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t . , 1C 12btwC, b Nbct Eur J Clin Pharmmacot (1985) 2$:301-3tD4 Steady-State Concentration of Cotinine as a Measure of' Nlicotine-Ihtake by Smokers . R L,. Galeaai'„P.©aenens ; and' M: Gugger' 'Department of Medicine, lnselspital Bern; University of Berne. Berne„S witxerland and I 'Depanment of Toaieoiogy; University of t.ouvain, Louvain, Belgium Summary. Measurement of plasma cotinine, the ma} jor metabolite of nicotine, is usually done'to deter- mine nicotine-intake in smokers. Cotinine is used in+ stead of nicotine because it has a much longer half-life than the mother substance andl its plasma concentrations are therefore less dependent on the exact times of blood sampling. Howeveq the (in- earity of the relationship between nicotine-intake and cotinine level in plasma has never been proven. Therefore cotinine was measurediin6 healthy volun« teers infused over 4 davs with several doses of nic+o- tine' i.v. up to 4'80ltglkg./day: Cotinine concxntra« tions in plasma were shown to be linearly and directly relatedito nicotine intake. The concentration of cotinine showed little variation during and fior up to 2'h after the last dose of nicotine. Therefore,,coti} nine can be used as an epidemiological marker of nicotine intake if it, is measured around the'time of the last cigarette of the day. Keywords: nicotine, cotinine: steady-state' plasma levels, smoking; cigarette consumption, nicotine in+ fusion. Nicotine, consumed primarily as inhaled cigarette smoke, is one'of the most widely abused drugs in the world. Its deleterious efflects on health are' well known_ As one attempt to reduce nicotine-intake, without affecting taste,,indust~ry has manipulated the design of cirarette filters. but, such' manoeuvres may notproduce the desired reduction in the level of nic- otine in the smoker [lJ1 The discrepancy betweenthe- orn and practice emphasizes the need for an, o'bjec- tic•e measure of true nicouine intake bv smokers. The deleterious effects o(l cigaretne smui:ing' on healnhideprnd muunVv on the amounvof tnr~ reachine European Journal o(' Clinical Pharmacology ®lSpringer-verlig,1985 the lung and the atnount, of nicotine reaching the cir- culation. As thettar-to-nicotine ratio is qµite constant in each brand of cigarette, determination of niicotine - intake could yield most of the data necessary, for study of the epidemiology of smoking. Nicotine, however, has a short half-life [2] in the body, in the range of only 2h [3Lso that its concen+ tration in blood is sensiciive to time of blood samplingg and is primarily a measure of the last few cigarettes smoked: This is of ' great concern as individuals smoke with, different itensities at different times. What is needed is a measure of'average cumulative nicotine intake over a suitable period of time, e.g. at lieast, the last, day: Cotinine, the principal metabolite of nicotine [2J, has a muchlonger half-life than~niicotine around'17'h [11;'1, The concentration of cotinine in plasma has therefore been used recently ['1, 5~-7J as a marker of nicotine consumption. However, the inference of' . nicotine-intake'from cotinine concentration can only be made if the relation between the 2 variables is a direct linear proportionality, i.e: metabolism of nico• tine and subsequent elimination of cotinine db not vary with concentration. In addition, nicotine intake wouldl have to be fairlv constant over at, least three da_vs, so that a steadw-state of cotinine can be achieved. The present study was performed to test the hy- pothesis of a direct linear proportionality between nicotine intake and ste:adv state cotinine_ Material and NLethods Six heaithv vollinteers. 2 females and 4 males. «•ere studied. Their mean age was _'3I.Svears (irangt ~(l ~S vears) and mean' weight 6i kg (rang.k ~'~-66ke9. The stud\ protocol %%as approved b%, the

302 TrN6 L IWeaa steady-state ooornenvstion (Wt) of niooane infusiau' R[: Caleam'et aL: Codnine Levd'and Niootine-tnuikrin Smotini . in ptzsma during the last dosing interval on ~the fourth day after four ditfetant Doscufniootine(}igJt6/dayl Subjeccs: (in Ng/Wipiection) 1 2 3 4 5 6 R(xsD) 120' (S) 79.1 l 1116 109 E5.1 71.1 1t4s 92.0 (lia). 2401(10) 168' 20 192 196 167 182 1t;S (1S.2)' 360' 05) 241 276' 292. 315 ' 249 291 276 (2d). 480 (20) 407' 413 415 377 312 361 381 ' (40.3)'. Stopc of cegiession line 0.769 0.836 0213 01642 0.667 0.763 0:7E3 (0.065)) 0' ...,afo ..{ o 20, 4.0 Moal~.la tr,.•,'-,4,^'). . tia.L Srtady-stste plauna oonoeotrstina af nicatine vs,dase of aico6ne. The nimdne dosa udiated anere pven ova a112 h4y- ouae period for4~aonseae6re daya:'n+e aonoanuaooo of bodnine was meaaumd sehent drnes durina the last'dasing interval on the foucth day. 7be',points s6oaa aee the meanwalua ( t SD), and'the meaa nepession line Ethical Committee of'the School of 'Medicine of the University of Beme. E.ach . vol unteer gave written in- fotmed consent to the inveshigation: Each of them was judged healthy by medical examination and bio- chemical and haematological screens. They were all smokers and'had consumed at least 2(Dcigarettes dai- ly for at least 12 months. They were not allowed' to smoke during the period of the study; butl they con- tinued'Ito smoke (the same brand and the same num+ ber of cigarett+rs) in between the experiments: During the experiment, subjects were hospital- ized. They received ordinary hospital meals and were permitted free access to coffee and tea. In. order too standardize the pH-dependent urinary elimination, of nicotine, urine pH was kept around 7 b!_v giving so- dium bicarbonate (NaHCOO by mouth. Nicotinras the free base was administered intra~ venously in sterile 0.9% sodium chioride solution, provided by the Pfiarmaceutical Development Cen- tey College of Pharmacy, Medical University of South Carolina, Chadeston, S.C. (thnottgh Dc T. D. Darby, USC School of MediCine, Coiilmbl S.C.);,in S'anl vials,eacl cotuaining nicotine l mg/ml. htitaotine S,1q;15 lor'2B 1tglkg bodyweight was given every 301min by continuous infusion orer .1l0min• with a Brauw Melsungen (FRG) precisiolt' pump- 'Ph'erust'infusioa of the dhy'was given at'7 a.1n. and the last at 7 p. aL Each dose level was given Ifor 4 oon- seattiire days (Monday to Tbursday). On each &y the 1230 pan. dose was aaissedAueto ltlncli.'Phe to- tat datl jr dnses wete nicotine 120, 240, 360 and 480 }sg/kg',bodyweight. Befone'starting the fust infusion in the morning and aRer the last' infusion of the day,1 mf infusion liquid!was saved for analysis of its nicotine content Blood samples (5 1 were : taken every day before the Crst daily dose. On the fourth, last day (Thurs- day) blood samples were taken befom in the middle . and at the end of the last'(7 p.m.) infusion„and then 5,10;,20, 30 and 45 min and 1,1.5, 2, 3, 4, 6,10,14,16, 20 and'24Ih thereafter. The blood samples wene cen- trifuged iimmediately'upon collection and the pliarsma 1 stored at -18 °C until analysed: Cotinine in plasma was determined by a newly developed' GC-SIM procedure (P,Daenens et al.. J. of Chromatography, in press). A deuterium-labelledd analogue of cotinine (3-cotinine+tnethyl-CDj) was synthesized for use as the internal standard. Briefay, to I ml plasma 100 ng, internal l standard and meth- ylene chloride was added. After shaking and cen- trifugation the organic fraction was withdrawn, dried over anhydhous sodidum snlphate and carefully evaporated tio, dryness at 40' `C. Just, prior to injec- tionl the tesidue was redissolved in 1000 methanol and a 1 µl aliquot was injected~ into the GC/MS sys- tem operating in the SIM mode (m/z 176 fior cotinine and m/z 179 fbr internal standtard~ A CPr'' wax 57 CB capillary column of 50 meters with ht.'lium (0.5 mI/min) as the canriergas was used with splitless injection, Temperatures: injection pon 260 °C; col- umn 40 °C for llmin, 40-150 °C at 35 °C/min.

Ft. L Galcarzi et at.: Cotininc Level and Nicotine,tntakc in Smoking 103. ~ O-O ~--~ *-* _._.~ .r~. n . c 5 a ~ „_s 20. -r -10 30 90 2 (mi°). Time ,-- 8 (h) Tabk,2.' , Mean nicotine content of 3 different brands of agarettes, using data collected by Gori'and Lynch (5I and'ealaulated by Eq. (_) Btand A B C' Cotinine leveU(n¢imll 301 2041 208 Cigarettes per dav 29.01 30:5 313 FTC-ratingr mg per cigarette 0.18 0.11 0A0 Calculated mean nicotine intake 0:861 0.55 0'S48 per cigarette (m¢] Ratio of mean nicotine per 137 1.01 1.0 ci¢arette to brand C a-. s T 24 303' Fte,2. Tune course of the plasma aonaen- tntion of cotinine during and after the iast intravenous infusion of nicotine. The,nico- tine doses indicated werc,given 24!times over a 12 h period during'the day for four consecutive days the mean difference was 0.28% (range - 53 to 127%) of the theoretical1 dose. There was no loss of nicotine in the injection flaid'overthe day;,the differ- ence between thecontxntration measured before the. first infusion in the morning and I after the last infu- sion at the end of the day was -0.59%, which: was not significantly different from zero. The'mean-steady state concentrations of cotinine are listed in Table 1. As can be seen from the same data„displayed in 1=<g:1, a'dear linear relationi exist- ed between the daily dose of nicotine for 4 days and the mean steadiv'state concentration of cotinine dur- ingthe last dosing interval. In no case was the inter- cept'of the regression line'significantly difFerent from zero, therefore each regression line was forced through zero. The mean regression line was 150-224 °C' at 15 'Clmin; DC/MS transfer line 225 'C; ion source 150 °C: The coefficient of variation for a 3-day validation study on a pooled plasma sample containing coti- nine 85':ng:<ml varied from 3.6 to 5.4%. Nicotine in the injectionfluid'wasdetetmined by U V-spectrophotometry. The area under the plasma cotinine cont:entra- tion - time curve (.aUCr) during the last dosing in+ terval (r) was determined by the trapezoidal, rule. The averaeeconcentration of cotinitue.at steady state (C;,) was calculated using the following formula C_ AUC-. p~) r Results The dose oC nicotiine injected. as measured bv the %obume deli.ered andlthr concentration in the infu- ;idrn Iluid', .iitlfcred' IGtulc tirom thr theoretical vailuc: Cotinine conc. (µg/'I) = 0:783'x Nicotine (gg/kg/day) (3) The smallest r= from the individual regressioni lines was 0.988. The individual slopes ranged from 0.667, to 0.842. The time-courses of the plasma concentration of' cotinine after, the different i.v.-doses of nicotine are__ shown in Fig.2: There was a clear plateau up to 2 h~' afrcerthe last' injecti on. ~. ©iscussion i W The results show that the a.erage plateau conLcntra- tion ofcotininewus dircctlv proportional tb, theM amounrof nicotine infused into the scstemic circulaAN . tion. This indicates thut thc kinrnics oficotininr is lin-Z 4

304'. ear up to the amount of nicotine used! and the : con- aentration of cotinine measured. The regression line can even be used as a standard curve in order to cal- culate the average daily nicotine intake over the last few days from a single measurement of ootinine con- centration in plasma. Although the analysis is based on an : average steady-state concentration, a single plasma~ concentration determination would' appear to be adequate for epidemiological use. The time course of eotinine in plasma during and after the last in jetxion i (or ciganette ~ in real life) is flat for a period ~ of about 2 h. Therefore, a single measurement at the end of a"smoking-&y" will yield.a reliable estimate of the average steadiystate ooncentration iof cotinine. Higher dbses of nicotine were not, given for ethi- cal I reasons. Therefore, the results are only applicable to concentrations of cotinine not exceeding 415 µg/l.. However, the majority of cotinine levels foundlin ep- idemiological studies (SJ Ido not exoeed t;hat' concen- tration. In addition, as there is no hint at all of a cur- vature in the relation between the input rate off nicotine and the plasma cotinine concentration, ex- trapolation to a higher concentration ~of ootinine may be undertalcen, although with less reliabt'lity. Strictly speaking, the regression line given hete,; Eq. (2), can only be used in,a. populatioti similar to that from which the present subjects were drawn, as the rates of transformation of nicotine to etxinine and of elimination of cotinine : in the population studied should be identical to the elimination rate in our sample. However, if comparative experiments are made, use of Eq. (2) will yield'at least relative re- sults. As an example of the application of Eq. (2) to ep- idemiological data, tFie : data of Gory and Lynch (51 were re-examined- They'measuried cotinine levels in a large group of smokers, given three different brands of'low-tar' cigarettes. The nicotine content of the cigarettes had previously been measured by a machine-smolung method, as required by the Feder- a1i Trade Commission: the nicotine contents are called the 'FTC'-ratings : Mean cotinine levels (mea- sured in the late att~ermoon)„ mean number of ciga- rettes smoked per day.,and the FTC-ratings; as given in the report (5) are shown in Table2 By applying Eq. (2)to the measuredlmeancotinine levei relatedito the differenr brands of cigarettes, it was possible to calculate the mean daily'nicotine intake (assuming a mean bodyweighrof 65 kg) by the smokers. Dividing this value by the mean number of cigarettes smoked per day, gives a mean nicotine content per cigarene. R L Galta» et al.: Cotinine Lzveland Ktaatine-tntake in Smoking As shown in Thble 2, the calculated value for mean nicotine oontent, per cigarette differed by al- most an order of'magnitude from the official FPC- rating, but it was in the same range as that found I by Benowitz and Jacobs (9J. However, as the ratiio of the calculated'mean nicotine content per cigarette be- tween the different brands was about the same as the ratio of the FIrC-ratings between, the different brands, the FTC-rating can probably be regarded as an appropriate rdative rating, at least in the low-tar group of cigarettes. It is considered that measurement of cbtinine and hence determinati'on of niootine-intake should be of great help in epidemiological studies of nicotine aon- sumption. Referenoes 1: Betnwitzl+tL, Hall sM, HbnniogRI„laeob l1I IP. Joaes ttT, Os+ taatt J1L (1983) Sttwkets of losr-yield ciearettes do not consume tess nicotine. N F.aaL1'Med 309:139-142 2: 6edtett AH, Gbtrod )W,lentter P (1971) The efftct of smoking on nicotine mctabolistn in viv!o tn man.1 P[tanttaoot'23'lSuppQ: 62S-67S 3. ttosenliattJ. Beno.dt: Nt.,Jacob P,Wikon KM (19&0) Dispo- sition Iunatics and effects of ittttavenous nioaitte. C]In Pitarma, cd Ther 2a: 517-522 4. Banowvitz B1i, KnytlF. Jaoob dIl P. Jones 1tT, OsmanAL (1983). Cotiitine disposition and etfeas Clin Phamtacol Ther 34: 604-611 S: Goti,GB;,Lyttch,CL(1983) Smokenintzke from cigarettes in the 1-mg fedenl Trade Coauniittiat tar dass., Reautat Toxicol Pfiannaoot 110-120 6 GciMER, Baer-Weiss V. Benowitz NL, Vatt! Vunakis H, Jau- vikM'P'(i981) Plasma nicotine and eotinine concentrations in ltabitud smokeless users. Clin Ptiumaeol Ther 30: 201-209~ 7. Matsukura S. Sakamotn N, Seino Y, TLmada T, Matsuyama H, Muranaka H(1979), Cotinine excretion and daily cigarette smokingin habituated smokers. Clin Pliarmacol T}ier 25: s53-16f 8: Beckett A4i, Gotnod U!W, Jettner P' (1972) 1 As possible relation between pKai and lipid solubility and the amounts excreted in urine of some tobacco alkalbids,given to man. I Plutrmacol 24: 115-120 9. Beno wit¢ ML, Jaoob Itl P(1!984) Daily inake of nicotine during cigarctte smoking: Clin Pharmaool IT1ter 35: 499-St14 Received: September 16; 1984 acoepted: December 14, 1964 R: L Gakana. M: D. University of Berne Department of, Medicine Inselspital CH-3000 Berne. Switurdand!