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was withdrawn was 6.9 cm rsther than the 30-cm length used in our previous studies.° EndbtMllal CGI1 Counts (Counts of AnuolMer EndotMlld Gll CarpsaM) The method of Iiladovec and' Rosemann„ was used. Nine millilitet7 of venous blood was collected'in a ailiconized centrifuge tube that irontained 1 mL of: 3.8% trisodium citrate and mised. Centrifugstion at 4•C and 396 g (middle of the tube) for 20 minutes removed the erythrocytes and ltukocytea. One milliliter of the auperrutant was rnixed with 0.2 mL of adenosine S'diphosphate, disodium salt (1 mg/mL ) andmechanieally shaken for ten minutes. Another centrifugation at 396 g for 20 minutes removed the platelet aggregstes. The supernatant was then centrifuged at 2100 g for 20 minutee. After suspension of the sediment in 0.1 mL of physiologic saline by s" with a ailiconized glass rod, four Neubauer cliambern were IIlled with the auspension, and the endothelial cells were counted using phaae-contrast microscopy. Resultu were expressed as the mean cell count of the four 0.9-µL chambers. On two occasions, we evaluated the possibility that the celL counted as endothelial cells might instead be megakarlrocyte 5ragrnenta. The cells were transferred from the saline suspensions to glass slides by centrilUgation (Cytospia, Shandon Southern Instruments, Inc, Birmingham, Ala). The slides were incubated for 45 minutes with mouse monoclonal anti-human platelet gly- coprotein lb antibody (Dako Corp, Santa Barbars. Calif) that was diluted in phosphate-bufferedaaline to a concentration of 16.5 µg of antibodyprotein per milliliter, washed with phosphate-buffered aaline, incubated' for 45 minutes with goat anti-mouse IgG fluo- reseein con,jugate (Boehringer Mannheim. Indianapolis) that was diluted to a concentration of 94 ~,.g of antibody protein per miililiter, and washed again. Fluorescence microscopy revealed no nuores- eence of the endothelial' cell preparations, while simultaneously processed alides on which human bone marrow aspiraus had been smeared showed strong IIuotereenee of large cells that were presumed to be megakaryocytea. P4esma flleotlne A portion of the platetet-rieh plasma, prepared in the first eentrifuption in the procedure for endothelial cell counting, was kept frozen u- 80'C until it was thawed for extraction and preparation for gas chromatography by the method of ihyersbend and Russelll" using an instrument that was equipped with a. nitrogen-phosphorus detector (Aerograph 14(l0, Varian Instru- menv Division,, Walnut Creek, Calif). The length of the column was 90 ctzi. Column temperature was lb0*C. The method was not otherwise modised' from the original.u The means of duplicate assays of each aample of plasma were used for statistiesl analyaia. Carbozytienwqbbin B, .d was taken into a heprrinised syringe for determination of the earbozyhemoglobin level by apeetrophotometry (11-2ffi CO•o>ometer, Instrumentation L+boriitaries, 1.tunito0. Yaas). Statletloai AeNyrsir Two-tailed Wileoxon algoed-rank tests wen used to determine the significance ofthe diffet+naa between4he means oJthe paired variablea ahown in the Table andin Figs 1 through 4. ConSdence intervals oftAe di.dereaoea were calculated aceordint to Gardner and Altman. p The Spearman rank eorrelation coefficient was used as a measure of the association between variables. RlfaJlTS The Table shows the mean values of each variable before and after the control period. No siQniScant differences occurred (P>.2 for each comparisonJ. Figure 1 shows that the platelet aggregate ratio of eacili of the ten subjects was lower after than before passive smoking. The mean values (_ 1 SD) were 0:87 = 0.06 before and 0.78 t-0.07 after peaive smoking, with a mean differ- ence of 0.09 and a 96% conIIdenee interval of 0.08 to 0.15. Figure 2 shows that the endothelial'cell count was always 11111111101 MIIaR t=kif 4100AMb!s*asntItir Cbilol +..,r.~ 14e+ ('a t t0) Yrelaele Mbn Aflr Prfalat appapats rfoo ON (=0.06) ON ( t0.0a) EnetansYil or ourt 2.2 CsoJ) 23 Plaarrr rroolkM ooviaMorf, 0 nw"%L 0 aoo0Gob=yfWq91p0YlAAl.'R 1.1(=o.e) 13 (=0.7) Fq t-Plate(et aqpnsqats ratios before antl afroer smokinQ- Itigber after thaa before passive smoking. Mean values ( z 1 SD) before and after were 2.8 ± 0.9 and 3.7 _ 1.1 per counting chamber, respectively, with a mean difference of 0.9 per cbamber and a 96% confidence interval of 0 to 1.8 per chamber. Figure 3 shows that nicotine was not detectable in the plaama of any subject before passive smoking and was present in the plasma of all but one subject after completion of the 20-minute period of passive smolrin`. The mean conrentration (=1 SD) after passive smoldn8 was 2.8= 1.2nV'mL. Figure 4 shows that the aarborybemoglobin level was higher after passive smoking in all but one subjeet, whore value was uncban*. Yean values ( s 1 SD) were 0.9'b ± 0.396 befott and 1.8%s 0:6% after passive smoking, with a mean difference of 0.4% and a 95% confidence interval of 0% to 0.8%. 11 After passive smoldnt, the percent carbozybemo82obia level did not correlate significantly (P> .60) with the platelet aggregate ratio or the endotLelial cell count. The correlation coeffioent between the chanae in the carboxyr- bemoglobin level from before to after passive smoking and the corr+eapondine eban8e in the endotDelial cell count was .78 (P<.01), while the change in the carbozyhemoglobia level was not aigstiscantly (P>.30) correlated with that of the platelet aar,e8ate ratio. Neither the plaams nicotine eonceatration after pe,saive smolriae nor its change from Arzh tham Med-wt 140, Fwnury 19dp Pesdve Smo"-Dev(s et sl 38T

7- J E 6- ~ c C7 U I S- 4- 3- 2- Paas" Shroluny Beforn After P - .002 V Fip 2.--EndothNial oell oounb be/oee and after passive smokinp. P.aerye 5rno+tr+p 8ebn ARter, 4- P- .004 be,Core to after pasai.e smoking was significantly (P>.20) correlated with the corresponding salues of the platelet aggnegste ratio or the endotbelial cell eoont. COMNElff Hladovec and Rossmann" described a simple method for the quantitation of anuclear careaases of endotbelial celis in blood. T'be identity of endothelial cells was based on Fig 4.--CarboYyfWmoqlobw lev.h Deion and attK passive anqk- ~ their morphologic similarity to cells that were scraped from the endotheliurn, with the exception that the celL isolated from blood had' no nuclei. Takahashi and Harker- added cultured human endothelial cells to blood and recov- ered: them from mononuclear cell fractions in which they were identified by the presence of factor VILI-related~ antigen as a cell marker. They detected no end'othelW cellr in mononuclear cell fractions of venous blood from ten normal subjecta. Using the method of Hladovec and Fiosa-mann," wC"' found endothelial cetls in the venous blood of normal men and women and men with coronary artery disease. Since, like Hladovec and Rosamann," we saw no nuclei in these cells, there is no conflict with the report of 'hkaFiaahi and Ha,rker- of the absence of endothelial cellY from mononuclear cell fractions. In a previous study,' we identified the anuclear cells as endothelial by their fluorei- cence after incubation with fluorescein-labeled anti-human factor VIII-related antigen antibody and by the lack of fluorescence of therpithelium in simultaneously incubated sections of human skin. The present studR showing that brief passive exposure to tobacco smoke under naturally occurring environmental conditions has consistent acute effects on the endothelium and plateleta, was limited to a group of ten healthy male nonsmokers. It seems likely thst this small group may be representative of the general population since we observed similar effects of active smoking on healthy male and female naive smokess,' healtby male" and female (.IW.D. and L.S., unpublished data, 1962) habitual smokers, and male hab9tual~ smokers with coronary artery disease.'.+' Other workers nsve shown that passive smoking by non- smokers lowers platelet sensitivity to the antiaggregstory effect of prost'acyelin.° Although a statirtically significant correlation between the change in the endothelial cell count and the change in the carboxybemoslobm level from before to after p.aai.e smoking waa found, there was not a sirnificant correlation between these variablee after passive smoking . We pre.i- ously found that smoking tobacco cigarettes had a much greater effect on both platelets and the endotbelinm than did smoking cigarettes that contained no nicotine.' The relative importanca of carbon monoxide, nibotine, and the many other components of tobaoco smoke as eauses of the 3M Ardf In1Kn k1ed-Vd 149, FeOruary 1989 Patsire SmokknQ--Devfa el al

observed effects on platelets and the endothelium remains unclear. The significance of enhanced platelet aggregate forma- tion and an increased'concentration of anuclear carcasaes of end'othelial' cells in blood' after passive smoking is not knoaPn. However, both platelet activationf and endothelial damage+ are prominent among the mechanisms thought to be involved in atherosclerosis and arterial thrombosis. Epidemiologic studies are needed to determine whether repeatad' episodes of passive exposure to tobacco smoke during a period of years enhance the development of atherosclerosis and its complications in nonsmokers. A large .iapanese studh-' indicated that nonsmoking wives of heavy smokers had a higher risk of developing lung cancer, while the husbands" smoking habit did: not affect their wives' risk of dying of ischemic heart disease. Stock' suggested that Japanese people may be protected from fatal coronary heart disease by a high dietary intake of eicosapentaenoic acid. We hope that our work will be an impetus to the development of epidemiologic investigations of a possible relationship ~ between passive smoking and vascular dieeases in Western countriea.. T3ii.ork se suppoKed by s 8rantbom tbe Ameriaan Heart Aseooation, 1Canese Aldliate, lbpeka, and by tke Vstentu Admtnistntion. IMhf'snOM I. Strcna JP, Richards YL: Ciauette amolcna aod stheioeeYaoris in wtopued men: AlAnwcleoai. 197a:23:451176. 2. Arono. W S: Eaea ot eiauette smoking and of' earEon monoade oo eotonary beart disease. Chest 1Y76:70a14-618. 9. Doll iR'. Peto R: Yortality in relation to amokitr. 20 yean' ab..rv.oon. oa malk Britiab doctors. B. Yd J Cliw Rea 1976;t 1S2S-1836. 4:, Sannd WB: Update on the toke of eaa.rette amo" in eoxvnary artery disease. Am fHlart J 1981:101:319-328. 5. Roe. R: The patborenesu of atEeo.eieroaib--an update. N 6wpl J l/ed 198e:914:4B8-b00. 6: Fluter V. Cbesebro,JH: Aatitbrombotic therapy: Role of plat.eiet- inhibitor drup: 1. Current eoneepe& of tLromlioaenesie: Role of plateleti. dfaNo Chw /°roe 1981c56:10p-112. 71 Plero.eky 1. Hladarse J: Suppression of tbe desqnamatittg eCeet of, smokitg onn the humaa endathelium by bydtosyethylrut4Mdea. Blood KaN4 1979:16:Y3s-240. 8: Davis JW, Sbelton L. Eiaenber* DA. st ak EQeets of tob.ceo and non- tobseeo ci8arette smo" on endotbebum aad'plYteiet.. C1iw PAarwacol Trr 198617t529-633. 9. De.is JW,' Shelton L. Eipnbera DA, et aL• Lack of eQeet of i.pirin on eianrette emoke-induced increase in atevlatinr endotEeLlal eells. Ba.+so- rlnsu 1B87i17:66-69. 10: Dnia JW, Shelton L. Hutman CR, et el: Smolbn8-iaducYd ehsnaes in endotbeiium and'platekru ue not asect.d by hydivsy.thyirntosid.s. Br J Esp PEtAb! 1996:67:766771. 11. Reoaud S. Blacbe D, Dumont E, et al' Platelet fLLOction after ciprette amokin( ia relation to niootine and'carban matw>ode. Cliw PAar.weol 2ae. 1984:36:38F396. 22 Davis JW, H••t•^-- CR. Lewis HD Jr, et aI: Cigarette smohng- iodueed enhancement of piataot timetion: Lack af prr.ention by aspirin in men with coronary artery disease. J LaE Clin 8fed'1986:106:479.483. 13. Ho.a UR: Cigarette smoke in elo..d sp.o... L'RVirvw B.aIU Arsp.er19722117-128. 14: , W nX$, Hosk JC: A ne+ metbod, for the quantitative detection of plateet aarepw in patientL with arterial iasuffidoqt lawc.t 1974:2.9Y4- 9E6. 1S. Rohr.r TF, PtSstQ B, Weber C. at aL V.liditT of'the Wu-Hoak method for the q•••n*»•••.e det.rminaLimn of plwlet aggre8aaon in .i.o. Blrr 1978:J6:IS~20. 16. Kohaaaa FH. Smith 1[H. S.1®ao EW: Do patienta.itb thromboem- botie di.ea.e bave circulating plas.letaar.gstr' B/aod 1984:64:206•200: 17. , Hl.dowe I Reesmann P: CiixuYatina endotbeli.l eelL isolated to- tetAer with plateiets and the experimental modifleation,of their counu ib, ran. TA.orwO /P.. 197aa:666-674: 18. Ftiy:rabend C. Rua.eU 1[AEi: Impra..d gsKbromataBrapbie method and mieroe:tr.etion technique for the measurement of nicvtine in biological fluids. J PAarm Pko++.oco1 1979:31:73-76. 19. Gardner !Lt Altmaa DG: ConSd.aee internis rather than P.ala.: Estimation r.thtr than hypotbsis tstialp Br ded J Cli. R.a lYBe1p2:748• 750. 20. Takahrbl H. Harker LA_ 1(eaamement of bnmsn endosEeli.l celL in wboie blood. 7T<+oab R.r 1iBa:91:1-1i 21. Bullrbuber OC. Pttnsensruber C, 9inanaer H', at alf Platelet sensi- tivity to prostaeyefin in smokm and oon amokera Cbat 1986:90:3f-38. Z2 Hireyams T: Noo-amo/ona wives of heavy amoken have a tii8har risk of lung caater. A study 6om Japan. Br N.d J C1iw B.r 1Y81382:18318b. 23. Stock SL: Passive smolong aod nicotine. Laweet 1981t:1044: PdrrMS Smo+tin¢-aaviY atal aM Arch trRrrn Mod- Vbi 140, Fobruaer 190

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