has been suggested that certain ammonium compounds [e.g., diammonium hydrogen phosphate (DAP) and urea], when used as tobacco additives, will increase “smoke pH”. It has also been suggested that the use of these additives will increase nicotine transfer from tobacco to smoke, increase the amount of nicotine in the vapor phase of smoke, and increase the bioavailability and “addictiveness” of nicotine (3-5). The scientific literature on the effects of ammonia and ammonia-forming additives on smoke chemistry and nicotine bioavailability has been reviewed by Dixon et al (2). This review concluded that these blend additives * To whom correspondence should be addressed. † Sycamore Lodge. ‡ British American Tobacco, Globe House. § Formally of Rothmans International, Group Science and Technology Centre, Tilbrook, Milton Keynes, UK. | British American Tobacco, R & D Centre.

contribute to the flavor properties of smoke but do not enhance nicotine transfer from tobacco to smoke or enhance the bioavailability of nicotine to the smoker by influencing the chemical properties of tobacco and mainstream smoke. The review also identified the requirement for a study in which plasma nicotine levels were measured after smoking cigarettes with different amounts of ammonia in mainstream smoke. Accordingly, the objective of this study was to assess the influence of the addition to tobacco of DAP or urea, as potential ammoniaforming compounds, on the retention of nicotine within the human respiratory tract and its uptake into venous blood under controlled inhalation conditions.

Materials and Methods Subjects. Ten male smokers were employees of Covance Laboratories Ltd. and were provided with details of all of the procedures used in the study prior to their giving consent to participate. They were given a financial payment for participation. The Covance ethical committee approved the study procedures and rates of reimbursement. Subjects were aged between 21 and 40 years and claimed to be inhaling smokers of 15-20 8-12 mg “tar” yield cigarettes per day. Regular smoking status was confirmed by saliva cotinine measurements exceeding 100 ng/mL (range of 223-564 ng/mL) in accordance with

10.1021/tx0340753 CCC: $27.50 © 2004 American Chemical Society Published on Web 03/16/2004

538

Chem. Res. Toxicol., Vol. 17, No. 4, 2004

Armitage et al.

Table 1. Blend Composition, Casing Ingredients, and Physical Specifications of the Experimental Cigarettes tobacco blend (% blend component)

casing ingredients (% ingredient in final blend)

flue-cured (31) burley (20.5) oriental (12) RTS (20)b expanded tobacco (11.5) shredded stem (5.0)

glycerin (1.4) propylene glycol (2.7) sugar (1.1) cocoa (0.4) chocolate (0.2) honey (1.4) licorice (0.4)

cigarette design specifications cigarette length, 83.5 mm circumference, 24.7 mm tobacco rod length, 56.5 mm tobacco rod weight, 661 mg tobacco rod density, 241 mg/cc paper permeability, 27 Coresta burn additive, 0.55% citrate filter type, cellulose acetate plasticizer, 7% triacetin filter pressure drop, 76 mmWg filter ventilation, 24% total pressure drop, 122 mmWg

a Note: three types of RTS used in the samples. Nonammoniated paper RTS containing 91% tobacco, 6% wood pulp, and 3% glycerin (20% inclusion in the control and urea-treated cigarettes). Ammoniated paper RTS containing 83.4% tobacco, 4% wood pulp, 3% glycerin, 5% sugar, 2.5% DAP, and 2.1% urea (8% inclusion in the DAP-treated cigarette). Ammoniated band cast RTS containing 84.1% tobacco, 3.8% glycerin, 1% sugar, 7.9% DAP, and 3.2% NH4OH (12% inclusion in the DAP-treated cigarette).

Table 2. Mainstream Smoke Data for Study Cigarettes type of cigarette NFDPM (tar) (mg/cig) Nicotine (mg/cig) CO (mg/cig) ammonia (µg/cig) pH of aqueous extract of smoke condensate

control

DAP-treated

urea-treated

9.6 0.67 10.1 16 5.6

10.2 0.70 10.0 26 5.5

9.3 0.65 10.1 38 6.1

Stirling et al. (6). The subjects were selected to be within normal limits for body mass index and respiratory function. Test cigarettes were provided to the subjects 2 days prior to experimental sessions to allow acclimatization to the tobacco blend style, which was different from their normal cigarettes. Subjects were asked to smoke only the study cigarettes during this period and to keep a record of their smoking activity. The use of nicotine gum and/or nicotine patches was prohibited. Subjects were asked to refrain from smoking for a minimum of 4 h before each test smoking session. Cigarettes. Three experimental cigarettes were used in the study. They contained the same blends of lamina, expanded and stem tobaccos, the same casings, filters, and cigarette papers, and had identical levels of filter ventilation. Details of the blend composition, casing ingredients, and physical specifications are contained in Table 1. The control (untreated) and DAP-treated cigarettes differed in the type of reconstituted tobacco sheet (RTS) used in their blends. Details of the compositions of the three types of RTSs are shown in the footnote to Table 1. The control cigarette incorporated a 20% inclusion of nonammoniated paper RTS in the final blend. The DAP-treated cigarette incorporated an 8% inclusion of ammoniated paper RTS and a 12% inclusion of ammoniated band cast RTS. The procedure used for the addition of DAP is typical of that used in normal cigarette production. The urea-treated cigarette incorporated a 20% inclusion level of the nonammoniated paper RTS, and 2.0% (w/w) urea was applied to the tobacco blend. Mainstream smoke yields for tar, nicotine, and CO were determined according to ISO methods (7-9) (one puff/min; puff duration, 2 s; puff volume, 35 mL; butt length, 35 mm) and are given in Table 2. Ammonia yields and the pH of aqueous extracts of smoke condensate (smoke pH) are also given in Table 2. The cigarettes were selected for weight and pressure drop (mean ( 5%) to reduce variability and conditioned at 22 °C, 60% relative humidity for a minimum of 24 h before use. Experimental Procedure. For each experimental smoking session, the control and DAP- and urea-treated cigarettes were smoked through a cigarette holder attached to a smoking analyzer, which recorded the subject’s puff volumes, puff durations, and puff times. This system was based on the method described by Creighton et al. (10). Each record was later used

Figure 1. Inhalation apparatus (A) and exhalation apparatus (B). to reproduce the subjects’ puff volumes and times on a smoking duplicator in order to determine the amounts of nicotine and solanesol in the smoke generated by the subjects in each smoking session. On completion of smoking, cigarettes were extinguished in solid CO2 and the butt length was measured. Subjects were asked to take seven puffs, at 60 s intervals, and to prevent any smoke escaping from the mouth after puffing and before exhalation (i.e., no waste or lost smoke). As required by the protocol for a given session, the smoke was either held within the mouth or a fixed volume of air was inhaled after each puff. The inhalation was taken from a collapsible anaesthetic bag containing the specified volume of air (Figure 1A), and the breath was held for the specified period. The experimenter timed the breath-hold duration and instructed the subject when to exhale. The first exhalation after each puff was directed through an acidified 92 mm Cambridge filter pad (Figure 1B) and was vacuum-assisted to overcome filter resistance; this facilitated a more natural exhalation pattern. All such exhalations from one cigarette were directed through one filter pad. The acidified Cambridge filter pads were prepared in a smoke-free environment by soaking in 1 N H2SO4 for 15 min. They were air-dried at room temperature for 24 h and stored in an airtight container. Inhalation and Mouth-Hold Manoeuvres. Inhalation volumes of 75 and 500 mL were taken from the collapsible bag to control the volume of the postpuff inhalation (75 or 500 mL). On each occasion, inhalation was followed by a 2 s breath-hold. Additionally, a mouth-hold condition was included by the subjects holding the smoke in their mouths for 2 s prior to exhaling. The 500 mL inhalation volume was chosen as it is within the typical range of “normal” postpuff inhalation volumes, i.e., 413913 mL (11). The 75 mL inhalation volume was intended to be a “dead space” inhalation, i.e., to allow the smoke to penetrate as far as the conducting airways but not into the gas exchange or alveolar region. Previous work on the inhalation of carbon monoxide (12) suggested that alveolar absorption does not occur with inhalation volumes less than 120 mL in adult males.

Blend Additives, Nicotine, and Solanesol Retention Determination of Nicotine and Solanesol Delivered to Human Volunteers. The puff volumes, durations, and intervals recorded during the smoking of each cigarette were reproduced using a custom built smoking duplicator at Rothmans International Group Science and Technology Centre, Milton Keynes, U.K. However, a standard “bell-shaped” puff was used instead of the exact shape generated by each subject. Creighton and Lewis (13) reported that there were negligible differences in nicotine delivery between puff shapes for a given puff volume and duration. The nicotine and solanesol deliveries from the duplicated smoking sessions were determined using ISO Method 10315:1991 (8) and ASTM Method D6271-98 (14), respectively. Determination of Exhaled Nicotine and Solanesol. After use, the acidified Cambridge filter pads were removed from the holder and extracted by shaking in alkali and di-isopropyl ether containing quinoline as the internal standard. The aqueous layer was separated from the organic layer and extracted twice more with internal standard solution. The combined extracts were stored at -20 °C awaiting analysis. Nicotine was analyzed by gas chromatography [based on ASTM Method D5075-96e1 (15)]. After evaporation of the extract to dryness and dissolution in methanol, solanesol was measured by HPLC [based on ASTM Method D6271-98 (14)]. Calculation of Nicotine and Solanesol Retention. The respiratory retentions of nicotine and solanesol were calculated as follows:

retention (%) ) 100 × (amount delivered amount exhaled)/amount delivered Blood Sampling. Blood samples were taken from each subject during the mouth-hold 75 and 500 mL inhalation conditions. A cannula was inserted into an antecubital vein, and 5 mL blood samples were taken at the following times: 1 min before the lighting puff (puff 1); 3.5 min after lighting (30 s after puff 4); 7 min after lighting (1 min after last puff); and 10, 15, 20, 40, and 60 min after lighting. Blood samples were collected into heparinized tubes and centrifuged. The plasma was transferred to polypropylene tubes and stored at -20 °C to await nicotine analysis, which was completed within 3 months. Additional 5 mL blood samples were taken presmoking and at 10 min after lighting. These were collected into EDTA anticoagulation tubes for carboxyhaemoglobin (COHb) determination. Determination of Plasma Nicotine. Nicotine was extracted from plasma samples by mixing with 35% ammonia solution, dichloromethane, and quinoline internal standard solution and analyzed by gas chromatography [based on ASTM Method D5075-96e1 (15)]. When nicotine levels were between the limit of detection (LoD, 2 ng/mL) and the limit of quantification (LoQ, 5 ng/mL), a value of 2.5 ng/mL (half LoQ) was assumed. This approach has been used previously (e.g., ref 16). These low plasma nicotine values occurred primarily in the mouth-hold condition. Determination of COHb in Whole Blood. All assays were performed within 1 h of sample collection using a blood gas analyzer (Instrumentation Laboratory Synthesis 35). Carboxyhemoglobin boost was determined by subtracting the presmoking value from the postsmoking value. Statistical Methods. Statistical evaluation was by one way ANOVA and additionally by Fisher’s test for the comparison of means (individual error rate 0.05) when a significant (p < 0.05) ANOVA result was obtained. Minitab Version 13.1 (Minitab Inc, State College, PA) was used. No outlier testing was undertaken. Pharmacokinetics. Pharmacokinetic parameters were derived from the plasma nicotine data using WinNonlin Version 1.5 (Pharsight Corporation, Mountain View, CA). Ammonia Analyses and pH Determinations of Aqueous Extracts of Condensate. The ammonia content of mainstream smoke was determined by smoking the cigarettes using a 35

Chem. Res. Toxicol., Vol. 17, No. 4, 2004 539 mL puff volume, 2 s puff duration, and 1 puff per minute machine smoking regime and trapping whole smoke into two sequential traps containing dilute sulfuric acid. The ammonia concentration was determined potentiometrically using an ammonia specific ion electrode. The pH of an aqueous extract of condensate (tar) was determined by machine smoking the cigarettes (same regime as above) and trapping the condensate using a Borgwaldt electrostatic trap. The condensate was dissolved in 5 mL of methanol and diluted with 5 mL of distilled water. The pH of the solution was measured using a pH electrode calibrated at pH 4 and pH 7.

Results Protocol Compliance. The protocol required familiarization sessions and 90 study sessions, which were conducted over a 5 week period. The following deviations from protocol occurred. Two subjects could not be cannulated on one occasion each. On three other occasions involving only single blood samples, it was not possible to perform the nicotine analysis. One smoking behavior record became corrupted and so could not be duplicated. It was essential to ensure that the subjects adhered to the protocol for each of the mouth-hold or inhalation procedures. Each subject was given a practice session during which they conducted all of the experimental maneuvers several times until they were fully familiar with the procedures. Blood samples and data were not collected during these practice sessions. The study operators carefully observed subject behavior and checked to ensure that smoke did not escape from the mouth during the postpuff period prior to subjects inhaling or exhaling through the test equipment. Many of the study sessions were videotaped and examined to ensure that subjects complied fully with the protocol. Influence of DAP and Urea on the Retention of Nicotine and Solanesol within the Mouth and Nicotine Absorption into Venous Blood under the Mouth-Hold Condition. There were no statistically significant differences (ANOVA, p > 0.05) between the three cigarettes for total puff volume (range of 381.7420.2 mL), nicotine deliveries to the mouth (range of 1038-1194 µg), solanesol deliveries to the mouth (range of 371-420 µg), or for solanesol retained in the mouth (range of 33.3-41.0%). However, nicotine retention in the mouth for the DAP cigarette (64.3 ( 10.5%) was significantly higher than for the control (46.3 ( 8.6%) or ureatreated cigarette (53.3 ( 11.3%). The difference between the control and the urea-treated cigarettes was not statistically significant. The mouth-hold retention data are shown in Figure 2A. Although considerable amounts of nicotine were retained during the 2 s mouth-hold condition, this did not result in any measurable increase in plasma nicotine over the presmoking levels for any of the three cigarettes (Figure 3A). Influence of DAP and Urea on the Retention of Nicotine and Solanesol within the Respiratory System, and Nicotine Absorption into Venous Blood, following a 75 and 500 mL Inhalation. With a 75 mL inhalation, there were no significant differences (ANOVA, p > 0.05) between the three cigarettes for total puff volume (range of 365.3-390.5 mL), solanesol and nicotine deliveries (range of 340-386 and 1029-1082 µg, respectively), and solanesol and nicotine retentions (range of 48.5-57.9 and 80.6-89.6%, respectively, Figure 2B).

540

Chem. Res. Toxicol., Vol. 17, No. 4, 2004

Armitage et al.

Figure 2. Effect of inhalation depth on mean ( SD retentions of solanesol (open bars) and nicotine (solid bars).

With a 500 mL inhalation (Figure 2C), there were no significant differences (ANOVA, p > 0.05) in the total puff volume (range of 411.7-435.0 mL), solanesol deliveries (range of 387-425 µg), and solanesol retentions (range of 65.1-70.7%) for the three cigarettes. Although nicotine retention was greater than 98% for all three cigarette types, the ANOVA test did indicate a statistically significant difference in these nicotine retention values (p < 0.01). The Fisher test revealed that the ureatreated cigarette produced a significantly higher mean nicotine retention value (99.6 ( 0.2%) than either the cigarette containing DAP (98.8 ( 0.6%) or the control (99.1 ( 0.5%). As the nicotine deliveries for the three cigarettes were very similar (range of 1116-1195 µg), it seems most unlikely that this small but statistically significant difference in % nicotine retention is of biological significance. However, there were no significant differences between the plasma nicotine profiles from the three cigarette types at either 75 or 500 mL inhalation volume (Figure 3B,C). Influence of Inhalation Depth on the Pre- to Postsmoking COHb Boosts. The mean presmoking COHb levels were in the range of 3.5-4.5% for the three inhalation conditions and the three cigarette types. The mean COHb levels fell slightly after the mouth hold

Figure 3. Effect of inhalation condition on the mean plasma nicotine values for the control (O), DAP-treated (0), and ureatreated cigarettes (4). Puffs taken during the 0-7 min period.

condition for the control (absolute change of -0.15% COHb), DAP-treated (-0.13%), and urea-treated cigarettes (-0.02%). None of these decreases were statistically significant. Mean COHb levels increased significantly after the 75 mL inhalations for the control (a boost of 0.39% COHb, p < 0.01), DAP-treated (0.37%, p < 0.05), and ureatreated cigarettes (0.45%, p < 0.05). The mean COHb boosts for the 500 mL inhalations were 0.83% for the control (p < 0.001), 0.81% for the DAP-treated (p < 0.01), and 1.05% for the urea-treated cigarette (p < 0.001). There were no statistically significant effects attributable to cigarette type on the COHb boosts for each of the three inhalation conditions.

Discussion A key finding of this study is that nicotine retention was greater than solanesol retention in all of the mouth-

Blend Additives, Nicotine, and Solanesol Retention

hold or inhalation conditions for the control and DAPand urea-treated cigarettes. This has implications for the mechanisms of retention of nicotine and solanesol during smoking. Solanesol does not evaporate from aerosol particles at body temperature, even at high dilution. It is, therefore, retained in the airways and lungs only by deposition of smoke particles caused by inertial impaction, Brownian diffusion, and gravitational sedimentation (2). The greater retention for nicotine demonstrates that it does not all remain in the aerosol particles with solanesol, otherwise retention of the two compounds would be similar. Therefore, our finding supports the hypothesis that nicotine evaporates from smoke particles and diffuses to and is absorbed onto the surface of the airways. Furthermore, our findings demonstrate that the evaporation of nicotine from the particles must be rapid. Indeed, exhaled smoke in the 500 mL condition contained solanesol but very little nicotine. This is consistent with the concept of evaporative gas deposition of nicotine proposed by Pankow (17, 18), with the data reported by Frost et al. (19) for a smaller number of subjects using similar methodology but with a Virginia blend cigarette, and by the in vitro experiments on the evaporation of nicotine from particles by Lewis et al. (20). Furthermore, nicotine retention was greater than solanesol retention following the mouth-hold maneuver. This demonstrates that some nicotine evaporation occurs initially within the mouth before inhalation has commenced. There have been concerns that the use of ammonia containing or forming compounds in commercial cigarettes will increase, what has loosely been termed in the literature smoke pH, and hence increase the proportion of nicotine in the nonprotonated (free) form (3-5). The Henderson-Hasselbalch equation (21) can be used to predict the proportions of nicotine in the protonated and nonprotonated forms from the pH of a dilute aqueous solution of nicotine at equilibrium. However, cigarette smoke is not a dilute aqueous solution nor is it at equilibrium. Additionally, as pointed out by Rodgman (22), smoke pH is an ill-defined quantity whose value is more dependent on the method used in its measurement than on some inherent chemical property of mainstream smoke. However, Rodgman (22) claimed that smoke pH measured in the laboratory may have value in the comparison of mainstream smoke from different cigarettes but that the absolute value, as measured by a pH meter or electrode, may have little relevance to the smoke pH values to which the cigarette smoker is exposed. Dixon et al. (2) concluded that the Henderson-Hasselbalch equation cannot be directly applied to a cigarette smoke, but laboratory-based pH measurements may provide an indication of the relative molar quantities of aqueous extractable acids and bases in tobacco smoke. We measured the pH of an aqueous extract of smoke condensate to compare the three cigarettes used in our study. Our results demonstrate that the untreated control and the DAP-containing samples were similar (pH 5.6 and 5.5, respectively) despite different mainstream smoke ammonia yields (16 µg/cig control; 26 µg/cig DAPtreated cigarette). These results agree with data from studies conducted by Ellis et al. (23) who examined the effects of the addition of ammonia compounds to reconstituted tobacco, at commercial application levels, on the pH of an aqueous extract of smoke, on nicotine yields, and on ammonia yields. The addition of the ammonia compounds in the Ellis et al. (23) study increased the

Chem. Res. Toxicol., Vol. 17, No. 4, 2004 541

ammonia content of the tobacco blend from 0.12 to 0.31%, and these figures are comparable to our current study where the ammonia content of the control cigarette was 0.09% and the DAP-treated cigarette was 0.24%. Ellis et al. (23) concluded that although the addition of ammonia compounds produced a small increase in mainstream ammonia yields, it did not change the pH of aqueous extracts of smoke or nicotine transfer efficiencies. Our results, and those of Ellis et al. (23), do not support the claim that ammonia-forming compounds, such as DAP, are used to increase smoke pH and enhance the amounts of nonprotonated or free nicotine in mainstream smoke (3, 24). The levels of DAP and other ammonia sources used in the two forms of ammoniated reconstituted tobacco incorporated into our DAP cigarette were comparable with levels used in some commercial U.S. products. Our experimental sample contained 2.5% DAP and 2.1% urea in the paper RTS [typical U.S. range for paper RTS: 2.53.5% for DAP and 1.75-2.25% for urea (25)]. The band cast RTS contained 3.2% NH4OH and 7.9% DAP [typical U.S. range for band cast RTS: 3-4% for NH4OH and 6-8% for DAP (25)]. The levels of nicotine retained in the mouth during the mouth-hold maneuver were surprisingly high for all three samples, and this raises the question as to whether these high retention values were genuine or occurred as a consequence of the subjects’ noncompliance with the protocol. An apparent high level of mouth retention could have been obtained if the subjects had lost significant amounts of smoke from the mouth prior to exhaling through the Cambridge pad. The experimenters observed each of the subjects specifically for the appearance of waste or lost smoke, and this was not detected in any of the subjects and hence was discounted as a source of error. A second problem could have been an inadvertent small inhalation during the mouth-hold period resulting in significant amounts of nicotine retention in the respiratory tract rather than in the mouth region. We have ruled out the possibility of inadvertent inhalation for the following reasons: (i) subjects practiced all maneuvers prior to the study sessions, (ii) the retention of nicotine was a lot less than in the 75 mL inhalation, which is already only a small inhalation, and (iii) the findings were consistent between subjects when not all would have inhaled. Furthermore, COHb levels fell marginally following the mouth-hold maneuvers on all three cigarette types. Inadvertent inhalations would have resulted in an increase in COHb levels as was the case for the shallow (75 mL) inhalations. Additionally, inhalations would have resulted in an increase in the venous blood levels of nicotine. Even the shallow (75 mL) inhalations resulted in increased blood levels of nicotine (Figure 3). However, the mouth-hold maneuvers were associated with small reductions in pre- to postsmoking blood levels of nicotine for all three cigarette samples. Thus, it is highly likely that the high retention levels of nicotine within the mouth are genuine and did not occur as a result of protocol noncompliance. Interestingly, the cigarette containing DAP produced a significantly higher retention of nicotine in the mouth than the untreated control sample during the mouth-hold condition, whereas mouth retention of the involatile particulate phase marker, solanesol, was similar for the DAP-treated and control samples. The mechanism whereby the addition of DAP increased mouth retention

542

Chem. Res. Toxicol., Vol. 17, No. 4, 2004

of nicotine is unclear. These results are consistent with the concept that the addition of DAP and other ammonia compounds increase the rate of evaporation of nicotine and when smoke is subjected to air dilution and an increase in temperature on entering the mouth, nicotine evaporates from the smoke particles and diffuses to the oral mucosa. The influence of air dilution on nicotine evaporation from the particulate phase of smoke was reported by Mariner and Frost (26) who measured nicotine evaporation from mainstream smoke by passing cigarette smoke through a denuder tube. In their study, nicotine vapor was selectively collected by an acidic lining on the denuder tube wall whereas nicotine that remained in the particulate phase passed through the denuder tube. It was shown that the amounts of nicotine evaporating and diffusing from the particulate phase of mainstream smoke were increased as the smoke was diluted with air. However, it is unlikely that an increase in smoke pH was a factor in the mechanism for the increase in mouth retention of nicotine because the control and DAP-treated samples had similar values for the pH of an aqueous extract of smoke condensate but different mouth retentions of nicotine. Also, the condensate from the ureatreated sample had the highest pH but was significantly lower than the DAP sample in mouth retention of nicotine. These findings with the urea-treated cigarette are somewhat surprising. The addition of 2% urea to the tobacco blend clearly increased both the pH of an aqueous extract of smoke condensate and the ammonia content of MS smoke, which could have affected the proportion of nonprotonated nicotine within the smoke particles. Pankow et al. (18) hypothesized that an increase in the fraction of nonprotonated nicotine in smoke condensate would increase the amounts of nicotine evaporating from the smoke particles and being absorbed as a gas into the linings of the respiratory tract. This hypothesis would predict higher nicotine retentions in the mouth from the urea-treated cigarette than from the other samples, but this was not observed. However, Cochran et al. (27) investigated the effect of increasing the pH of an aqueous extract of condensate, by the addition of urea to tobacco, on the proportion of nicotine in the gas phase of cigarette smoke. They used a denuder tube in these studies, and the amounts of nicotine initially present in the gas phase and amounts evaporating into the gas phase during the passage of the particles through the denuder tube were estimated. Adding urea (range of 0.5-2.5%) increased the pH of an aqueous extract of smoke condensate but did not produce a systematic increase in either the initial amounts of nicotine in the vapor phase or in the amounts evaporating from the smoke particles. Our results showing a lack of an effect of urea and pH on nicotine retention within the mouth are consistent with the findings of Cochran et al. (27). The urea-treated cigarette produced a higher mainstream smoke ammonia yield than the DAP-treated cigarette but was lower than the DAP cigarette in mouth retention of nicotine. Hence, the ammonia yield does not seem to be the reason for the enhanced mouth retention of nicotine associated with the DAP-containing cigarette. Further work will be required to identify the mode of action of DAP in enhancing mouth retention of nicotine. In addition, it would be relevant to determine if increased mouth retention of nicotine is perceived by smokers as increased strength, as proposed by Benowitz (39).

Armitage et al.

Although significant quantities of nicotine were retained within the mouth during the 2 s mouth-hold maneuver on all three cigarettes, venous nicotine levels did not increase following the introduction of smoke into the mouth, showing that mouth exposure resulted in minimal systemic absorption of nicotine. These results are consistent with the findings of Gori et al. (28) and Zacny et al. (29) who did not observe increases in venous nicotine levels in smokers who took cigarette smoke into their mouths but did not inhale it; however, neither group measured nicotine retention. Our data are consistent with work using inhaled nicotine vapor, where the nicotine is found primarily in the mouth, esophagus, and stomach (30, 31). These and related studies (e.g., refs 3234) indicate that nicotine transfers poorly from the mouth to the systemic circulation. There was no evidence that the addition of DAP and other ammonia compounds to the reconstituted tobacco enhanced the amounts of nicotine retained within the respiratory tract following the 75 or 500 mL inhalation maneuvers. Similarly, the uptake of nicotine from the mouth or respiratory tract, as assessed from the plasma nicotine profiles, was not enhanced by the addition of DAP or other ammonia compounds to the RTS. The high application level of urea was chosen specifically to elevate the pH of an aqueous extract of smoke condensate for the purposes of this study and resulted in a condensate smoke pH value (6.1) that was higher than either the control or the DAP-containing sample. Increasing pH by the addition of urea to the blend did not increase the amounts of nicotine absorbed into the systemic circulation. Our study did not examine the influence of ammonia containing or forming additives on arterial levels of nicotine. One widely held view is that nicotine is very rapidly absorbed from the lung, has a very short (5-20 s) transit time to the brain (35, 36), and produces very high peak concentrations within arterial blood. Rose et al. (37) measured arterial levels of nicotine following cigarette smoke inhalation and intravenous nicotine administration. They observed peak arterial nicotine levels, which were far lower than would have been predicted from the rapid (1-2 s) absorption of all of the nicotine in a puff of smoke into the pulmonary circulation. Additionally, Rose et al. (37) observed similar arterial: venous nicotine concentrations for matched doses of nicotine from the inhalation of smoke and intravenous administration. They hypothesized that the arterial concentrations of nicotine were lower than had been predicted because nicotine is initially deposited and distributed into lung tissue, thus slowing entry into the arterial circulation. Our results from the shallow (75 mL) inhalation maneuver show that 80-90% of the nicotine was retained within the respiratory tract, which is consistent with the hypothesis that significant quantities of nicotine are deposited into respiratory tissue during smoke inhalation and would ultimately enter the bronchial circulation and venous return rather than gaining direct access to the pulmonary circulation via the alveolar region of the lung. The fact that the addition of ammonia producing ingredients did not significantly change the levels of nicotine retention during the shallow inhalation or enhance the venous levels of nicotine following the shallow and moderate (500 mL) inhalation suggests that these ingredients are unlikely to enhance arterial levels of nicotine following smoke inhalation. However, a fur-

Blend Additives, Nicotine, and Solanesol Retention

ther study using similar techniques to those described by Rose et al. (37) would be required to address this issue. Our results are clearly contrary to the unsubstantiated claims (4, 5, 38) that the addition of DAP or urea to a cigarette tobacco blend results in an enhanced uptake of nicotine from the respiratory system into the systemic circulation during smoking. Our findings are, however, in broad agreement with Benowitz (39) who claimed that most of the nicotine inhaled in cigarette smoke is absorbed irrespective of pH and that differences in pH do not affect bioavailability but may influence the perceived strength of the cigarette.

Chem. Res. Toxicol., Vol. 17, No. 4, 2004 543

(7)

(8)

(9)

(10)

Conclusion The addition of ammonia containing or forming ingredients to tobacco increased mainstream smoke ammonia levels. The pH of an aqueous extract of smoke of a ureatreated cigarette was higher than that of either an untreated control or DAP-treated cigarette. Although there was a small but significant increase in nicotine retention in the mouth-hold maneuver for the DAP-treated cigarette, nicotine retention following smoke inhalation was similar, and almost complete, for all three cigarettes. Nicotine uptake into venous blood was also unchanged by the addition of DAP or urea to the cigarettes. Solanesol retentions within the mouth and respiratory tract were consistently lower than nicotine retentions for all cigarettes and inhalation conditions. This indicates that the kinetics of nicotine evaporation from smoke particles and nicotine’s subsequent behavior as a gas in the respiratory tract are an important aspect of the deposition and absorption process.

(11)

(12)

(13)

(14)

(15)

(16) (17)

(18)

Acknowledgment. The study was funded by British American Tobacco, Philip Morris (Europe), and Rothmans International, prior to the latter’s merger with British American Tobacco. It was undertaken at Covance Laboratories Ltd., Harrogate, U.K., under the direction of Mark Bentley with the exception of the duplication of the subject smoking behavior records and determinations of nicotine and solanesol delivered to subjects. These were performed at Rothmans International, Group Science and Technology Centre, Milton Keynes, U.K. The experimental cigarette samples were prepared for the study by Brown & Williamson Tobacco Corporation. We thank Drs R. Baker, R. Dempsey, J. Seeman, and K. St. Charles for their helpful comments during the preparation of this paper.

(19)

(20)

(21) (22) (23)

References (1) Leffingwell, J. C. (1999) Basic chemical constituents of tobacco leaf and differences among types. In Tobacco: Production, Chemistry and Technology (Davis, D. L., and Nielsen, M. T., Eds.) pp 265-284, Blackwell Science Ltd., Oxford. (2) Dixon, M., Lambing, K., and Seeman, J. I. (2000) On the transfer of nicotine from tobacco to the smoker. A brief review of ammonia and “pH” factors. Beitrage Tabaksforschung 19 (2), 103-113. (3) Pankow, J. F., Mader, B. T., Isabelle, L. M., Luo, W., Pavlick, A., and Liang, C. (1997) Conversion of nicotine in tobacco smoke to its volatile and free-base form through the action of gaseous ammonia. Environ. Sci. Technol. 31, 2428-2433. (4) Hurt, R. D., and Robinson, C. R. (1998) Prying open the door to the tobacco industry’s secrets about nicotine. J. Am. Med. Assoc. 280, 1173-1181. (5) Bates, C., McNeill, A., Jarvis, M., and Gray, N. (1999) The future of tobacco product regulation and labeling in Europe: implications for the forthcoming European Union directive. Tobacco Control 8, 225-235. (6) Stirling, E. M., Collett, C. W., and Ropss, J. A. (1996) Assessment of nonsmokers exposure to environmental tobacco smoke using

(24)

(25) (26)

(27)

(28)

personal-exposure and fixed location monitoring. Indoor Built Environ. 5, 112-125. International Standards Organization (1991) ISO 4387: Methods for Chemical Analysis of Tobacco and Tobacco Products. Determination of Total and Nicotine-Free Dry Particulate Matter Using a Routine Analytical Smoking Machine, ISO. International Standards Organization (1991) ISO 10315: Methods for Chemical Analysis of Tobacco and Tobacco Products. Determination of Nicotine in Smoke Condensate of Cigarettes (GasChromatographic Method). International Standards Organization (1995) ISO 8454: Methods for Chemical Analysis of Tobacco and Tobacco Smoke Products. Determination of Carbon Monoxide in the Vapour Phase of Cigarette Smoke (NDIR Method). Creighton, D. E., Noble, M. J., and Whewell, R. T. (1978) Instruments to measure, record and duplicate human smoking patterns. In Smoking Behaviour (Thornton, R. E., Ed.) pp 277288, Churchill Livingstone, Edinburgh, London and New York. U.S. Department of Health and Human Services (USDHHS) (1988) The Health Consequences of Smoking: Nicotine Addiction, Public Health Services, Washington, DC. Guyatt, A. R., Holmes, M. A., and Cumming, G. (1981) Can carbon monoxide be absorbed from the upper respiratory tract in man? Eur. J. Respir. Dis. 62, 383-390. Creighton, D. E., and Lewis, P. H. (1978) The effect of smoking pattern on smoke deliveries. In Smoking Behaviour (Thornton, R. E., Ed.) pp 289-314, Churchill Livingstone, Edinburgh, London and New York. American Society for Testing and Materials, D6271-98 (1998) Standard Test Method for Estimating Contribution of Environmental Tobacco Smoke to Respirable Suspended Particles Based on Solanesol. American Society for Testing and Materials D5075-96e1 (1996) Standard Test Method for Nicotine and 3-Ethenylpyridine in Indoor Air. Nehls, G. J., and Ackland, G. G. (1973) Procedures for handling aerometric data. J. Air Pollut. Assoc. 23, 180-184. Pankow, J. F. (2001) A consideration of the role of gas/particle partitioning in the deposition of nicotine and other tobacco smoke compounds in the respiratory tract. Chem. Res. Toxicol. 14 (11), 1465-1481. Pankow, J. F., Tavakoli, A. D., Luo, W., and Isabelle, L. M (2003) Percent free base nicotine in the tobacco smoke particulate matter of selected commercial and reference cigarettes. Chem. Res. Toxicol. 16 (8), 1014-1018. Frost, B. E., Mariner, D. C., and Sinclair, N. M. (1998) Factors relating to nicotine physio-chemistry and retention in human smokers. Proceedings of the CORESTA Congress, October 1998, Brighton, U.K., pp 211-218. Lewis, D. A., Colbeck, I., and Mariner, D. C. (1994) Dilution of mainstream tobacco smoke and its effects upon the evaporation and diffusion of nicotine. J. Aerosol Sci. 26, 841-846. Morie, G. P. (1972) Fraction of protonated and unprotonated nicotine in tobacco smoke at various pH values. Tob. Sci. 16, 167. Rodgman, A. (2000) Smoke pH: A review. Beitrage Tabaksforschung 19, 117-139. Ellis, C. L., Cox, R. H., Callicutt, C. H., Laffoon, S. W., Podraza, K. F., Seeman, J. I., Kinser, R. D., Farthing, D. E., and Hsu, F. H. (1999) The effect of ingredients added to tobacco in a commercial Marlboro Lights cigarette on FTC nicotine yield, “smoke pH” and Cambridge filter trapping effiency. Proceedings of the CORESTA Smoke and Technology meeting, Innsbruck, Austria, September 5-8. CORESTA Bull. 3, 108. U.S. Food and Drug Administration (1995) Notice in Federal Register, August 11. Regulations Restricting the Sale and Distribution of Cigarettes and Smokeless Tobacco Products to Protect Children and Adolescents: Proposed Rule and Analysis Regarding FDA’s Jurisdiction over Nicotine Containing Cigarettes and Smokeless Tobacco Products, U.S. Food and Drug Administration, Washington, DC. Dr. Kelley St. Charles, personal communication. Mariner, D. C., and Frost, B. E. (1998) Determination of nicotine evaporation from mainstream smoke using denuder tubes. In CORESTA Proceedings, pp 206-210, Brighton, England, 11-15 October 1998. Cochran, E. W., Joseph, M. J., Stinson, S. L., and Summers, S. S. (2003) Application of a diffusion-denuder method for the investihgation of the effects of ‘smoke pH’ on vapour phase nicotine yields from different types of cigarettes. Beitrage Tabaksforschung 20 (6), 365-372. Gori, G. B., Benowitz, N. L., and Lynch, C. J. (1986) Mouth versus deep airway absorption of nicotine in cigarette smokers. Pharmacol. Biochem. Behav. 25, 1181-1186.

544

Chem. Res. Toxicol., Vol. 17, No. 4, 2004

(29) Zacny, J. P., Stitzer, M. L., Brown, F. J., Yingling, J. E., and Griffiths, R. R. (1987) Human cigarette smoking: effects of puff and inhalation parameters on smoke exposure. J. Pharm. Exp. Ther. 240, 554-564. (30) Lunell, E., Bergstrom, M., Antoni, G., Langstrom, B., and Nordberg, A. (1996) Nicotine deposition and body distribution from a nicotine inhaler and a cigarette studied with positron emission tomography. Clin. Pharmacol. Ther. 59, 593-594. (31) Bergstrom, M., Nordberg, A., Lunell, E., Antoni, G., and Langstrom, B. (1995) Regional deposition of inhaled 11C-nicotine vapor in the human airway as visualized by positron emission tomography. Clin. Pharmacol. Ther. 57, 309-317. (32) Russell, M. A. H., Jarvis, M. J., Sutherland, G., and Feyerabend, C. (1987) Nicotine replacement in smoking cessation. Absorption of nicotine vapor from smoke-free cigarettes. J. Am. Med. Assoc. 257, 3262-3265. (33) Schuh, K. J., Schuh, L. M., Henningfield, J. E., and Stitzer, M. L. (1997) Nicotine nasal spray and vapor inhaler: abuse liability assessment Pyschopharmacology 130, 352-361. (34) Molander, L., Lunell, E., Anderson, S.-B., and Kuylenstierna, F. (1996) Dose released and absolute bioavailability of nicotine

Armitage et al.

(35) (36) (37)

(38)

(39)

from a nicotine vapour inhaler Clin. Pharmacol. Ther. 59, 394400. Russell, M. A. H., and Feyerabend, C. (1978) Cigarette smoking: a dependence on high-nicotine boli. Drug Metab. Rev. 8, 29-57. Royal College of Physicians of London (2000) Nicotine Addiction in Britain: A Report of the Tobacco Advisory Group of the Royal College of Physicians, pp 36-37 Rose, J. E., Behm, F. M., Westman, E. C., and Coleman, R. E. (1999) Arterial nicotine kinetics during cigarette smoking and intravenous nicotine administration: implications for addiction. Drug Alcohol Depend. 56, 99-107. Henningfield, J. E., and Fant, R. V. (2000) Nicotine delivery systems: Implications for abuse potential, medications development, and public health. In Nicotine and Public Health (Ferrence, R., Slade, J., Room, R., and Pope, M., Eds.) pp 229-247, American Public Health Association, Washington, DC. Benowitz, N. L. (1996) Report of Canada’s Expert Committee on Cigarette ModificationssConference Proceedings March 1996, pp 54, 80.

TX0340753