Concentration of C-reactive protein, magnesium and calcium in children with acute bronchoconstriction before and after therapy with salbutamol

Introduction: In childhood, bronchoobstruction is mostly caused by respiratory viral infections. The aim of this study was to find out possible changes in serum concentration of magnesium and calcium (participants in bronchoconstriction), and concentration of C-reactive protein (marker of inammation) in children with moderate and severe bronchoconstriction, caused by viral respiratory infections. 
Materials and methods: The study included 32 children with acute bronchoconstriction caused by a viral respiratory infection. Inhalation of salbutamol was administered according to the severity of bronchoconstriction. Blood sampling was done on admission and the third day of salbutamol administration. 
Results: Therapy with salbutamol led to a relief of dyspnea within 36 hours, and symptoms of viral infection have relieved. During follow-up period, magnesium concentration was higher in children with moderate (but not in severe) bronchoconstriction than in healthy children, with consequently higher magnesium to calcium ratio. Concentration of C-reactive protein decreased spontaneously by gradual disappearance of signs and symptoms of viral infection. 
Conclusion: Determination of serum magnesium and calcium concentrations and determination of their ratio are not sufficient enough to follow-up on effects of therapy. Additional studies of ionized forms of magnesium and calcium, their intracellular content, as well as concentration in exhaled breath condensate are needed.


In tro duc tion
Bron cho con stric tion is the con stric tion of the airways due to the tig hte ni ng of sur roun di ng smooth mus cle, and cli ni cal con sequen ces of su ch sta te are coug hi ng, whee zi ng, and shor tne ss of brea th (1). It can be cau sed by di e re nt cli ni cal con di tio ns, i.e. chro nic ob struc ti ve lu ng di sea ses (as thma, chro nic ob struc ti ve pul mo na ry di sea se -COPD, bron chiec ta sis), vi ral in fec tio ns, in ter sti tial lu ng disea se, extratho ra cic le sio ns (pha ri nx, larynx, and up per trac hea) and in trat ho ra cic le sio ns (lower trac hea). In chil dhood, bron choob struc tion is mostly cau sed by res pi ra to ry vi ral in fec tio ns and asthma. In sen si ti ve in di vi dua ls, the in am ma tion is usual ly in du ced by cau sal al ler ge ns lea di ng to the sympto ms of as thma (bron cho con stric tion, cou gh, che st tig htne ss). Ad di tio nal ly, res pi ra to ry in fec tions, es pe cial ly res pi ra to ry vi ral in fec tio ns (inclu di ng rhi no vi ru ses, in uen za, pa rain uen ce, res pi ra to ry syncytial vi ru ses, co ro na vi ru ses, ade no vi rus) can trig ger as thma tic sympto ms, su ch as whee zi ng and che st tig htne ss (2).
Deter mi na tion of se rum eo si nop hil ca tio nic protein (3), and re cen tly, de ter mi na tion of se rum CRP (usi ng hi gh sen si ti ve met hod) can be used to mo ni tor in flam ma tion in pa tien ts wi th as thma (4). Howe ver, im por tan ce of CRP, as a mar ker of chro nic, la te nt in flam ma tion is not enough tes ted.
Bo th, in ves ti ga tio ns in vit ro (5) and in vi vo (6) demon stra ted that mag ne sium ac ts on bron chial smo oth mus cle re laxa tion. It is known that many me tabolic fun ctio ns of mag ne sium and cal cium are tig htly in te r-re la ted. As in tra cellu lar ca tion, mag ne sium ac ts as a cal cium chan nel bloc ker (7). This is con sis te nt wi th the fa ct that mag ne sium also ac ts as a cal cium an ta go ni st. Cal cium is known to act as a broncho con stric tor. Ad di tio nal ly, magne sium is al so in vol ved in the pro ces ses of in amma tion (8) and oxi da ti ve stre ss (9). Hypo mag nesae mia may re su lt in re du ced pul mo na ry fun ction, es pe cial ly in pa tien ts wi th as thma (10). In pre vious in ves ti ga tio ns we ha ve poi nt out at the sig ni ca nt re dis tri bu tion of mag ne sium be tween plas ma and leu ko cytes du ri ng acu te as thma in chil dren (11). Pa tien ts wi th acu te as thma (12) and even tho se with chro nic as thma (13) ha ve in crea sed con cen tration of CRP.
The aim of this stu dy was to nd out pos sib le chan ges in se rum con cen tra tion of mag ne sium and cal cium (as par ti ci pan ts in bron cho con striction), and con cen tra tion of CRP (as a mar ker of inam ma tion) in chil dren wi th mo de ra te and se ve re bron cho con stric tion, cau sed by vi ral res pirato ry in fec tio ns. CRP, mag ne sium and cal cium con centra tio ns we re de ter mi ned on hos pi tal ad mis sion and at dis char ge from the hos pi tal, i.e. be fo re and af ter ad mi nis tra tion of sal bu ta mol (sho r t-ac ti ng β 2 -ad re ner gic re cep tor ago ni st, bron cho con striction re lie ver).

Ma te ria ls and met ho ds Pa tien ts
The stu dy in clu ded 63 chil dren, aged 1-16 yea rs (6 ± 4 yea rs), di vi ded in to two grou ps: chil dren wi th acu te bron cho con stric tion cau sed by a vi ral res pira to ry infec tion (N = 32), and cli ni cal ly heal thy children re fer red for syste ma tic me di cal che ck-up as a con trol group (N = 31). All pa tien ts we re re fer red from pri ma ry heal th ca re o ces to hospi tal between De cem ber 2008 and Ja nua ry 2009. Ba sed on GINA gui de li nes (1) mode ra te bron cho constric tion was con r med in 20 chil dren, and se ve re bron cho con stric tion in 12 chil dren (Fi gu re 1).
Pre vious ly, in 13 chil dren mo de ra te per ma ne nt asthma was diag no sed ac cor di ng to the cri te ria recom men ded by the PRACTALL (14) and GINA 2006 (1). Du ri ng res pi ra to ry syncytial vi rus in fec tion (conr med in na sop ha ryngeal wa sh spe ci me ns), mode ra te bron cho con stric tion was con r med in 10 chil dren, and se ve re bron cho con stric tion in 9 children (Fi gu re 1). Al ler gic di sea se was not evi den ced in other 13 chil dren.
Sal bu ta mol so lu tion (150-200 μg in a sin gle do se) wi th an ECO NO neb ne bu li zer System (Me dix Li mited; Lut te rwor th, UK) was ad mi nis te red at di e rent ti me in ter va ls, ac cor di ng to the se ve ri ty of broncho con stric tion. Diag nos tic wo r k-up and the ra py we re per for med ac cor di ng to the sta ndardi zed pro ce du re, in li ne wi th et hi cal prin cip les, Hel sin ki Dec la ra tion on Hu man Rig hts from 1975 and Seoul amen dmen ts from 2008. Stu dy ap pro val was ob- Non-asthmatics N = 10 tai ned from the Hos pi tal Et hi cs Com mit tee. An infor med con se nt in wri ti ng was ob tai ned from the pa ren ts. Blood sam pli ng was do ne on ad mis sion (Day 1) and the thi rd day of sal bu ta mol ad mi nis tration (Day 3).

Met ho ds
The se rum con cen tra tio ns of mag ne sium and calcium we re de ter mi ned usi ng stan dar di zed spectrop ho to met ric met ho ds, and CRP (hi gh sen si ti vity C-reacti ve pro tein) con cen tra tion was de ter mined by im mu no tur bi di met ric met hod on la tex par tic les (15), on an Olympus AU 400 se lec ti ve autoa na lyzer (Olympus, To kyo, Ja pan). Rea gen ts from the sa me ma nu fac tu rer we re used. Com ple te blood ce ll cou nt was ana lyzed using Sysmex XT-1800i blood ce ll coun ter (Sysmex. Cor po ra tion, Kobe Hyo go, Ja pan).

Sta tis tical ana lysis
The va riab les we re des cri bed as mean and stan dard de via tion (x ± SD) if they had nor mal dis tri bu-tion, or me dian and in te rquar ti le range (IQR) if not. Nor ma li ty was tes ted usi ng D'Agos ti no-Pear son test. Stu de nt's t-te st (for nor mal dis tri bu tion) and Ma nn-Whit ney te st (for asymmet ric dis tri bu tion) were used for com pa ri son of in de pen de nt samples, whi le va rian ce ra tio, F-te st was used for compari son of de pen de nt va riab les (16). Va lues of P < 0.05 we re con si de red sta tis ti cal ly sig ni ca nt. Corre la tion of the stu dy va riab les was expres sed by coe cie nt of cor re la tion and Spear ma n's coe cient of ra nk cor re la tion, res pec ti ve ly (r). Da ta pro cessing was per for med usi ng Med Ca lc sof twa re (Medi sof twa re, Ma ria ker ke, Bel gium).

Re sul ts
In ha la tion of sal bu ta mol led to a re lief of dyspnea wit hin 24 to 36 hou rs. Al so, sig ns and sympto ms of vi ral in fec tion ha ve gra dual ly re lie ved.
Du ri ng the fol low-up pe riod, con cen tra tion of magne sium was sta tis ti cal ly sig ni can tly hig her than in con trol group of chil dren (Tab le 1). Sta tis ti cal diffe ren ces be tween mag ne sium con cen tra tion be- CRP -hi gh sen si ti ve C-reac ti ve pro tein; IQR -in te rquar ti le ran ge; da ta are pre sen ted wi th arit hme tic mean ± stan da rd de via tion or wi th me dian (in te rquar ti le ran ge). Sta tis ti cal sig ni can ce: 1 = Mo de ra te (Day 1) vs. Con trol; 2 = Mo de ra te (Day 3) vs. Con trol; 3 = Se ve re (Day 1) vs. Con trol; 4 = Se ve re (Day 3) vs. Con trol; 5 = Mo de ra te (Day 3 vs. Day 1); 6 = Day 1 (Mo de ra te vs. Se ve re); a < 0.0001; b < 0.001; c = 0.002; d = 0.005; e = 0.020; f = 0.030; g = 0.040 TAB LE 1. Va lues of mag ne sium, cal cium, mag ne sium to cal cium ra tio and CRP, in 32 chil dren wi th bron cho con stric tion be fo re (day 1) and af ter the ra py wi th sal bu ta mol.
Do dig S. et al. CRP, mag ne sium and cal cium du ri ng the ra py wi th sal bu ta mol tween 1 st and 3 rd day we re not sig ni ca nt, neit her in chil dren wi th mo de ra te nor seve re bron cho constric tion. The re was no sta tis ti cal ly sig ni ca nt diffe ren ce in plas ma cal cium con cen tra tio ns be tween in ves ti ga ted grou ps of chil dren. Mag ne sium to calcium ra tio was si gni can tly hig her on 3 rd day of thera py in com pa ri son wi th the group of heal thy chil dren (P = 0.04).
Af ter ad mi nis tra tion of sal bu ta mol, in the ma jo ri ty of pa tien ts wi th se ve re bron choob struc tion (10/12), con cen tra tion of mag ne sium in crea sed, and calcium dec rea sed, re sul ti ng in in crea sed va lues of mag ne sium to cal cium ra tio in mo st chil dren. In the sub group of chil dren wi th mo de ra te broncoob struc tion, con cen tra tion of mag ne sium increa sed in 5/10 of chil dren, dec rea sed in 10/20 of chil dren, and re mai ned un chan ged in 5/20 of children. Si mul ta neous ly, con cen tra tion of cal cium increa sed in 8/20, dec rea sed in 6/20 of chil dren, and re mai ned un chan ged in 6/20 of chil dren. Magnesium to cal cium ra tio dec rea sed in 11/20 of children, in crea sed in 1/20 of chil dren and re mai ned un chan ged in 8/20 of chil dren.
CRP con cen tra tion was sta tis tical ly sig ni can tly higher in pa tien ts wi th bron choob struc tion in compa ri son wi th group of con trol chil dren (Day 1 vs. con trol: P < 0.001; Day 3 vs. con trol: P < 0.001) (Table 1). Al so, CRP was hig her pret he ra peu ti cal ly than on the thi rd day of drug ad mi ni stra tion in chil dren wi th mo de ra te bron choob struc tion (P = 0.002), but not in chil dren wi th se ve re bron choob struction. At ad mis sion, CRP was hig her in chil dren wi th mo de ra te bron choob struc tion than in tho se wi th se ve re bron choob struc tion (P = 0.020). The highest CRP va lue (44.0 mg/L) was re cor ded in no nas thma tic chi ld (aged 3 yea rs) wi th mo de ra te broncho con stri tion.
Mag ne sium and cal cium showed sig ni ca nt cor rela tion in pa tien ts at ad mis sion (r = 0.545; P = 0.002) but not af ter the ra py (r = 0.353; P = 0.052). Al so, cor re la tion was con r med be tween mag ne sium and cal cium con cen tra tion in sub group of pa tien ts wi th mo de ra te bron choob struc tion (r = 0.528; P = 0.021) as we ll as in patien ts wi th se ve re bron choconstriction (r = 0.641; P = 0.043) (Fi gu re 2). The re was no cor re la tion be tween mag ne sium, cal cium, mag ne sium/calcium ra tio and con cen tra tion of CRP. Al so, se lec ted bio mar ke rs we re re la ted to neit her leu ko cyte num ber nor num ber of lympho cytes and neut rop hi ls (da ta not pre sen ted).
Rate of as thma tic was higher in group of chil dren wi th se ve re bron cho con stric tion.

Dis cus sion
Stu dy re sul ts ha ve shown in crea sed se rum concen tra tion of CRP du ri ng three days fol low-up in chil dren wi th bron cho con stric tion cau se by res pira to ry vi ral in fec tion in com pa ri son to co ntrol group of chil dren. That in crea se be fo re sal bu ta mol ad mi nis tra tion (but not on the thi rd day of the rapy) was grea ter than in chil dren wi th mo de ra te bron cho con stric tion. Du ri ng fol low-up pe riod, magne sium con cen tra tion was hig her in chil dren wi th mo de rate (but not in se ve re) bron cho con stric tion than in heal thy chil dren, wi th con sequen tly hig her mag ne sium to cal cium ra tio. We hypot he si ze that CRP va lues mig ht be in cor re la tion wi th deg ree of in am ma tion cau sed by vi ral in fec tion. Fur thermo re, mag ne sium and mag ne sium to cal cium ratio we re pos sib ly in crea sed due to the re laxa tion of bron chial smoo th mus cle fa ci li ta ted by mag nesium.
Vi ral in fec tio ns, es pe cial ly tho se cau sed by ade novi ru ses and res pi ra to ry syncytial vi rus, cou ld sig nican tly in crea se se rum CRP va lues (17,18).
In vi ral in fec tio ns le ve ls of CRP cou ld be hig her than 40 mg/L, and in so me chil dren even hig her than 80 mg/L (19). In the pre se nt stu dy, the hig he st CRP va lue oc cur red in no n-as thma tic chi ld wi th mo de ra te bron cho con stri tion.
Hypo mag ne semia cou ld lead to bron chial smoo th mus cle con trac tion or la ck of bron chial smoo th mus cle re laxa tion (20). In the pre se nt stu dy hypomag ne se mia was not re cor ded in eit her of pa tients. Con tra ry, ini tial se rum mag ne sium con cen tration was hig her in chil dren wi th mo de ra te broncho con stric tion (but not wi th se ve re bron cho constric tion) than in heal thy chil dren. It cou ld be presu med that the se pa tien ts ha ve mo de ra te bronchoob struc tion ju st be cau se of a se rum mag nesium le ve ls su cie nt to re dis tri bu te in tra cel lu lar ly.  C D E Con cen tra tion of mag ne sium in va rious ce ll types is a bet ter in di ca tor of mag ne sium ho meos ta sis or re dis tri bu tion in pa tien ts wi th bron cho con striction than serum mag ne sium con cen tra tion (11,21). Al so, mag ne sium to cal cium ra tio was hig he st on thi rd day of sal bu ta mol ad mi nis tra tion. Tig ht calcium to mag ne sium cor re la tion re cor ded in subgrou ps of chil dren wi th mo de ra te and se ve re bron choob struc tion and in as thma tic chil dren with mo de ra te bron choob struc tion in di ca ted their close in te r-reac ti vi ty. Our pre vious in ves ti ga tion has shown the mag ne sium to cal cium ra tio to be changed in exha led brea th con den sa te in chil dren wi th asthma in com pa ri son to heal thy chil dren (22).
Al thou gh su e ri ng from so me li mi ta tio ns (sma ll num ber of chil dren wi th se ve re bron chcon striction; de ter mi na tion of mag ne sium and cal cium in se rum, not in cel ls, de ter mi na tion of to tal se rum mag ne sium and cal cium, and not their io ni zed forms) our stu dy de mon stra ted the con cen tra tion of mag ne sium, as a mar ker of smoo th mus cle re laxation, to un de rgo chan ges du ri ng sal bu ta mol ad minis tra tion, es pe cial ly in chil dren wi th mo de ra te bron choob struc tion cau sed by vi ral infec tion. CRP, as a bio mar ker of nonspe ci c im mu ni ty, dec reased spon ta neous ly by gra dual di sap pea ran ce of sig ns and sympto ms of vi ral in fection. De ter mi nation of se rum mag ne sium and cal cium con cen tratio ns and de ter mi na tion of their ra tio are not sucie nt enou gh to fol low-up on e ec ts of the ra py. Ad di tio nal stu dies of io ni zed forms of mag ne sium and cal cium, their in tra cel lu lar con te nt, as we ll as con cen tra tion in exha led brea th con den sa te are nee ded.