Effects of growth and aging on the reference values of pulmonary nitric oxide dynamics in healthy subjects

J. B ea h Res. 11 (2017)047103 h ps://doi.o g/10.1088/1752-7163/aa7957 PAPER E ec s o g ow h and aging on he e e ence alues o pulmona y ni ic oxide dynamics in heal hy subjec s M Högman 1 , A Tho nad sson 1,2 , P Li 2 , T Hua-Huy 3 , A T Dinh-Xuan 3 , E Tu esson 4 , H D essel 5 , C Janson 1 , K Koskela 6 , P Oksa 6 , R Sauni 6 , J Ui i 6 , E Moilanen 7 and L Leh imäki 8 1 Dep . o Medical Sciences, Respi a o y, Alle gy and Sleep Resea ch, Uppsala Uni e si y, Uppsala, Sweden 2 Cen e o Resea ch and De elopmen , Uppsala Uni e si y/Region Gä lebo g, Sweden 3 Dep . o Respi a o y Physiology, Medical School, Pa is Desca es Uni e si y, Pa is, F ance 4 Dep . o Clinical Sciences Lund, Respi a o y Medicine and Alle gology, Lund Uni e si y, Sweden 5 Epidemiology, Bios a is ics and P e en ion Ins i u e, Di ision o Occupa ional and En i onmen al Medicine, Uni e si y o Zu ich, Zu ich, Swi ze land 6 The Finnish Ins i u e o Occupa ional Heal h, Tampe e, Finland 7 The Immunopha macology Resea ch G oup, Facul y o Medicine and Biosciences, Uni e si y o Tampe e School o Medicine and Tampe e Uni e si y Hospi al, Tampe e, Finland 8 Alle gy Cen e, Tampe e Uni e si y Hospi al; and Facul y o Medicine and Biosciences, Uni e si y o Tampe e, Tampe e, Finland E-mail: [email p o ec ed] Keywo ds: b ea h es , ni ic oxide, ma hema ical model, heal h, pulmona y gas exchange Supplemen a y ma e ial o his a icle is a ailable online Abs ac The lung jus like all o he o gans is a ec ed by age. The lung ma u es by he age o 20 and age- ela ed changes s a a ound middle age, a 40–50 yea s. Exhaled ni ic oxide (F E NO)has been shown o be age, heigh and gende dependen . We hypo hesize ha he ni ic oxide (NO)pa ame e s al eola NO (C A NO), ai way flux (J aw NO), ai way di using capaci y (D aw NO)and ai way wall con en (C aw NO) will also demons a e his dependence. Da a om heal hy subjec s we e ga he ed by he cu en au ho s om hei ea lie publica ions in which heal hy indi iduals we e included as con ol subjec s. Heal hy subjec s (n=433) anged in age om 7 o 78 yea s. Age-s a ified e e ence alues o he NO pa ame e s we e significan ly di e en . Gende di e ences we e only obse ed in he 20–49 age g oup. The esul s om he mul iple eg ession models in subjec s olde han 20 yea s e ealed ha age, heigh and gende in e ac ion oge he explained 6% o a ia ion in F E NO a 50 ml s −1 (F E NO 50 ),4% in J aw NO, 16% in C aw NO, 8% in D aw NO and 12% in C A NO. In conclusion, in his s udy we ha e gene a ed e e ence alues o NO pa ame e s om an ex ended NO analysis o heal hy subjec s. This is impo an in o de o be able o use hese pa ame e s in clinical p ac ice. In oduc ion The use o non-in asi e me hods o diagnose espi a- o y diseases and moni o ea men is ad an ageous o bo h pa ien s and heal hca e p o essionals. Exhaled ni ic oxide (F E NO)has been used ex ensi ely since i s disco e y in human b ea h [1], especially in as hma whe e clinical p ac ice guidelines ha e al eady been published [2]. The pulmona y ni ic oxide dynamics models ha e he ad an age o being a mo e p ecise assessmen o ni ic oxide (NO)dynamics, bu hei applica ion has been limi ed [3]. The echnical de elop- men has apidly e ol ed and oday we ha e NO analyse s adop ed o clinical use, bo h in specialized espi a o y medicine andp ima y ca e [4,5]. F E NO om one single exhala ion will gi e a mea- su ed alue o NO p oduc ion om he en i e espi a- o y sys em. A mo e de ailed insigh can be gained h ough he use o he ma hema ical wo-compa men model (2CM)o pulmona y NO dynamics, which di - e en ia es he NO exchange o he pe iphe al and cen- al pa s o he lung and explains he flow dependence o F E NO [6,7].Inb ie , he2CMconsis so anal eola compa men comp ising he pe iphe al gas exchanging pa s o he lung ( espi a o y b onchioles and al eoli) and an ai way compa men comp ising he conduc i e OPEN ACCESS RECEIVED 1 May 2017 REVISED 13 June 2017 ACCEPTED FOR PUBLICATION 14 June 2017 PUBLISHED 13 Sep embe 2017 O iginal con en om his wo k may be used unde he e ms o he C ea i e Commons A ibu ion 3.0 licence. Any u he dis ibu ion o his wo k mus main ain a ibu ion o he au ho (s)and he i le o he wo k, jou nal ci a ion and DOI. © 2017 IOP Publishing L d ai ways la ge han espi a o y b onchioles. Gas in he al eola compa men holds a ce ain concen a ion o NO (C A NO). Du ing exhala ion, al eola gas a els h ough he b onchial compa men and mo e NO di - uses om he b onchial wall in o he luminal gas (ai - way NO flux, J aw NO)[8].C A NO and J aw NO can be es ima ed based on a linea model i F E NO is measu ed a h ee flow a es a o leas 100 ml s −1 [9].I aflow a e less han 30 ml s −1 is used oge he wi h a median and a high flow a e,i.e.100and300mls −1 , hen a non-linea model can be applied which also es ima es he ai way wall concen a ion o NO (C aw NO)and he di using capaci y o NO om he ai way wall o he gas s eam (D aw NO)[8,10]. In es iga ions ha e used he 2CM wi h in e es ing esul s, especially o C A NO whe e inc eased alues ha e been ound in se e e as hma [11], al eoli is [12], and ch onic obs uc i e pulmona y disease [10,13] and ea ly scle ode ma [14].C A NO has been specifically used o iden i y scle ode ma pa ien s a high isk o lung unc ion de e io a ion and ad ancing disease, wi h 5.3 ppb being sugges ed as he cu o alue [15]. Re e ence alues a e necessa y o any new me hod o be accep ed in clinical p ac ice, and e e - ence alues o F E NO a he ecommended flow o 50 ml s −1 (F E NO 50 )ha e been published [16,17]. Heigh , age and gende ha e been shown o influence F E NO 50 . Re e ence alues o NO pa ame e s om ex ended NO analysis a e limi ed o wo publica- ions, one wi h 89 adul s [18]and one wi h 66 child en [19]. The lung ma u es by he age o 20 in ega d o closing olume [20]and in olde age he di using capaci y declines in a linea ashion wi h inc easing age [21], and hese changes in pulmona y physiology migh also a ec NO pa ame e s. The aimo hiss udywas oes ablish e e ence alues o NO pa ame e s in heal hy subjec s anging om young o old age. Me hods Subjec s Da a om heal hy non-smoking subjec s we e ga he ed by he cu en au ho s om hei ea lie publica ions in which heal hy indi iduals we e included as con ol subjec s [10,14,18,19,22–30]. In he majo i y o hese s udies measu emen s o lung unc ion and symp om ques ionnai es e ified he heal h s a us. Gende , age and heigh we e no ed. The exhaled flow oge he wi h co esponding exhaled NOle elswe e collec ed. NO analysis F E NO 50 and F E NO alues om exhala ion wi h flows o 5–500 ml s −1 o he ex ended NO analysis we e ga h- e ed. All da a we e ecalcula ed ei he wi h he linea model (Tsoukias & Geo ge, TG)[9]using h ee flow a es o a leas 100 ml s −1 o wi h he non-linea model (Högman-Me iläinen Algo i hm, HMA)[10,22]using a low flow a e o 5, 10 o 20, a median a e o 100 and a high flow a e o 300, 400 o 500 ml s −1 . Da a we e ed in o an algo i hm in a s anda d Mic oso ® Excel en i onmen , a ailable as supplemen a y in o ma ion, o he es ima ion o he NO pa ame e s (s acks.iop. o g/JBR/11/047103/mmedia). When gene a ing NO pa ame e s om he linea model [9],Pea son’s - alue was no ed. Wi h he use o NO pa ame e s om he non-linea model [10,22]aplo o flow wi h co esp- onding NO alues can be gene a ed; a a flow o 50 ml s −1 , a NO alue was no ed and compa ed o he measu ed F E NO 50 o a quali y con ol o he es ima ion o he NO pa ame e s. Wi h he non-linea model he e is also a buil -in quali y es o he cu e [10].Thisisin line wi h he fi s guidelines o he ex ended NO analysis [31]. S a is ical analysis Due o aging o he lung, he subjec s we e di ided in o h ee age g oups, <20 yea s, 20–49 yea s and 50 yea s. Desc ip i e da a o he subjec s a e p esen ed as equency o as medians and qua iles whe e app o- p ia e. The dis ibu ions o he NO pa ame e s, s a ified by age g oups, a e p esen ed as an a i hme- ical mean o geome ical mean ( o skewed dis ibu- ed da a)and as 2.5 h, 5 h, 25 h, 50 h, 75 h, 95 h, 97.5 h pe cen iles. A K uskal-Wallis es and one-way ANOVA we e used o es o di e ences in he dis ibu ion o NO pa ame e s be ween he age g oups. In he case o significan di e ence be ween age g oups, pos -hoc es s we e pe o med using a pai wise Mann-Whi ney U- es . Pea son Co ela ion was used o es co ela ions o C A NO. Spea man’s ank o de co ela ion was used o he o he NO pa ame e s. Gende -s a ified simple eg ession models we e fi ed wi h he loga i hms o F E NO 50 ,C aw NO, D aw NO, and J aw NO, espec i ely, as he dependen a iable, and wi h age as an independen a iable. Loga i h- mically scaled eg ession lines we e e ans o med back in o na u al scale and all eg ession lines we e hen plo ed along wi h hei co esponding 95% e e ence in e als. Mul iple eg ession modelling was pe o med on da a whe e subjec s younge han 20 yea s we e exclu- ded, as child en di e om adul s in ega ds o he ela ionship be ween age and heigh , which made i di ficul o fi obus s a is ical models. The models we e specified wi h he C A NO in na u al scale, he loga i hms o F E NO 50 ,C aw NO, D aw NO, and J aw NO, espec i ely, as he dependen a iable, and wi h age, heigh and gende , including in e ac ion e ms be ween gende * heigh and gende * age, as indepen- den a iables. Fo all he models, ANOVA chunk es s we e pe o med, join ly es ing i he wo in e ac ion e ms con ibu ed significan ly o he models as com- pa ed o omi ing hem om he model. As his was no he case o any o he NO pa ame e s, he models we e efi ed wi hou he in e ac ion e ms. To 2 J. B ea h Res. 11 (2017)047103 M Högman e al accoun o a po en ial clus e e ec in he da a, we also con olled o s udy cen e and es ima ion me hod (TG e sus HMA). To help he in e p e abili y o eg ession coe ficien s, he a iables age and heigh we e cen ed and age was scaled o a uni o 10 yea s and 10 cm espec i ely [32]. Fo he ac o gende , B ep esen s he expec ed a io in geome ical means be ween a male and a emale, keeping all o he a i- ables fixed. Fo he loga i hmically ans o med pa a- me e s, eg ession coe ficien s ha e been e ans o med o na u al scale using he exponen ial unc ion. The boo s ap p ocedu e p oduces op i- mism-co ec ed es ima es o R 2 , wi h a co ec ion ac- o based on he a e age di e ence, in o e 5000 boo s ap samples, be ween he R 2 o he model fi o he boo s ap da a and he R 2 o he boo s ap model applied o he o iginal da a. Model assump ions o no mali y and homo- scedas ici y o esiduals we e assessed om g aphs. A p- alue <0.05 was conside ed s a is ically significan . Excel (Mic oso R O fice 2011)was used o calcula- ions o he NO pa ame e s. S a is ical analyses we e pe o med using SPSS, . 22 (SPSS Inc., Chicago, MI, USA), and R [33]using he ms package [34]. Resul s Heal hy subjec s (n=433)aged be ween 7–78 yea s we e analysed. The e we e mo e men (n=268) han women (n=165)( able 1). The e was no di e ence in F E NO 50 be ween he s udy cen es (p=0.37). The NO pa ame e s we e es ima ed using he lin- ea model TG (n=87)wi h an - alue om 0.90 o 1.0, and wi h a median alue o 1.0 (0.99, 1.0). In he non-linea model HMA (n=346), all passed he buil - in quali y es . The di e ence in measu ed and es i- ma ed F E NO 50 anged om −5.0 o 5.0, wi h a median alue o 0.3 (−0.6, 1.3)ppb. NO pa ame e s in he di e en age g oups The e we e s a is ically significan di e ences in he dis ibu ion o he NO pa ame e s be ween he young, middle and olde age g oups ( able 2).F E NO 50 was highe in he olde age g oup compa ed o he young age g oup (p<0.001)and he middle age g oup (p=0.001), and F E NO 50 was highe in he middle age g oup han he younge age g oup (p<0.001).J aw NO was lowe in he young age g oup compa ed o he middle age (p<0.001)as well as he olde age g oup (p<0.001).C aw NO was highe in he olde age g oup compa ed o he young age g oup (p<0.001)and he middle age g oup (p<0.001), and C aw NO was highe in he middle age g oup han in he younge age g oup (p<0.001).D aw NO was lowe in he olde age g oup compa ed o he young age g oup (p=0.023)and he middle age g oup (p=0.001).C A NO was lowe in he middle age g oup compa ed o he young age g oup (p=0.001)and he olde age g oup (p<0.001). NO pa ame e s in he di e en age g oups by gende The e was only a di e ence be ween gende s in he middle age g oup in F E NO 50 (p<0.001),J aw NO (p<0.001),C aw NO (p<0.001)and C A NO (p=0.027)bu no in D aw NO ( able 3). Reg ession analyses Rela ionships be ween age and he NO pa ame e s (J aw NO, C A NO, D aw NO and C aw NO), wi h uni a ia e eg ession lines and es ima ed 95% e e ence in e als, a e shown in figu e 1.F E NO 50 is shown in he supplemen a y ma e ial, a ailable online. The mul iple eg ession analyses, wi h he boo - s ap alida ion s ep, showed in he age g oups abo e 20 yea s ha age, heigh and gende in e ac ions oge- he explained 6% o a ia ion in F E NO 50, 4% in J aw NO, 16% in C aw NO, 8% in D aw NO and 12% in C A NO ( able 4). Age was a significan p edic o in all models (p<0.001)excep o J aw NO (p=0.18) ( able 4). The associa ion was posi i e o F E NO 50 and all NO pa ame e s. Gende con ibu ed as a significan main e ec o C aw NO and C A NO only. Mul iple lin- ea eg ession models poo ly p edic ed he la ge a ia- ions in F E NO 50 and NO pa ame e s. In he age g oup <20 yea s he e we e only 83 sub- jec s and he e o e mul iple eg ession models we e no applied. Age co ela ed posi i ely o F E NO 50 ( =0.31, p=0.005)and o J aw NO ( =0.32, p =0.003).The e we e s onge co ela ions be ween heigh and F E NO 50 ( =0.45, p <0.001),andheigh andJ aw NO ( =0.41, p=0.001), while no co ela ions we e ound be ween heigh and C A NO, C aw NO and D aw NO. Table 1. Subjec cha ac e is ics in he di e en age g oups p esen ed by gende . Age g oup <20 y s 20–49 y s 50 y s Gende Female Male Female Male Female Male Subjec s, n 41 42 82 113 42 113 Age, yea s 10 (9, 11)10 (8, 12)33 (26, 40)39 (30, 44)53 (52, 60)56 (52, 65) Heigh , m 1.39 (1.32, 1.47)1.37 (1.31, 1.49)1.68 (1.64, 1.71)1.81 (1.75, 1.85)1.67 (1.63, 1.69)1.76 (1.72, 1.80) Weigh , kg 34 (30, 38)32 (28, 39)60 (55, 68)80 (73, 87)70 (61, 76)79 (73, 88) BMI, kg/m 2 17 (16, 19)17 (16, 19)22 (20, 23)25 (23, 26)25 (23, 27)26 (24, 28) Da a a e gi en in median (25, 75 pe cen ile). 3 J. B ea h Res. 11 (2017)047103 M Högman e al Discussion In his s udy we ha e gene a ed e e ence alues o NO pa ame e s om an ex ended NO analysis o heal hy subjec s. By pooling he heal hy subjec s’da a om ea lie published da a he alues o NO pa a- me e s o a la ge g oup o subjec s can be p esen ed. We ha e ound ha age influences F E NO and all he NO pa ame e s, while gende a ec s NO pa ame e s only in he middle age g oup. Mul iple linea eg es- sion models poo ly p edic ed he la ge a ia ions in F E NO 50 and NO pa ame e s. In he See e al pape (n=13.275)abou 10% o he a ia ion in F E NO was explained by a a ie y o a iables [35], and his is in line wi h he cu en esul s (n=433)whe e abou 6% o he a ia ion in F E NO 50 was explained by age, heigh , gende , NO model and s udy cen e. Lung de elopmen In he <20 age g oup, F E NO 50 and he ai way NO pa ame e s J aw NO and C aw NO we e lowe han in he o he age g oups. This could possibly eflec an inc eas- ing mucosal su ace a ea wi h inc easing heigh and g owing lung olumes. This was also p esen in he s udy by Jacin o e al whe e he F E NO 50 inc ease b eakpoin appea ed a ound 14 yea s in gi ls and 16 yea s in boys [17]. This is in line wi h he g ow h o he body, and mo e specifically he de elopmen o he b onchial ee. Ageing In he middle and olde age g oups pulmona y aging seems o inc ease C A NO. This possibly eflec s dec eased di usi i y o gases in he dis al po ion o he lung, as C A NO is de e mined no only by ac o s p oducing NO in he lung pe iphe y bu also by how much al eola NO can di use in o he pulmona y ci cula ion whe e i is apidly sca enged by haemoglo- bin. In olde age, he di using capaci y declines in a linea ashion wi h inc easing age [21]and in elde ly heal hy subjec s he e is a dec ease in s eady-s a e ans e capaci y o ca bon monoxide (CO)[36]and NO [37]. The e is also an inc ease in esidual olume Table 2. Mean alues and pe cen ile dis ibu ion o F E NO 50 and NO pa ame e s in he h ee age g oups. Pe cen ile dis ibu ion Age g oups Mean p- alue * 2.5 5 25 50 75 95 97.5 1 F E NO 50 , ppb <20 y s 10.8 <0.001 4.7 4.9 7.1 10.5 15.9 25.6 27.0 20–49 y s 16.0 6.6 7.4 12.0 15.3 20.9 38.0 45.5 50 y s 18.2 7.7 8.5 13.2 18.2 25.3 36.5 44.9 1 J aw NO, nl/s <20 y s 0.40 <0.001 0.08 0.10 0.26 0.38 0.66 1.36 1.60 20–49 y s 0.76 0.31 0.34 0.53 0.70 1.08 2.01 2.51 50 y s 0.81 0.27 0.31 0.52 0.83 1.23 2.00 2.23 1 C aw NO, ppb <20 y s 64 <0.001 19 21 34 58 123 208 439 20–49 y s 105 30 35 65 105 160 309 441 50 y s 155 40 51 89 150 267 491 535 1 D aw NO, ml/s <20 y s 7.5 0.002 0.9 1.3 4.8 8.7 13.1 25.6 26.9 20–49 y s 7.8 1.3 2.5 5.3 8.3 12.7 19.1 21.6 50 y s 5.7 1.0 1.2 3.5 6.2 10.3 17.1 20.8 C A NO, ppb <20 y s 2.07 <0.001 0.11 0.61 1.52 2.05 2.73 3.59 3.88 20–49 y s 1.72 0.21 0.29 1.13 1.61 2.23 3.66 3.93 50 y s 2.2 0.33 0.51 1.48 2.25 2.85 3.77 3.88 1 Da a wi h skewed dis ibu ion a e gi en in geome ical mean, * K uskal-Wallis es o di e ence in mean alues be ween age g oups. Table 3. F E NO 50 and NO pa ame e s in he di e en age g oups p esen ed by gende . Age g oup <20 y s 20–49 y s 50 y s Gende Female Male Female Male Female Male 1 F E NO 50 , ppb 11 (8, 16)10 (7, 15)13 (10, 17)18 (13, 23) * 17 (12, 23)19 (14, 26) 1 J aw NO, nL/s 0.43 (0.28, 0.66)0.37 (0.23, 0.66)0.63 (0.44, 0.83)0.87 (0.60, 1.25) * 0.75 (0.49, 1.14)0.84 (0.54, 1.26) 1 C aw NO, ppb 76 (48, 130)54 (30, 84)77 (54, 115)126 (77, 211) * 121 (71, 173)163 (100, 288) 1 D aw NO, ml/s 6.5 (4.0, 10.9)8.7 (5.5, 17, 4)8.8 (6.2, 12.8)7.3 (4.8, 12.7)7.1 (4.8, 11.4)5.4 (3.2, 9.8) C A NO, ppb 2.12 (1.78, 2.39)1.98 (1.25, 2.33)1.99 (1.22, 2.39)1.52 (1.07, 2.06) * 2.44 (1.25, 2.92)2.20 (1.45, 2.83) Da a a e gi en in median (25,75 pe cen ile). 1 Geome ical mean. Mann-Whi ney U- es o gende di e ences, * p<0.05. 4 J. B ea h Res. 11 (2017)047103 M Högman e al Figu e 1. Rela ionship be ween age and he NO pa ame e s, ai way NO flux (J aw NO), al eola NO (C A NO), ai way di using capaci y (D aw NO)and ai way wall con en (C aw NO), wi h uni a ia e eg ession lines and es ima ed 95% e e ence in e als. Since child en di e ma kedly om adul s, in pa icula ega ding he associa ions be ween heigh and age, he young age g oup was ea ed sepa a ely. 5 J. B ea h Res. 11 (2017)047103 M Högman e al [38] eflec ing obs uc ion o he dis al pa o he ai ways ha could possibly con ibu e o he inc ease in C A NO seen in his s udy. Thus, he e is an accumula ion o NO om he al eola egion oge he wi h he inhaled NO om he ai ways ha inc eases wi h age, and bo h can con ibu e o he inc ease o C A NO. Howe e , he up ake o NO in pulmona y capilla ies is e y high [39], and he inc ease in C A NO could also be due o o he causes. One o hese o he causes a ec ing C A NO may be ha clinically heal hy olde subjec s ha e an al e ed inflamma o y cell p ofile and can ac ually ha e a low-g ade inflamma ion in he lowe espi a o y ac [40]. This could be due o he mac ophages becoming less e ficien in sca enging in ading mic oo ganisms in olde age g oups [41,42]. This could be an explana ion o he inc eased exhaled F E NO 50 and NO pa ame e s, i.e. J aw NO, C aw NO and C A NO, in ou olde subjec s. In s udies wi h olde unheal hy pa ien s, i is impo - an ha he con ol subjec s be ma ched o hem by age un il he e is enough da a o his age g oup. The e o e, he inc eased C A NO ha has been ound in COPD pa ien s should be e-e alua ed since hey ha e been compa ed in some s udies o younge indi iduals [10,13]. Howe e , in o he s udies, e.g. in cases o sys- emic scle osis o al eoli is, he C A NO alues a e su ely inc eased since he e we e no age di e ences be ween he pa ien s and con ol subjec s [12,14,43].Ma ching by gende should also be aken in o accoun o he mid- dle age g oup, since C A NO inc eases ea lie in emales. This is possibly explained by a dec ease in he capilla y blood olume o he lung [44]causing an impai ed gas exchange in women in he middle age g oup. D aw NO dec eased wi h inc easing age. This is in e es ing, as D aw NO is he o al di usi i y o NO om b onchial mucosa o luminal ai , and i can be assumed o eflec bo h he o al su ace a ea a ailable o di usion and also he physical p ope ies o he mucosa a ec ing he di usi i y o gases. As indi iduals g ow so do hei b onchial ees, and one would assume ha D aw NO inc eases wi h inc easing heigh , bu we did no see his. Ins ead, we ound ha C aw NO inc eased and his explained he inc ease in J aw NO and F E NO 50 du ing he g ow h pe iod. The dec ease o D aw NO ound in olde age migh eflec he physical changes occu ing in he b onchial mucosa o he aging lung. Gende I was only in he middle age g oup whe e a gende di e ence could be ound in F E NO 50 ,J aw NO, C aw NO and C A NO. In he eg ession model only he a ia ions in C aw NO and C A NO we e significan o gende . Olin e al ound F E NO 50 o be highe in men han in women a ound 50 yea s o age wi h 18 esp. 15 ppb espec i ely, bu when compa ing F E NO 50 be ween he sexes wi h simila heigh s and ages no di e ence was ound [16]. Jacin o e al ha e shown a gende di - e ence in he same age g oup wi h men sligh ly abo e 15 ppb and women a ound 12 ppb [17]. The co esp- onding alues o F E NO 50 in he p esen s udy wi h he young age g oup excluded a e 16 ppb o men and 15 ppb o women, which a e in line wi h he alues ob ained by Olin e al using he same analysing me hod, namely chemiluminescence. A limi a ion in his s udy is ha da a we e pooled, which esul ed in mo e men han women, especially in he old age g oup. In addi ion, he c oss-sec ional design o he s udy is no op imal o assess he ela ion be ween age and NO pa ame e s. Howe e , long enough longi udinal s udies would equi e decades o ollow-up. I would be in e es ing o pu lung unc ion in ela ion o he NO pa ame e s, bu un o una ely we did no ha e lung unc ion da a om all o he sub- jec s. We did check ha he e was no significan di e - ence in he mean F E NO 50 alues be ween he di e en cen es, which sugges s ha he me hodology was simila enough o allow o he pooling o he da a. In conclusion, in his s udy we ha e gene a ed e e ence alues o NO pa ame e s om an ex ended NO analysis o heal hy subjec s. This is impo an in o de o be able o use hese pa ame e s in clinical p ac ice. We ound ha pulmona y aging seems o inc ease C A NO, which is possibly a eflec ion o a dec eased di usi i y o gases in he gas exchange a ea. The impai ed immune de ence sys em ha occu s wi h old age could also explain he inc ease in all NO pa ame e s excep D aw NO ha was dec eased in his g oup. Fu he s udies o addi ional pooling o da a a e needed be o e we can p o ide e en be e age- ela- ed e e ence alues o he NO pa ame e s and Table 4. Reg ession coe ficen s (B)and p- alues o he mul iple eg ession models o NO- a iables. The R 2 is he unadjus ed coe ficien o de e mina ion o he models and R2boo is he co esponding op imism-co ec ed R 2 alues as es ima ed by boo s apping. In e cep Age Heigh Gende (male) B B p- alue B p- alue B p- alue R 2 R 2 boo F E NO 50 ppb 15.8 1.07 <0.001 1.04 0.29 1.12 0.13 0.08 0.06 J aw NO nl/s 0.77 1.03 0.18 1.05 0.33 1.10 0.26 0.07 0.04 C aw NO ppb 86.6 1.16 <0.001 0.87 0.04 1.70 <0.001 0.19 0.16 D aw NO ml/s 8.6 0.88 <0.001 1.21 0.01 0.69 0.01 0.11 0.08 C A NO ppb 1.95 0.2 <0.001 −0.03 0.68 −0.24 0.09 0.15 0.12 6 J. 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