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r Urinary cotinine measurement in patients with Buerger's disease - Effects of active and passive smoking on the disease process Misahiro Matsushita, MD, Shigchiko Shionoya, MD, and Takatnshi Matsumoto, MD, NeB%a, J" Although :Buergcr's disease is knowa to be closely related to smoking, no objectivc analysis of the smoke-associated problems has been pcrformed+ In this study, cotiniac, the major metabolite of nicotine, was used as a scasitivc marker to measure levels of active smoking and the exposure of nonsmokers to tobacco smoke because it has a relatively long half-life and because cotinine kvels canbe determined by noninvasive means in urine. According to urinary eatininc levels, 40 patients with Buerger's diseasc were classified as (1) smokrrs: those with urinary eotinine levels abotc 50 ng/mg etritinure; (2) passive smokers: those with Icvcls bttx•ecn 10 and 50 nglmg ereatinine; and (3)1 nonsmokers who did not experience noticeable passive smoking: those with levels bcJow 10 ng/mg creatinine. Tberc were 10 smol~srs, 9 passivc smokers, and 21 nonsmokers. The course of the discase, after the initial trcatment at oer hospital, was studied retrospectively. Seven of the 10 smokers, none of the 9 passrt smokers, and 4 of the 21 nonsmokers ezpcrien.;ed aggr•~avation of the diseue. Of the four nonsmokers who exprrienced aggravation; t3irre had still been smokns and one had been exposed to tobacco smoke in the workplace at the time ofrelapse.'Ibere was a significant differcncc in the aggravation rate betwcen the smokers' group and the other two groups. Among the smokers, the seven patients whose conditions worsened showed signi5cant3y higher cotininc levels than the three remaining patients who were in the stage of remission: The conclusions were: (1)~a very dose relation between active smoking and the course of Buerger's disease was established, and (2') effects of passive smoking on the disease process were still incondusive. (J Vnsc Suae 1991;14:53-8.) Buerger's disease is chanctcrizcd by peripheral u~t+•:al occlusion of the extremities most frcqucnth- :.^ ynung adult male smokers.'= In general, all patients with Buergcr's disease have a history of smoking, and smoking is also known to be closeh• related to exacerbations of the diseasc.' •' The outlook in rcgard'to the effects on the limbs of a patient -Aitli Buerger's disease is favorablc if he stops smoking, but the disease gets progressively worse if he continues to smokc.'•' tiowevcr, we have occuionallr found that the d4assc recurrcd in patients who stated that thn• had abstained from smoking. Many of them may have been lying about thcir smoking habits: some wIerc From the First Depara»ent of Surgery, Nan, Unnsrsin• Sdwd ofModicnc (Drs. Mnsushin andShionorna) and tfie Depart- menv of Surgery, Nagoya Second Red Cross Hospital (Dr. riatstrrnoto). Rr?.rinr requesa: Muattiro Matsushita, MD; The First Depart- it of Surgery, Nagoya University SchooF of Mrdianc, Tiu-urnai-cho, snuwa-ku, Nagoya, Japan. u/i/yg4p8 _ . ~...,..,K dccmed to have denied themselves the pleasure of smoking but had been exposed to tobacco smoke in the home and workplacc. Because there is no objective test to evaluate smoking, previous studies have had to depend on paticnts' testimony of smoking habits. An objective method oferaluationof the degree of active and passive smoking is necessary to elucidate the relationship bcn+xcn smoking and Buerger's discuc. By measuring urinary conccntrauon of cotininc, the major menbolite of ni¢otinc, we found a correlhtion between smoking and the natural course of Bucrger's disease in trtrospectire study. PATIENTS AND METHODS Urine samples were colleaed for: measurement of nicotine and cotinine levels from 50 volunteers (23 smokers and 27 nonsmokers) without noticeable passive smoking and whose statements of smoking histories were regarded as reliablc. The time pattcrn of nicotine and cotiniite excretion was studied to judge whcncer alkaloid is suinblc as the marker for 53

54 Matswcbisa, SbionerS and Maax,noro smoking from the stindpoint of the half lifc of cotininc in the human body. For this purpose, urinary nicotine and cotinine levels of a healthy nonsmoker (one of the authors) were measured after hc had smoked one eigaratc and after he had been placed in a passive smoking environment. For our passive smoking experiment, the subject was placed in an airtight room (19.1 m=) and espose& to sidc-strcam tobacco smoke fmm a total of 40 cigarettes for 3 hours. Urine samples from 40 patients with Bucrgds disea.se were cvllocted~ (one for each case) when the patients came to our cTtnic. Each paDent's statement about aurcnt smoking status and inz~olunnn• expo- sune to smoking was trquested, at cach visit. Our clinical criteria for the diagnosis of Buerger's disease are: (1 ), hisrory of smoking; (2) onset before the age of 50 years; (3)~ infrapoplitcal arterial occhuivc disczsc;, (4) either uppcrdimb involvement or phlc- bitns migrans; and (5) absence of athcroscJtrroac risk facaora other than smoking:'Ihe clinical diagnosis of Buerger's disease was made when all five require- mcnts were mct.'•' Infrzpoplitcal obstruction was confirmed by arteriognphyin eachcase, and arterio- graphic findings such as tapering or abrupt occlusion, corkscrew or rootGkc appcarance of collarcnls, and corrugated appearance served as supporting evi- d:ncc. All of the patients had a history of smoking aforc the onset of the disease. Ar onset, the age of these 40 patients ranged from 26 to 49 vears (maan, 37 years). There uere 38 men and 2womcn. All 40 patients had been treated in our insotution for more than~ I rcar, andr their case histories were reviewed retrospectively. The initial treatments of these pa- ticnu were bypass grafting and synpatheteRomy, in 2; bypass grafting in 4, syrnpathercctom}' in 24, and medical'trcatment only in 110. The follow-up period ranged from 1 to 22 years, s~itfi a mean of 8.3 y,cars, In case of recurrence of pain at rest, ischemic ulceration, or graft failure (except early failure, less ttlan 30 days), which wcre eonfirmcd by follow-up surveillance, the patient %,ras considered clinicalli• to have "aggnvation~of the diseasc." Utinary nicotine and cotinine k,xls were derer- mined by high-pcrforrnancc liquid chromatography (HPLC) according to Mizobuchi's mcrhod` wi ti some modifications. Wc changed the extraction procedures in order to assess ven• low levels of these alkaloids. Urine samples were stored at -20° C until! analysis. Ten milliliters of urine was centrifugcd: Afrer the addition of 4 gm sodium chloride, 0.1 ml 2586 anunonium hydroxide, and 2 ml chloroform, the urine samples were shaken for 10 minutes and centrifuged at 12,000 rpm for 10 minutes. The chloroform layer was colJccted and then shaken with 5 ml1 of 0.1 N hydrochioric acid for 10 minutes and eentrifugcd at 12,000 rpm for 10 minutes. The resulting aqueous layer was shaken with 2 gm sodium, hydrochloride, 0.2 ml ammonium hydroehloridc, and 1 ml chloroform, and then centrifugcd'at 12;000 rpm. Fifty microliters of this chloroform~ layer was used for the HPLC. 1t,verage total recoveries were 98% for nicotine and 85% for eotinine. The detec- tion limits of nicotine and cotinine were 2 ng/ml and3 ng/ml, respectively. Urinary rucoanc and cotlninc values were normalized by ercatininc excretion and expressed as nanograms per milligram of ereatinine. Statistical sigrai5canct was assessod by Studeat's t test or ehi-square analysis, and the results were considered significant ar p< 0.05. RESULTS For the healthy control subjects, urinary nicotine levels were 576 ± 474 ng/mg acatininc (mcan value t standard dcviation) in the smokers, an& 5.2 = 3:8 ng/mg creatanine in the nonsmokers who did not havc perceptible involuntary exposure to -~ tobacco smoke (p < 0.01). Urinary cotinine levels for these two groups were also signi5cantly different (859 ± 814 ng/tng creatiiune in the smokers vs 5,6 :t 2.3 ng/mg cscatininc in the non- smokers, p< 0.01).. Urinary cotinine levels dis- criminated berv'eenthc smokers and the nonsmokers more distinct)y than nicotine levels. Therefore those with urinary, cotinine levels above 50 ng/rng ereati- nine may be rrgardcd, as smokers (Fig. 1): In smokers, urinary excretion of cotinine roughy cor- related to sclf-reponc&cigarettc consumption (Fig. 2). Fig. 3 shows urinary nicotine and cotnninc levels in a healthy nonsmoker after he had smoked one cigarette and after he had been exposed to side-stream smoke. Urinary cotinine elevation after active smok- ing lasted for 60 hours. The urinary cotinine level after passive smoking was lower compared with the level after active smoking, but it showed the same rise and fall as thc level after active smoking. The disappearance of nicotinc from the urine was faster than that of cotinine. Because of this, only the urinary cotanine level was used for studies on the paacnts.. Fig. 4 shows the urinary cotininc levels in patients with Buergcr's disease. All, three patients who eon, fesscd themselves to be current smokers had cotinine levels that were higher than 50 ng/mg ereatininc. Of the 37 patients who assertcdthat they were nor active smokers, seven (19%) had cotinine levels above 50 ng/mg crcatininc. According to our definition, these 2Q2:35Z1858

~ .4Manc 14 ..ienbc ] t~fi.. 1991 Orinitrr aioatine Urinarr coiiaiae sN/.l ctaitioiee al/nt posti.iae r<a.t1 . 3a00 Mi, 3000, 2000 2Q00, / 1000 1000 4I ~ 500 5a0 ~ - 1 200 100 200 160 . 50 • 50 20 , 20 10 i 5 10 5 3 3 2 2 ~ 1 ~ 1 NOllsmol'ers' Smoters NonsmoLers Sslolsers Fig. 1. Urinary tsicotine levc3s (kfl) and cotininc kvels ~ (rsqht) of nonsmokers and smokcrs, in haalthy, control 5:,hjec•tis. T2sere wetr sigtuficant diffcrrnces betwcen the r.: o groups (p < 0.01): Gorinine kncis disaitninate tx- twecn smokers and s+onsmokers more distinctly than } nicotine kwLs do. seven patients were considered active smokers, whereas the other 30 patients .vere regarded' as ezsmokers. The 30 ezsmokcrs were then di-%ided into rwo groups, on the grounds of self-reported invol- ay exposure to smoking: Urinancotirune levels ;_: c 10.2 ± 4.2 ng/mg creatinina in those who wer•c imolluttarily exposed to smoking and 6.1 = 3.5 ng/mg creatinine in those .vho were not a.posed (p < 0.01) (Fig, 5). On the basis of these resuha, we decided that for this study, those with urinary eotinine levels between 10 and 50 nglmg aeatanine would be identified as nonsmokers «tith noticeable passiv' smoking (passive smokers) and those with :°'1s below 10 ng/ing creatinine would be identified i+ i.onsmokcrs without perceptible passive smoking ( Fig. 5). The 40 patients were classified into three groups: (T) those with urinary cotinine levels above 50 ng/mg erztininc (active smokers), (2) those with cotinine lcvels between 10 and 50 ng/mg creatinine (passive smokers), and (3) those with cotinine levels below 10 ngJmg ereaunine (nonsmokers without noticeable Pascive smoking). Eventually, 10 patients west dos- >itied as active smokers, 9 as passive smokers, and 21 as nonsmokers. Z?le disease worsened in 7(70%) of Lhiwsry aonnine .uanrnwenr sn1 Bre.er} Iiruu 5 S a=/ot cre3tinice 3000E 0 . ~ 2000 ~ 0 C .. ~ 0 0 !I . . 0 -! T0-11 20-29 30~- Cq~rettt//ity (N=3) (N~6) (N=9) (N~S) Fig. 2. Urinary, corinine levels in smokers. Smokers wen dassitied' into four groups on the basis of self-reported cigarctre consumption. Urinary corirutx kvels roughly conxlated to daily cgarettc consumption. the 10 smokers; in none (0%) of the 9 passiwc smokers; and' in 4 (19%) of the 21 nonsmokers. Trherc were significant differences in the rlre of the aggnvation of the disease between the smokers and the passive smokers (p < 0.01) and brn-een the smokers and the nonsmokers (p < UAI ); However, no signi5cant diffcrences in the rate of aggravation .vere found between the passive smokers and the nonsmokers (Fig. 6). Of the four c.xsmokers who expericnced worxning of the disuse, three admitted that they had'still been active smokers at that time. The other one stated that he had been involuntirily exposed to noticeable smoking in the .vorkplace all day at the time of rraurence. This patient had sympathetectomx and bypass operation of the Ic.frkg for the initial matmenc: Four years later, fe:morocru- ra1 bypass grafting, in the right kg was necessary beause of right popIiteal artery ocdusion that was a resuh of a skip lesion. Thereafter, howe.=, he has kept away from tobacco smoke in the workplace and he has been doing well for 2 years (Fig. 6). ,•lmong the 10 current smokers, the mean cotininc kvrl' for the seven patients who had aggravation of the disnu was sigtu5cantly higher than the level for the thm patients who did not expetience rdapses (1208 ± 734 ng/mg creatnnine vs 147 = 79 ng/mgg ctatininc, p < O.DS).

66 MRmtAbYta, SnonoYS, axd IHLMdwOto Iq/rst crt7tinint ---Micetiae -Cotiuine 12 2/ 36 48 60 12(hours) After Wive smokiap -- Micetiae -- Catikiu : 10 u c a 0 A . . ,.---~-, ° Betore ~ 12 2/ 36 48 60 72 (hours) Atter passive smokirGY jwTsW d VA.SCZJLAR SURGERY Fig. 3. Utinus nicotine (b.okm tiru) and coQninc (wl:d Jinr) ezavaon over time. After active smoking (abovr). and after high involuntary exposure to smoke (bclcm), in a healthy nonsmokcr. Cotiainc levels decreased more slo..-tv than ~ nicotine kvel4 did.. DISCUSSION Carboayhernoglobimor nicotine concentrations have been used as indicators of smoking.° In vascular surgery, carboayhcmoglobin has been used to determine smoking habits of paoents who had arterial recon.structnvc opcrations,` ° and Vi'iscman et al,° reporte& that the median concentration of earbox)fiemoglobin was significantly higher in those patients whose grafts had failcd than in those whose grafts were patent. Ho..'ever, blood carboxyhcmo• globin concentrations have not proved to be markers specific to smoking, and nicotine measurements have been regarded as providing more accurate assess- ments.1D Recenth•, cotinine has been considered a more sensitivc marker of smoking because it has a much longer plasma half-life than nicotine does (about 30 hours vs about 30 minutcs);,''" In this study, ururary cotininc levels dearly discriminated between smokers and nonsmokers. Bv measurement of cotinine leve1s,10 patients were identified as active smokers, although seven of them daimed to have quit smoking. Of these 10 active stnokcrs, seven experi- encc6aggravation, and there was a significant differ- ence in the rate of aggravation between active smokers and exsmokers. It was confirmed that active smoking was very closely related to recurrences of Buerger's disease. Three former smokers, ho.veva, experienced worsening of the disease even though their urinary cotinine level remained within a non- smoker's or a passive smoker's range. Since the urinary cotinine elevation aftcr smoking hstcd; for only 60 hours, our assessments of smoking were limited to a very short period. Past smoking habits cannot be estimated by ill-tirne& measurement of eotinine, a short-term markcr, when paocnts have abstained from smoking. Serial examination of uri, nary cotininc levels should be performed to solve ttus problem, Bontue the number of cigarcttes smoked roughly correlatcd witti the urinary cotii•iinc kvd,1u'" thiss levd may reflcct the intensity of smoking. Howeva, there was considerable variation in cotnnine exeartion, 4 -F, among subjects who smoked approxirnately the sune . number of cigarettes. These variations were assumed to be caused by differences in nicotine eontent per ~ eigarettc and in the manner of smoking (inhiling or' ~ pufbng, frcqucncy,Jcn.gttr of cigarettes smoked). "" '' In this study, among the patients who continued to .r~. smoke, those who experienced aggravation of the disease had signi5cant]y higher cot3nine levels t3tan 20235118FQ :~:47L

lhiinary Gaitwint 1wcaArrs+wewt An1 Brn7er} luttrr 57 0 c ., ~ 3000 2000 1000 aj/br tnitieiu 5a0 0 0 0 e 100, ~ 200, 50 20 10 5 3 2 1 t . ~ . 0 0 Ersaokers CGrrat s.okers (N'-31)' (It-t) r_. 4. Urinary cotinine ltvels. PaDentswere ditiided into t+ro groups according to tkteir statementr about ttieir smoking, habits. R'egardlets of their claims, they Rrrc classi5ed as smokers if they had urinary eotinine levels above b0Ing(mg creatininc. those who.rerc in remission, Hown•er; even among those who experienced aggravation, the urinan• Ls4+H:nc li:vels varied w•ideiy. It scems impossiblt to ct which patient will become worse. judging Irum~the number of cigarettes that were smoked. Recent studies have indicated that im•oluntan• vposurc to smoking may be as harmful as active smoking.'s Sinzinger and Kefalides'° reported that passivc smoking reduced platelet sensiti%•irn• to anti, aggregaton, prostaglandins (Es, 12, Di), and the reduction in sensitivity was much more sntrc in Ma*Smokers than in smokers. Passi.•e smoking might ete;t a poor influence on the cardiovascular system fur nonsmokers. In this study, the influence of involuntary exposure to smoking on Buergcr's dis- ease was studied'~ by measuremenr of urinary eotuune ltvels, but no significant relationship betv~rat utvol- untary exposure to smoking and recurrence of the diseue w•as found. However, there was one patient who had aggravation of the disease, who testified to liav=' abandoned smoking habits, and this person had tl.c _rinary cotininc lcvel of a nonsmoker. Because he had been involuntarily exposed to noticeable smok- ing at the time of worsening of the disease and is not ai/.E c S0 rtstiiiu 30 ~ 20 ~ 8 = j~ ~ 10 ~ __" T T a c 5 L 111 I 0 r r Z 3 U i N.t aposed Espesed (1-13) (1-17) Fig. g. Urinary codninr kvels of nonsmokers wittiout involuntar)•, exposure to smoking and nonsmokers with involuntary exposure to smoking, Thcra was a significant difference between the two groups (p < 0.01). 't/eE er t~tisise 3000 . 2000 : 1000 500 . Smo kers ~ 200 0 .~E 0 100 ~ . w ~ 50 20 s Pass ive serokers 0 10 ---- ~--- . 5 2 ~. 0 ~ Noas ootie smok mokers •iteoyt eablr-oassire' ing 1 . .~ Nbl auraYned Aurartted' (r-i9) (r-ll) Fig. 6. Urinuy cotininc kveis and ~ the course of Buergct's disease. There were significant differences in aggtavation between the smokers' group and the other two groups, but no significant differences were found betx•een passive smokers and nonsmokers .vithout noticeable passive smok- ing. Three (arreriskJ) of the four nonsmokers uith aggra- c•ated conditions stated that thet• had been srnoking,at the time of worsening of the discasc. t

58 Maaacsfiirg,Sbioroys, sa1.tlsnre.anv ezposcd to tobacco smokc now, the worsening of his disca_u may be associated with the pssr imrolunnry exposure to smoking. The incorrect tinung of uruiaty cotinine measurcmcnt mat<cxplain Mh;• no significant rdationstup x•as f'ound bcmmcn passive smoking,and the worsening of thc disease in this study. A cooperative epidemioiogic and eiinialistudy that is based on the long-tcrm and umdy c,zluatton of cff'eas on hcalth of involuntary exposure to smoking may pro.idc the evidence to support the hypothesis that passive smoking can influcncc the occurrence of Buerger's disease and the worsening of the disease process. -In conc)usion, coaninc is a sensitive but short- cerm marker of smoking. Smofdng tobacco was very closely related to tfie course of Bucrget's disease, but no signi5cant corrclarion bctween pa.ssivc smoking and the disease process has been found' yet REFERENCFS 1. Shinono}a S. What is BUcrgees disast? Worid~ J Surg 1983;7:rs44-51_ 2. Mcl:usick \'& Harris tY'S, Ottesen OE, Gfladman R?vl; Shclln• 16?ci; Bloodwclli RD. Buetgers disease: a distinct clinical usd parNologic cncn•: JAMA 1962;181:93-100. 3. Shionoya S. Buerga's diseuc (tluombouigiics ob6tesans). Inl Ruthcrford RB, ed: Vssculzr Issgay: 3rd ad. rhitadelphia: , l1B Saunders. 1989:207-1 7. 4. Shionos•i S, Baa 1, Nakata Y. ct al. Vascular reaonamxtion in BUer¢er's disctsc. Br )iSurg 19r'6;63tS41~6. S. Mizobuchi 1\S- F;iuda Y, Tamasc K. Sssafi M, Ueda Y. Simulnneous dererminaaon of nieoanc and eotininc in tiuman unnc bs fsigh-pcrfocmancc liquid chromatography. )wrtss! d VASGZJ1JlR SUAGY3CY' Annual Rcport of the Nan Prdeaural Irssanm of Public Hcahti 1985;20:60-6. 6. Maav!'nsra S, Taminato T, Kinrw N, a al Effoca of envircxuncncal tobacco smokc on urinary ooonine cartion in nonmsoktrs: evidcrscs for pusivc smoking. N Engl J Med 1984;314-:828-32. 7. Hocscausn M. Ssmpic high-perforttunce liquid c3fromato- graphic medsod for rapid dctasninauon of nicoenc and ooanine in urine. J Osrornacogr 1985;344:391-6. 8. Gnsnhalgh RM, Laing sr, Colc FV, T:ybr GW. Smoking arsd sttrsiaJ rcooasansaion. Br I Surg 1981;64:605-7: 9. Wisertun S, ICrnckungcoo C, Dain R, a aL Iatiuerscc of smoking usd plisma faaon on the parvscy of fe:noropoplianl' vtin grafts. Br Med 1 1959;299:643-6: 10. Rusadl MAH, Fevaabead C: Blood and usin:ry nieodne in non4mokrrs. hncer 1975;,1:179-8L. 11. Langonc f1, Gijika HB, Van Vunakis H. Niaoonc atd its rnerabolias: tsdioimmursoisays for nicaone aud: mtirsi>'sc- Biocherniisay 1973;12:5025-30. 12. Zeidenberg r, Jaffc TH, Kuvler M, l.evitt MD, Laingone T1, Van Vunaltis H. NfcoDne: conNnc le+vJs in blood during czssaoon of smoking. Gompr Psychiac•1• 1977;18:93-101. 13. Masukun S,,Sakamoro N; Scino Y, Tamada T, Mamryami H, Muruuka H., Cooninc ezaroon and diily ag:rertr smoking in habituated smokers. C1in Pharmaml'Tlxr 197'9; 25:555-61. 14. Wikos RG, Hugtsa ); Roland J. Vcri6aoon of smoking history in patients aftar infuction using urinary nicotinc and mtinirx meanurmena. Br Med ), 1979;27:1026-8. 15. Ficlding JE, rhrnow K1; Mcdical progras-hnJdh cffeca of invohurury Imoking. N Engi 1 Med 1988;319:1452-60. 16. Shirssingcr H, l:cfilides A. Passive smoking seuercly doQeases plan:kt scnsio.irr to antiaggrcgarory prnataglusdins. Lanca 1982;2:392-1 Submirted;Sept. 26. 1990; ,acccprcd )an. 30, 1991. t cn ~ I/~~~ ~.J R