nture publishing group Popultion Study Self-reported smoking sttus nd plsm cotinine concentrtions mong pregnnt women in the Norwegin Mother nd Child Cohort Study Liv G. Kvlvik 1, Roy M. Nilsen 2, Rolv Skjærven 1, Stein Emil Vollset 1, Øivind Midttun 3, Per Mgne Uelnd 4, Kjell Hug 1 Introduction: Underreporting of smoking in epidemiologic studies is common nd my constitute vlidity problem, leding to bised ssocition mesures. In this prospective study, we vlidted self-reported tobcco use ginst nicotine exposure ssessed by plsm cotinine in the Norwegin Mother nd Child Cohort Study (MoB). Methods: The study ws bsed on subsmple of 2,997 women in the MoB study who delivered infnts during the period 2002 2003. Self-reported tobcco use (test vrible) nd plsm cotinine concentrtions (gold stndrd) were ssessed t pproximtely gesttionl week 18. Results: Dily smoking ws reported by 9% of the women, occsionl smoking by 4%, nd nonsmoking by 86% of the women. Sensitivity nd specificity for self-reported smoking sttus were clculted using cotinine cut-off estimted from the study popultion (30 nmol/l). Plsm cotinine concentrtions 30 nmol/l were found in 94% of self-reported dily smokers, 66% of occsionl smokers, nd 2% of nonsmokers. After the numbers of self-reported nonsmokers with cotinine concentrtions bove the cut-off limit were dded, the dily smoking prevlence incresed from 9 to 11%. The sensitivity nd specificity for self-reported dily smoking, using 30 nmol/l s the cut-off concentrtion, were 82 nd 99%, respectively. Discussion: These findings suggest tht self-reported tobcco use is vlid mrker for tobcco exposure in the MoB cohort. Although the prevlence of mternl smoking during pregnncy hs declined during the pst 20 y (1 3), smoking remins strong environmentl risk fctor for dverse pregnncy outcomes nd complictions. In 2008, 16% of pregnnt Norwegin women reported smoking dily t the beginning of pregnncy, nd 8% reported smoking t the end of pregnncy (3). Therefore mny pregnnt women continue to be exposed to tobcco smoke. This underscores the need for further epidemiologic reserch nd public helth strtegies to prevent smoking during pregnncy. In ddition, there is concern bout whether women report their true smoking sttus in epidemiologic studies. By compring self-reported smoking sttus with cotinine mesurements, study on pregnnt women in the west of Scotlnd found 25% underestimtion of true smokers from selfreported smoking hbits (4). In Swedish study, 6% of selfreported nonsmokers were probbly smokers, nd 3% hd cotinine concentrtions suggestive of pssive smoking (5). Such underreporting my constitute serious vlidity problem, leding to bised ssocition mesures (6). In ddition, missing dt on self-reported smoking seem to be common problem. A recent study from Norwy found tht 12% of pregnnt women hd not reported smoking hbits to the popultion-bsed Medicl Birth Registry of Norwy (2). In Sweden, dt on smoking hbits during pregnncy were missing for 9% of the women, nd in Denmrk 4% of bbies hd mothers whose smoking hbits hd not been registered (7,8). Cotinine is the primry metbolite of nicotine nd is sensitive mrker of tobcco smoking s well s use of snuff nd nicotine replcements. It is commonly used s biomrker for environmentl tobcco smoke exposure (9,10). In this study, we vlidted self-reported tobcco use in the subjects in the Norwegin Mother nd Child Cohort Study (MoB) ginst mternl plsm cotinine. For this purpose, we lso evluted plsm cotinine cut-off to seprte ctive smokers from pssive smokers nd nonsmokers. Results Popultion Chrcteristics Of the 2,997 women who were the subjects of this study, the men mternl ge t delivery ws 29.8 y (SD, 4.6; rnge, 15 43), 44% of the subjects were pregnnt for the first time, nd 96% of the subjects were mrried or cohbiting (Tble 1). A totl of 63% of the subjects hd prepregnncy BMI between 18.5 nd 24.9 kg/ m 2, nd 42% of the subjects hd eduction of 12 y. Self-Reported Nicotine Exposure Of the 2,997 subjects, 263 (8.8%) reported dily smoking nd 126 (4.2%) reported occsionl smoking during pregnncy (Tble 1). A totl of 1,491 subjects (50%) reported ever smoking 1 Deprtment of Public Helth nd Primry Helth Cre, University of Bergen, Bergen, Norwy; 2 Centre for Clinicl Reserch, Hukelnd University Hospitl, Bergen, Norwy; 3 Bevitl AS, Bergen, Norwy; 4 Section for Phrmcology, Institute of Medicine, University of Bergen, Bergen, Norwy. Correspondence: Liv G. Kvlvik (Liv.Kvlvik@isf.uib.no) Received 20 June 2011; ccepted 07 Februry 2012; dvnce online publiction 4 April 2012. doi:10.1038/pr.2012.36 Volume 72 Number 1 July 2012 Peditric Reserch 101
Kvlvik et l. Tble 1. Mternl tobcco exposure during pregnncy ccording to mternl chrcteristics mong 2,997 women in the Norwegin Mother nd Child Cohort Study, 2002 2003 Chrcteristic Totl Nonsmoking (n = 2,586) Occsionl smoking (n = 126) Dily smoking (n = 263) Missing (n = 22) Pssive smoking (n = 472) Smokeless nicotine products (n = 27) n (%) % % % % % % All women 2,997 (100) 86 4.2 8.8 0.7 16 0.9 Mternl ge (y) <25 379 (13) 76 8.7 14 0.8 33 25 34 2,152 (72) 89 3.4 7.3 0.7 14 1.1 35 456 (15) 84 4.2 11 1.1 12 0.9 Prity 0 1,303 (44) 87 4.5 8.0 0.6 20 1.2 1 1,116 (37) 87 3.8 8.6 0.7 12 0.6 2 435 (15) 84 5.1 10 1.1 12 0.9 3 133 (4) 84 2.3 13 0.8 16 0.8 Mritl sttus Single 80 (3) 61 13 24 2.5 43 2.5 Cohbitting 1,342 (45) 83 5.1 12 0.6 20 1.3 Mrried 1,539 (51) 91 2.9 5.3 0.8 11 0.5 Prepregnncy BMI (kg/m 2 ) <18.5 81 (3) 75 8.6 16 17 2.5 18.5 24.9 1,876 (63) 88 3.9 7.9 0.6 14 0.8 25 29.9 609 (20) 87 4.6 8.0 0.7 17 0.8 30.0 315 (11) 85 3.5 11 0.6 21 0.6 Mternl eduction (y) 12 1,254 (42) 77 6.1 16 1.0 26 0.9 13 16 1,166 (39) 92 3.3 3.9 0.6 9.4 1.1 17 500 (17) 95 1.8 3.2 0.2 4.8 0.6 Informtion on pssive smoking, mternl ge, prity, mritl sttus, prepregnncy BMI, nd mternl eduction ws missing for 20, 10, 10, 36, 116, nd 77 women, respectively. nd 698 (23%) reported dily smoking during the 3 mo before becoming pregnnt. Dily smoking during pregnncy ws more common mong the youngest subjects, mong those with higher prity, nd mong those who were single. Also, subjects with low prepregnncy BMI nd low eduction smoked more thn others. Similr profiles were observed for occsionl smokers. Further, pssive smoking ws reported by 472 subjects (16%). In the entire smple, 216 subjects (7.2%) reported pssive smoking t work, 194 (6.5%) reported pssive smoking t home, nd 62 (2.1%) reported pssive smoking both t home nd work. Of the 472 subjects reporting pssive smoking, 111 reported being dily smokers, 38 reported occsionl smoking, 321 reported being nonsmokers, nd dt on smoking sttus were missing in 2 subjects (i.e., 3.7, 1.6, 11, nd 0.1% of the totl popultion, respectively). Overll, 27 subjects used smokeless nicotine products during pregnncy. Of these, 15 used chewing tobcco or snuff, 9 used nicotine chewing gum, 1 used nicotine dhesive ptch, nd 2 used nicotine inhler. Cotinine Concentrtions nd Self-Reported Smoking Sttus Plsm cotinine concentrtions incresed significntly with incresing cigrette consumption in both dily smokers nd occsionl smokers (Tble 2). Overll, plsm cotinine ws correlted (Spermn) with both number of cigrettes per dy (r = 0.51; 95% confidence intervl (CI): 0.42, 0.60) nd number of cigrettes per wk (r = 0.48: 95% CI: 0.32, 0.61). Among those reporting nonsmoking but pssive smoking, cotinine concentrtions were low (geometric men, 1.9 nmol/l). Subjects using smokeless nicotine products hd plsm cotinine concentrtions of ~100 nmol/l. Cotinine Cut-Off nd Self-Reported Smoking Plsm cotinine concentrtions of >0 nmol/l were found in totl of 963 (32%) subjects. A density plot of these cotinine concentrtions showed two distinct distributions (Figure 1). Using kernel density estimtion nd bootstrp method, we estimted tht the lowest point between the two distributions of log plsm cotinine concentrtions corresponded to geometric men of 29.8 nmol/l (95% CI: 20.0, 56.0) (Figure 1,b nd Tble 3). To vlidte reported dily smoking, we excluded from the nlyses occsionl smokers nd users of smokeless nicotine products (n = 148) s well s those for whom dt on smoking hbits were missing (n = 22). The corresponding overll men sensitivity nd specificity for self-reported dily 102 Peditric Reserch Volume 72 Number 1 July 2012
Cotinine nd self-reported smoking Tble 2. Plsm cotinine concentrtions ccording to tobcco exposures during pregnncy mong 2,997 women in the Norwegin Mother nd Child Cohort Study, 2002 2003 Plsm cotinine (nmol/l) Tobcco exposure Totl number of subjects Geometric men 95% Confidence intervl Dily smokers, cigrettes per d All b 263 346 295 406 1 4 69 135 92.1 199 5 9 103 430 344 538 10 14 64 520 413 656 15 22 645 505 824 Occsionl smokers, cigrettes per wk All c 126 49.7 32.7 75.8 1 4 44 16.7 8.2 33.8 5 9 27 78.4 40.5 152 10 14 24 99.2 39.8 247 15 19 244 116 511 Pssive smokers All 472 8.3 6.5 10.5 At work 216 3.1 2.4 4.1 At home 194 18.1 12.1 26.9 Both t home nd t work 62 23.3 11.3 48.2 Dily smoking only 152 329 263 410 Both dily nd pssive 111 371 294 468 smoking Occsionl smoking only 88 52.6 31.3 88.3 Both occsionl nd pssive 38 43.7 20.7 92.3 smoking Pssive smoking only 321 1.8 1.6 2.1 Smokeless nicotine products 27 96.9 35.8 262 Geometric men; i.e., ntilog of men of logrithmic vlues. b Includes five women who reported dily smoking but provided no informtion on the number of cigrettes smoked per dy. c Includes 12 women who reported occsionl smoking but provided no informtion on cigrette dose. smoking were estimted to be 81.9% (95% CI: 77.3, 86.4) nd 99.4% (95% CI 99.1, 99.7), respectively. The uncertinty in terms of percentile vlues ppered lrger in estimtes of sensitivity thn in those of specificity (Figure 1c,d nd Tble 3). The plsm cotinine cut-off vlue (30 nmol/l) ws lso used to vlidte self-reported dily smoking ccording to bckground vribles, such s mternl ge, prity, mritl sttus, prepregnncy BMI, nd mternl eduction (Tble 4). A totl of 296 subjects hd cotinine concentrtions 30 nmol/l. Of these, 242 (82%) reported dily smoking, nd 54 (18%) reported nonsmoking. Among the 54 subjects who reported nonsmoking but hd plsm cotinine levels 30 nmol/l, 45 reported ever smoking, 30 reported dily smoking during the lst 3 mo before pregnncy, nd 13 reported pssive smoking. Adding the number of self-reported nonsmokers with cotinine concentrtions bove the cut-off level to the number of self-reported dily smokers resulted in n increse in dily smoking prevlence from 8.8% (263/2,997) to 11% (317/2,997). Cotinine nd Other Nicotine Exposures Among the 121 occsionl smokers (excluding subjects using smokeless nicotine products), 80 (66%) hd cotinine concentrtions 30 nmol/l. The sensitivity for combined self-reported occsionl nd dily smoking ws 86% (95% CI: 82, 89) nd the specificity ws 98% (95% CI: 97, 98). Among the 27 subjects who used smokeless nicotine products during pregnncy, 16 reported nonsmoking, 5 reported occsionl smoking, nd 6 reported dily smoking. A totl of 21 (78%) hd cotinine concentrtions 30 nmol/l. Dt on self-reported smoking hbits during pregnncy were missing for only 22 (0.7%) subjects; in these subjects, the cotinine concentrtions were <1 (n = 13), <5 (n = 6), 76.1 (n = 1), 475 (n = 1), nd 597 (n = 1) nmol/l. Discussion Principl Findings This prospective study vlidted self-reported smoking sttus ginst mesured plsm cotinine concentrtions in 2,997 pregnnt women in the MoB study. Our clcultions suggested tht plsm cotinine concentrtion of 30 nmol/l (5.3 ng/ml) would be the optiml cut-off vlue to seprte ctive smokers from pssive smokers nd nonsmokers. By lso using this cut-off vlue in the clcultion of sensitivity nd specificity, we found tht self-reported smoking sttus hd sensitivity of 82% nd specificity of 99%. Strengths nd Weknesses The strengths of the study were (i) it comprised subsmple of 2,997 pregnnt women in the MoB study, llowing for precise estimtes overll s well s in subgroups; (ii) we hd detiled self-reported dt on dily nd occsionl smoking nd on pssive smoking; (iii) dt on self-reported smoking were missing for only 0.7% of the subjects; nd (iv) we lso hd the opportunity to exmine smokeless nicotine exposure, such s snuff, nicotine chewing gum, nicotine dhesive ptch, nd nicotine inhler. Our study lso hd some limittions. One limittion is the time difference between returning the completed bseline questionnire nd the blood smpling, with respect to some of the subjects. Approximtely 85% of the subjects returned the questionnires within 4 wk from the time of blood smpling, wheres for the other 15% this intervl ws longer. Becuse smoking behvior my chnge round the time of the ultrsound screening, such differences in time intervls could hve led to misclssifiction of smokers nd nonsmokers. Furthermore, subjects for whom the time spn between smoking nd blood smpling ws lrge could hve tested s nonsmokers bsed on cotinine nlysis. Such misclssifiction would be more common mong occsionl smokers with vrible time spn since the lst cigrette. The subjects were informed tht their blood smples nd those of their children would be used for reserch purposes, Volume 72 Number 1 July 2012 Peditric Reserch 103
Kvlvik et l. 0.4 b 2,000 Frequency Density 0.3 0.2 0.1 0.0 c 1,500 1,000 500 0.01 0.1 1 10 100 1,000 Plsm cotinine (nmol/l) 1,500 1,000 500 0 d 1,500 1,000 500 Frequency Frequency 10 20 30 40 50 60 70 80 Cotinine cut-off (nmol/l) 0 65 70 75 80 85 Sensitivity 0 90 95 100 98.5 99.0 99.5 100.0 Specificity Figure 1. Distributions of log plsm cotinine, plsm cotinine cut-offs, nd sensitivity nd specificity for self-reported dily smokers, bsed on 10,000 resmples from the Norwegin Mother nd Child Cohort Study, 2002 2003. () A density plot of log plsm cotinine concentrtions obtined using kernel density estimtion. (b) The distribution of the cut-offs from the 10,000 resmplings. (c,d) The distributions of the sensitivities nd specificities estimted for ech cut-off from the 10,000 resmplings. Tble 3. Summry sttistics of cut-off between ctive smokers nd nonsmokers/pssive smokers mong 2,997 women in the Norwegin Mother nd Child Cohort Study, 2002 2003 Percentile Men 2.5th 25th 50th 75th 97.5th Cut-off (cotinine units: nmol/l) 29.8 20.0 26.5 29.6 32.2 56.0 Sensitivity (%) b 81.9 77.3 80.4 82.0 83.5 86.4 Specificity (%) b 99.4 99.1 99.3 99.4 99.6 99.7 Estimtes found by 10,000 resmplings of the observed dt (nonprmetric bootstrp method). Antilog of logrithmic vlues. b Sensitivity nd specificity for self-reported dily smoking: informtion on smoking hbits during pregnncy ws missing for 22 women nd ws excluded for 27 women using other kinds of nicotine products. but they were not told tht the blood smples would be tested for nicotine exposure. The ttending nurses were lso unwre of the purpose of the blood smples. It is therefore unlikely tht knowledge of specific blood test(s) could hve cused chnges in smoking behvior before blood smpling or ffected selfreported smoking sttus. Cotinine Cut-Off Levels in Pregnnt Women In our study popultion, we observed bimodl distribution of log plsm cotinine concentrtions (Figure 1), suggesting seprtion of ctive smokers from pssive smokers nd nonsmokers (11,12). By identifying the lowest point between the two distributions, we suggest cut-off vlue of 30 nmol/l (5.3 ng/ml) (95% CI: 20, 56) for plsm cotinine. Erlier studies in nonpregnnt subjects hve indicted serum cotinine levels of 80 85 nmol/l s cut-off for identifying ctive smokers (12 14). Studies on pregnnt women hve used cutoff between 57 nd 99 nmol/l (10 18 ng/ml) (4 5,15 19). On the other hnd, study bsed on representtive smple of the US popultion recommends 17 nmol/l (3 ng/ml) s the overll cut-off (20). All these results re outside our estimted confidence limits. Therefore generl cotinine cut-off in pregnnt smokers hs yet to be estblished. Selection Bis A recent study compred women prticipting in the MoB study to ll women giving birth in Norwy, using dt from the Medicl Birth Registry of Norwy for the period 2000 2006 (21). The cigrette consumption t the end of pregnncy ws significntly lower in the MoB subjects thn in the totl popultion (6.9 vs. 7.4 cigrettes). There were lso fewer subjects for whom the smoking hbits were not known in the MoB study thn in the totl popultion. Becuse the MoB study group is selected smple, smokers in the study my lso hve lower nicotine intkes thn the generl popultion. This my hve resulted in lower plsm cotinine cut-off in our study thn those mentioned in other pregnncy-relted studies. Vlidtion nd Underreporting of Smoking Sttus Precise nd vlid monitoring of smoking sttus in pregnncy hs significnt public helth implictions nd is essentil in epidemiologic reserch. A recent study showed higher nondisclosure rtes of tobcco smoking mong pregnnt women thn mong nonpregnnt women (22). The stigm ssocited with smoking during pregnncy might ffect the vercity of self-reporting nd thereby reduce the overll 104 Peditric Reserch Volume 72 Number 1 July 2012
Cotinine nd self-reported smoking Tble 4. Sensitivity nd specificity for self-reported dily smoking ccording to the geometric men plsm cotinine cut-off t 30 nmol/l mong 2,997 women in the Norwegin Mother nd Child Cohort Study, 2002 2003 Chrcteristic Nonsmokers Dily smokers <30 nmol/l 30 nmol/l <30 nmol/l 30 nmol/l Sensitivity Specificity n n (%) n n (%) % 95% CI % 95% CI All women 2,516 54 (2.1) 15 242 (94) 82 77 86 99.4 99.0 99.6 Mternl ge (y) <25 283 6 (2.1) 5 49 (91) 89 78 95 98 96 99 25 34 1,856 37 (2.0) 9 144 (94) 80 73 85 99.5 99.1 99.7 35 369 11 (2.9) 1 47 (98) 81 69 89 99.7 98.5 100 Prity 0 1,100 22 (2.0) 9 93 (91) 81 73 87 99.2 98.5 99.6 1 940 25 (2.6) 5 90 (95) 78 70 85 99.5 98.8 99.8 2 362 2 (0.5) 1 40 (98) 95 84 98.7 99.7 98.5 100 3 106 5 (4.5) 0 17 (100) 77 57 90 100 97 100 Mritl sttus Single 47 2 (4.1) 2 15 (88) 88 66 97 96 86 99 Cohbitting 1,064 33 (3.0) 12 144 (92) 81 75 86 98.9 98.1 99.4 Mrried 1,378 19 (1.4) 1 77 (99) 80 71 87 99.9 99.6 100 Prepregnncy BMI (kg/m 2 ) <18.5 57 2 (3.4) 0 13 (100) 87 62 96 100 94 100 18.5 24.9 1,600 33 (2.0) 9 137 (94) 81 74 86 99.4 98.9 99.7 25 29.9 514 11 (2.1) 4 43 (92) 80 67 88 99.2 98.0 99.7 30.0 263 5 (1.9) 0 33 (100) 87 73 94 100 98.6 100 Mternl eduction (y) 12 932 31 (3.2) 11 181 (94) 85 80 90 98.8 98 99 13 16 1,050 17 (1.6) 2 43 (96) 72 59 82 99.8 99.3 99.9 17 466 6 (1.3) 2 13 (87) 68 46 85 99.6 98.5 99.9 Informtion on smoking hbits during pregnncy ws missing for 22 women nd ws excluded for 27 women using other kinds of nicotine products. CI, confidence intervl. CIs of sensitivity nd specificity were clculted by the Wilson procedure without correction for continuity. relibility of self-reported smoking in helth studies. In our study, the lowest sensitivity for self-reported smoking hbits ws found in subjects with the highest level of eduction. There is uncertinty bout the dded vlue of mesuring cotinine levels to ssess smoking sttus (15). We found tht ~94% of dily smokers who did not use smokeless nicotine products hd cotinine concentrtions 30 nmol/l. Also, ~98% of nonsmokers who did not use smokeless nicotine products hd cotinine concentrtions below the cut-off vlue. A study mesuring cotinine in cord serum found cotinine concentrtions bove the cut-off level (80 nmol/l (14 ng/ml)) in 88% of the subjects who were self-reported dily smokers, nd cotinine concentrtions below the cut-off level in 96% of the selfreported nonsmokers (19). However, other studies hve found tht 23 26% of women who were smokers ccording to their cotinine concentrtions vlues did not report themselves s smokers (4,6,22). A study showed n overestimtion of the odds rtio for the occurrence of smll-for-gesttionl-ge deliveries s well s for smoking-relted reduction in birth weights, when compring results before nd fter reclssifiction of smokers bsed on cotinine mesurement t pproximtely gesttionl week 28. However, it did not lter the directions of the ssocitions between the prmeters nd the outcomes (6). Other studies in pregnnt women tht compred self-reported smoking sttus with cotinine vlues hve found self-report to be poor indictor of smoking sttus, with sensitivity of 47.4 nd 89.5%, respectively, nd specificity of 94.9 nd 65.3%, respectively (23,24). Occsionl Smokers A Norwegin study tht estimted umbilicl cord serum cotinine t delivery s mesure of fetl exposure to tobcco products indicted considerble interindividul vrition in fetl nicotine exposure mong newborns of occsionl smoking mothers, with 46% hving cotinine vlues bove the chosen cut-off (19). In our study, 66% of the occsionl smokers hd cotinine concentrtions bove the cut-off. This might be explined by vritions in the time elpsed since the lst cigrette in occsionl smokers. Volume 72 Number 1 July 2012 Peditric Reserch 105
Kvlvik et l. Conclusions In conclusion, in substudy of lrge cohort study, we showed tht self-reported smoking sttus in pregnncy hs sensitivity of 82% nd specificity of 99% t plsm cotinine cut-off level of 30 nmol/l. Our results indicte tht self-reported smoking is vlid mrker for tobcco exposure in the MoB cohort. Methods Setting MoB is prospective popultion-bsed pregnncy study estblished by the Norwegin Institute of Public Helth (21,25,26). Prticipnts were recruited during the period 1999 2008, nd 38.5% of the women who were invited consented to prticipte (27). The cohort includes 108,639 children, 90,725 mothers, nd 71,574 fthers. Blood smples were obtined from both prents during pregnncy nd from mothers nd children (umbilicl cord) t birth. Follow-up is conducted through questionnires t regulr intervls nd by linkge to ntionl helth registries including the Medicl Birth Registry of Norwy. This study is bsed on version 3 of the qulity-ssured dt files relesed for reserch in April 2007. Study Popultion For the purpose of this study, we used subsmple of 3,000 mothers with bbies born during the period July 2002 December 2003. These women were selected rndomly from mong those who hd donted blood smples t the ultrsound screening nd who where registered in the Medicl Birth Registry of Norwy. In ddition, they hd returned completed bseline questionnire nd food frequency questionnire during the second trimester (28). We excluded three women for whom there were no plsm cotinine dt; dt from 2,997 women were vilble for nlysis. Informed consent ws obtined from ech subject, nd the study ws pproved by the regionl committee for medicl reserch ethics. Blood Smpling nd Cotinine Anlysis The blood smples (nonfsting) were collected from the subjects t weeks 17 18 into EDTA tubes, which were centrifuged within 30 min fter collection, nd plced in the refrigertors in the hospitls (4 C). Smples were shipped by mil overnight to the biobnk of MoB. On the dy of receipt, usully 1 2 d fter the blood smple ws drwn, EDTA plsm ws liquoted onto polypropylene microtiter pltes (300 μl per well, 96-well formts), seled with het-seling foil sheets, nd stored t 80 C. Plsm cotinine concentrtions were nlyzed t Bevitl AS (http:// www.bevitl.no) using liquid chromtogrphy tndem mss spectrometry method published erlier (29). The limit of detection of the method ws 1 nmol/l (0.18 ng/ml). For subjects with plsm cotinine vlues below limit of detection (n = 111), the vlues were imputed by ssigning rndom plsm cotinine vlue between 0 nd 1 nmol/l to ech of the subjects. The coefficients of vrition were 2.3 2.9% (within-dy) nd 5.5 6.2% (between-dy). Cotinine hs hlf-life of ~9 h in pregnnt women (30). A serum cotinine cut-off vlue of 17 nmol/l (3 ng/ml) ws previously recommended to distinguish smokers from nonsmokers (20). Nicotine Exposure The informtion on nicotine exposure ws extrcted from the bseline questionnire (http://www.fhi.no/mob). It included informtion bout exposure to pssive smoking t work or t home nd bout whether the subjects hd ever smoked, whether they smoked before pregnncy nd during the second trimester, nd whether they used smokeless nicotine products. Of note, most of the subjects returned the completed bseline questionnire t bout the time of blood smpling. Subjects who did not return the questionnire were sent reminder 3 4 wk fter the ultrsound exmintion. Consequently, self-reported smoking sttus ws vilble t men gesttionl ge of 19.0 wk (SD, 4.0), wheres the men gesttionl ge t blood collection for plsm cotinine mesurement ws 18.2 wk (SD, 2.1). Accordingly, in 15% of the subjects, there ws difference in gesttionl ge of >4 wk between blood smpling nd self-reporting of smoking sttus. Covrites Dt on mternl ge t delivery (<25, 25 34, 35 y), mritl sttus (mrried, cohbiting, single, other/missing), nd prity (0, 1, 2, 3 previous deliveries) were obtined from the Medicl Birth Registry of Norwy, wheres dt on smoking hbits, prepregnncy BMI (<18.5, 18.5 24.9, 25.0 29.9, nd 30 kg/m 2 ), nd mternl eduction were obtined from the MoB bseline questionnire. Eduction ws mesured s highest level of completed eduction nd ctegorized s 12, 13 16 or 17 y. Sttisticl Anlyses Sttisticl nlyses were crried out using SPSS (Sttisticl Pckge for the Socil Sciences) version 15 nd SAS (Sttisticl Anlysis System) version 9.2 (SAS Institute, Cry, NC). R version 2.8.1. (The R Foundtion for Sttisticl Computing, http://www.r-project.org) softwre ws used for grphicl illustrtions. Plsm cotinine concentrtions were log-trnsformed to chieve less skewed distribution of dt nd were reported s geometric mens, i.e., ntilog of mens of the logrithmic vlues (31). Spermn s correltion coefficient with 95% CI ws used to estimte the ssocition between plsm cotinine nd numbers of cigrettes smoked. Active smokers were seprted from pssive smokers nd nonsmokers by estimting the lowest point between two distinct distributions of log plsm cotinine. This ws performed using nonprmetric bootstrp method (the SURVEYSELECT procedure in SAS). Briefly, we resmpled rndomly 10,000 times from the totl popultion, creting 10,000 lterntive dt sets. For ech set, we locted the lowest log plsm cotinine point between two peks using kernel density estimtion. Finlly, from the 10,000 point estimtes, we estimted the geometric men, which ws used s the plsm cotinine cut-off between ctive smokers nd pssive/nonsmokers. The corresponding 95% CI ws constructed by extrcting the 2.5th percentile nd 97.5th percentile of the 10,000 estimtes. Bsed on this bootstrp procedure, we simultneously estimted the overll sensitivity nd specificity for self-reported dily smoking. The uncertinty ws ddressed by extrcting the 2.5th, 25th, 50th, 75th, nd 97.5th percentiles. Using the cotinine cut-off s determined by the bootstrp method, we lso estimted sensitivity nd specificity of self-reported dily smoking ccording to bckground vribles. The estimted plsm cotinine cut-off ws considered to be the gold stndrd, nd selfreported informtion ws considered the test in sensitivity nd specificity clcultions. Sensitivity is the percentge of subjects with plsm cotinine concentrtions bove the cut-off tht re correctly identified s dily smokers by self-report. Specificity is the percentge of subjects with plsm cotinine concentrtions below the cut-off tht re correctly identified s nonsmokers by self-report. The 95% CIs of sensitivity nd specificity for self-reported dily smoking ccording to bckground vribles were clculted using the Wilson procedure without correction for continuity (32). ACKNOWLEDGMENTS Those conducting the Norwegin Mother nd Child Cohort study re grteful to ll the prticipting fmilies in Norwy who re tking prt in this ongoing cohort study. STATEMENT OF FINANCIAL SUPPORT The Norwegin Mother nd Child Cohort Study is supported by the Norwegin Ministry of Helth nd the Ministry of Eduction nd Reserch, Ntionl Institutes of Helth/Ntionl Institute of Environmentl Helth Sciences (contrct no. NO-ES-75558), Ntionl Institutes of Helth/ Ntionl Institute of Neurologicl Disorders nd Stroke (grnt nos. 1 UO1 NS 047537-01 nd 2 UO1 NS 047537-06A1), nd the Norwegin Reserch Council/ Functionl Genomics (grnt no. 151918/S10). This work ws supported by the Foundtion to Promote Reserch Into Functionl Vitmin B12 Deficiency. Disclosure: The uthors declre no conflict of interest. 106 Peditric Reserch Volume 72 Number 1 July 2012
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