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315 II. I4C TRACER STUDIES TO DEVELOP SAMPLING PROTOCCILS FOR QUANTITATIVE NICOTINE DOSIMETRY FOLLOWING SMOKE EXPOSURE J. E. Caton, R. A. Jenkins, M. P. Maskarinec G. M. Henderson, and R. W. Harvey The ability to determine cigarette smoke dose by performing a simple analysis on a sample obtained non-destructively from the smoking subject has long been a goal of smoking and health studies. The estimation of the levels of nicotine and0or its metabolites has always been considered a possible approach to the estimation of smoke uptake because (1) the only source of nicotine is tobacco; (2) nicotine is a major component of both the tobacco and the smoke aerosol and (3) nicotine is readily absorbed into the biological system. Because of these properties (angular origin, abun- dance, and efficiency of adsorption), the level of nicotine and its meta- bolites in the serum or urine of a smoking subject would appear to be an excellent indication of the dose of cigarette smoke retained. However, there is some question concerning the reliability of nicotine measurements as a quantitative indicator of smoke uptake because nicotine is rapidly metabolized and distributed throughout the biological system. In order to address these concerns, a collaborative effort between Borriston Research Laboratories at ORNL was established to determine the utility of analyzing either the serum or urine from a smoking dog for nicotine and/or its meta- bolites in order to estimate cigarette smoke uptake by the animal. The experimental approach to this study was to expose a dog to two successive Code 32 (SEB IV) cigarettes labeled with 14C-nicotine bitartrate and continuously monitor the blood and urine both during and for some period~ of time after the smoke exposure. Continuous withdrawal of blood was accomp- lished by connecting a peristaltic pump to a cannula in the jugular vein and pumping at 0.5 ml/min. In practice flexible cannulae were inserted into

316 both jugular veins and sutured in place. This presented a second source of blood in the event of a blockage by clotting or irreversible bending of the flexible cannula. After the first preliminary study on August 11, 1977, all dogs were given heparin I.V. prior to exposure in order to minimize blockage caused by clotting. Urine was collected by bladder catheterization. Three preliminary studies were carried out on single dogs on August 11, 1977, October 10, 1977, and December 15, 1977. Based on these preliminary studies the following general procedure was established: Blood was collected at the rate of 0.5 ml/min starting at least 2 min. before smoke exposure and continu- ing for 45 min after the termination of smoke exposure. The blood was frac- tionated at 2-min intervals. Urine collection was also started before smoke exposure with the blank sample being the urine collected during catheteriza- tion. The collection interval for each sample was 15 min for the first 6 hr. The dog was then placed in a metabolic cage and the urine was collected on a 24-hr basis for 4 days. Five dogs were exposed according to the above procedure on March 20-22, 1978. After exposure, one of these dogs refused to cooperate and no signifi- cant samples were obtained. Excellent sets of samples were obtained from the remaining four dogs; the results are presented in Tables III-1 and 111-2. The 14C-activity measured in the blood increased quite rapidly after the i,nitia- tion of exposure to the labeled cigarette. The level of activity in the blood reached a maximum at the end of the exposure to the second cigarette. After smoke exposure is completed there is a sharp decline in the activity detected in the blood (Figure III-1). This sharp decline slows significantly at 10 to 12 minutes after smoke exposure as indicated in Figure III-1. The 14C-activity in the urine is summarized in Table 111-2. Significant amounts of 14C-activity begin to appear in the urine about 1 hr after the beginning of exposure to smoke. Elimination is very rapid during the next 3 hr. However, significant amounts are eliminated over a 3-day period as indicated in Figure 111-2.

317 TABLE III-1 Concentration of Nicotine in Dog Mood as Function of Time After Start of Smoke Exposure to Smoke Containing 14C-Nicotine Bitartrate Nicotine Concentration, ng/m1a Dog g No. Timeb, rs~n n. 5249 5244 5172 5272 0 0 0 0 0 2 8 16 13 24 4 64 54 38 34 6 132 73 46 41 8 123 60 83 40 10 114 55 97 59 12 124 62 112 14 120 87 138 16 129 83 109 18 127 78 98 20 102 102 57 97 22 102 90 86 24 79 90 78 26 64 85 77 28 62 72 56 70 30 57 74 37 69 32 53 69 41 65 34 48 69 33 61 36 44 71 33 57 38 40 71 38 59 40 41 63 32 57 42 41 54 30 57 44 39 60 32 53 46 36 54 28 55 48 40 60 28 60 50 35 53 28 48 52 35 54 21 47 54 34 54 48 56 31 50 45 58 32 52 42 60 37 44 33 aNicotine concentration is that amount of nicotine that should be present if all the material being traced by the 14C-activity were still present as nicotine. This is certainly not the case. Thus, the numbers in this Table represent concentrations of nicotine plus its metabolites expressed in terms of nicotine. bTime that the exposure to the second labeled cigarette ended.

318 TABLE 111-2 Total Amount of Nicot4ne Eliminated in Urine as a Function of Time After Start of Exposure to Smoke from Cigarettes Labeled with i4C-nicotine Bitartrate Plicotine Eliminated in Urine, ug a Dog No. Timeb, h.5249 5244 5172 5272 0 0 0 0 0 0.25 0.7 0.1 0 0.5 2 2 0.1 7.6 0.75 28 2 0.3 113 1.0 120 22 0.4 206 1.5 278 377 298 425 2.0 435 641 426 477 2.5 604 772 488 596 3.0 737 929 726 862 3.5 814 1072 819 1051 4.0 888 1171 895 1148 4.5 1018 1275 952 1369 5.0 1060 1317 991 1376 5.5 1386 1027 1462 6.0 1489 1133 1625 20 1341 1796 1260 2048 30 1821 1315 2176 40 1444 1848 1357 2229 50 1928 2240 70 1473 1950 2243 80 1523 1370 90 1532 aNicotine eliminated is actually the amount of 14C-tracer eliminated expressed in terms of the nicotine originally in the cigarette. Much of this tracer is now present as some metabol i te of ni coti ne. bFor comparison purposes sampling times have been rounded-off.

FIGURE III-1 NIC®TINEaIN BLOOD e: Dog 5172 CI: Dog 5249 t: Dog 5272 91 `j~ (I GL o: Dog 5244 ~ ~ N -1.00 12.33 25.67 39.00 52.33 65.67 TIME ELAPSEQ SINCE START OF SMQKING' 79.00 (minutes) a{Vicotine concentration is amount indicated by i`~C activity and actually ~ represents sum of nicotine plus metabolites. ,~ bEnd of exposure to the second of two labelled cigarettes is taken as ~ zero timeU

32© FIGURE 111-2 NICOTINEaIN URINE 0 C'7 ® ~ N ~ CM 0 0 0! B U ~ ~co 1 ® LQ 0 C~1 e ~ E- ~ zQ ~ 6 L4 ~ ~ LO ~ ® ~ L ~ z ~ ~ ° ®~ ~ z lb A: Dog 5172 0 ® Q: Dog 5249 ~ +: Dog 5272 Oc ° o: Dog 5244 ~ E-1 v+ ® 9 Q Q ~ 0 0.00 16.67 33.33 50.00 66.67 83.33 100.00 TIME ELAPSED SINCE START OF SMOKING (Hours) a7otal nicotine is amount indicated by 1'*C activity and actually represents sum of nicotine plus metabolites.

321 Final evaluation of the results have not been completed. Preliminary indications from the blood tracer 1evels suggest that the nicotine/cotinine levels in serum may be too transient to obtain meaningful samples. 14C- levels in urine indicate that a very large fraction of the total nicotine dose is excreted in the first few hours following smoke exposure. In a chronic exposure situation, animals are often exposed over a 10-12 hour span each day. The data suggest that to obtain a meaningful sample, all urine voided during the day, including that eliminated during smoke exposure, should be collected. Also, from Figure 111-2, the elimination of the 14C label undergoes a sharp change in slope when the animal is placed in the metabolic cage. This is somewhat unexpected, since a smoother approach to a steady state condition would be the more usual case. This suggests that another mechanism is controlling the apparent excretion of the 14C label. It is entirely possible that when the animal excretes the urine on the surfaces of the metabolic cage, a significant fraction of the nico- tine (or its metabolites) is lost, either due to volatility across the large surface area or a reaction with either the stainless steel surface or a contaminant on that surface. Further experiments will be necessary to confirm these hypotheses.