Relationship between Exhaled Nitric Oxide and Childhood Asthma by klutzfu51


									Relationship between Exhaled Nitric Oxide and
Childhood Asthma
North West Lung Centre, Wythenshawe Hospital, and Royal College of General Practitioners Manchester Research Unit,
Parkway House, Manchester; and Molecular Medicine Unit, St. James University Hospital, Leeds, United Kingdom

               The purpose of the study was to determine if exhaled nitric oxide levels in children varied according
               to their asthmatic and atopic status. Exhaled nitric oxide was measured in a sample of 93 children at-
               tending the North West Lung Centre, Manchester, United Kingdom, for the clinical evaluation of a
               respiratory questionnaire being developed as a screening tool in general practice. The clinical assess-
               ment included full lung function, skin prick testing, and exercise challenge. Children were said to be
               asthmatic either by consensus decision of three independent consultant pediatricians, who reviewed
               all the clinical results except the nitric oxide measurements, or by positive exercise test. Atopic asth-
               matic children had higher geometric mean exhaled nitric oxide levels (consensus decision, 12.5 ppb
               [parts per billion] 95% CI, 8.3 to 18.8; positive exercise test, 12.2 ppb 95% CI, 7.6 to 19.7) than did
               nonatopic asthmatic children (3.2 ppb 95% CI, 2.3 to 4.6; 3.2 ppb 95% CI, 2.0 to 5.0), atopic nonasth-
               matic children (3.8 ppb 95% CI, 2.7 to 5.5; 5.7 ppb 95% CI, 4.1 to 8.0), or nonatopic nonasthmatic
               children (3.4 ppb 95% CI, 2.8 to 4.1; 3.5 ppb 95% CI, 3.0 to 4.1). Thus, exhaled nitric oxide was raised
               in atopic asthmatics but not in nonatopic asthmatics, and these nonatopic asthmatics had levels of
               exhaled nitric oxide similar to those of the nonasthmatics whether atopic or not. Frank TL, Adisesh
               A, Pickering AC, Morrison JFJ, Wright T, Francis H, Fletcher A, Frank PI, Hannaford P. Rela-
               tionship between exhaled nitric oxide and childhood asthma.
                                                                                                AM J RESPIR CRIT CARE MED 1998;158:1032–1036.

Inducible nitric oxide synthase is active in asthmatic airways                     exhaled NO (eNO) levels, nasal NO (nNO) levels, and asthma
(1) and nitric oxide (NO) can be measured in exhaled air (2–                       in a community-based group of children 5 to 15 yr of age.
6). So far, however, there have been few studies (7, 8) of NO
levels in childhood asthma and none in a community-based                           METHODS
population.                                                                        In September 1995 the parents or guardians of 3,290 children were
    The Wythenshawe Community Asthma Project (WYCAP)                               sent a postal questionnaire designed to identify the 1-yr period preva-
is a long-term prospective study of the natural history of                         lence of designated respiratory symptoms (APPENDIX 1). Nonrespon-
asthma in two general practice populations on a housing es-                        dents were sent reminders at 4 and at 8 wk after the initial mailing.
tate in Manchester, United Kingdom. In 1993 and 1995, postal                       Completed questionnaires were received back for 2,434 children,
questionnaires were sent to all adults and children registered                     which after allowance for an estimated 5.5% of children no longer be-
with the two practices asking about respiratory symptoms and                       lieved to be living at the mailing address, gave an adjusted response
asthma-related conditions (9–11). The children’s question-                         rate of 78%. A stratified weighted random sample of respondents at-
naire was based on the ISAAC questionnaire (12). In order to                       tending one of the general practices was personally invited by their
                                                                                   general practitioner to attend The North West Lung Centre, Wythen-
test a hypothesis that children with three or more positive
                                                                                   shawe, Manchester, for clinical review. The sampling method was de-
responses to five key questions might have asthma, detailed                        signed to yield a high number of asthma cases. Children were not ex-
clinical assessments have been made on a random sample of                          cluded if they had a previous diagnosis or were receiving treatment
respondents to the 1995 questionnaire. These examinations                          for asthma. The clinical assessment involved a full medical history,
provided the opportunity to examine the relationship between                       physical examination, and investigations including exercise challenge,
                                                                                   spirometry with reversibility to 2-agonists, a 1-wk electronic peak
                                                                                   flow diary record, skin prick testing to house dust mite, grass pollen,
                                                                                   cockroach, dog, and cat. Atopy was defined as a skin wheal 3 mm
(Received in original form July 28, 1997 and in revised form March 31, 1998)       greater than that of the negative control. The mean wheal size for
Supported by Glaxo-Wellcome UK, Royal College of General Practitioners, Zen-       each allergen was defined as the mean of the maximal wheal diame-
eca Pharmaceuticals, North West Lung Centre, and Manchester Airport Authority      ters at 90 degrees to each other. Five allergens were studied and the
PLC.                                                                               mean wheal diameter was calculated for the five allergens together. A
Correspondence and requests for reprints should be addressed to Dr. T. L. Frank,   positive exercise test was defined as a fall in FEV1 15% from a pre-
North West Lung Centre, Wythenshawe Hospital, Southmoor Rd., Manchester,           exercise value after 6 min of free running exercise. Exercise tests were
M23 9LT, UK.                                                                       performed outdoors in the summer months.
Am J Respir Crit Care Med Vol 158. pp 1032–1036, 1998                                  The eNO was measured using a chemiluminescence analyzer
Internet address:                                              (Model LR2000; Logan Research, Rochester, UK), sensitive to NO
Frank, Adisesh, Pickering, et al.: Exhaled Nitric Oxide and Childhood Asthma                                                                                 1033

from 1 to 5,000 parts per billion (ppb) by volume, and with a resolu-           the necessary equipment was not available when they at-
tion of 1 ppb, which was designed for on-line recording of exhaled NO           tended. Three children were unable to make any satisfactory
concentration. The response time (10 to 90%) was 0.6 s. In addition             recordings. Of the 94 remaining children one could not per-
to NO, the analyzer also measured CO2 (resolution, 0.1% CO2; re-                form the eNO measurement and another refused to allow the
sponse time, 200 ms), with sample pressure and volume in real time.
                                                                                nNO measurement to be taken. Two of these 94 children re-
The sampling rate of the analyzer was 250 ml/min for all measure-
ments. The analyzer was calibrated daily using a certified NO mixture           fused a skin prick test; 89 of the remaining 94 children were
(114 ppb) in nitrogen (BOC Special Gases, Guilford, UK).                        able to perform an exercise test. The three consultants agreed
    Measurements of eNO were made by using a sidearm sampling                   on the diagnosis in 55 children, two of the three agreed on the
technique as described by Kharitonov and colleagues (6, 13). The NO             diagnosis in 20 children, and in 19 children there was no agree-
value corresponding to the plateau of the end exhaled CO2 reading               ment between the consultants.
( 5% CO2) was recorded.                                                            Children with probable asthma had significantly higher
    The nNO was measured with a Teflon tube inserted into one of the            geometric mean eNO levels (8.3 ppb) (Table 1) than did those
nares while the subject held his or her breath, i.e., with no active exha-      who were nonasthmatic (3.6 ppb) (p 0.05, Tukey-HSD test).
lation for at least 30 s (6, 13). Two readings of exhaled and nasal air
                                                                                The mean nNO levels for children with probable asthma were
were taken, with results being expressed as a mean value.
    Measurements of NO were made by the clinical investigator TF
                                                                                also greater than those in the other groups, but the differences
who had been trained by AA. Readings from the stored data were                  did not reach statistical significance. Atopic children had sig-
made by AA who was unaware of the clinical results or of the expert             nificantly higher geometric mean eNO levels (7.8 ppb) than
opinion. The data had been stored with only an identifying case num-            did nonatopic children (3.4 ppb) (p 0.05, one-way ANOVA);
ber. The expert consensus opinion had not been obtained at the time             they also had higher mean nNO (996 and 658 ppb, respec-
the readings were taken.                                                        tively) (p    0.05, one-way ANOVA). Children with positive
    Three independent consultant pediatricians with an interest in              exercise test results had significantly higher geometric mean
asthma were then supplied by post with all the information from the             eNO levels (8.5 ppb) than did those with negative exercise test
clinical assessment except the NO findings. After reviewing this the            results (4.4 ppb) (p      0.05, one-way ANOVA) and higher
pediatricians were asked to rate into four categories the probability
                                                                                mean nNO levels (1,031 and 793 ppb, respectively) (p 0.05,
that each child had asthma:        90% (probable asthma), 50 to 90%
(possible asthma), 10 to 50% (asthma unlikely) or          10% (nonasth-        one-way ANOVA) (Table 1).
matic). When one of the experts disagreed with his colleagues, the fi-             The atopic probable asthmatics had significantly higher geo-
nal rating used was the consensus view of the two agreeing physicians.          metric mean eNO levels (12.5 ppb) than did the nonatopic prob-
When all three consultants disagreed the child was rated according to           able asthmatics (3.2 ppb), the atopic nonasthmatics (3.8 ppb),
the middle opinion.                                                             and the nonatopic nonasthmatics (3.4 ppb) (p 0.05, Tukey-
                                                                                HSD test) (Table 2 and Figure 1). Atopic probable asthmatics
Statistics                                                                      also had significantly higher mean nNO levels (1,108 ppb)
The eNO data followed an approximate log-normal distribution, so                than did nonatopic nonasthmatics (671 ppb) (p 0.05, Tukey-
they were converted to natural logarithms for analysis. The data were           HSD test).
reconverted to the original units for presentation; nNO data followed              Atopic children with positive exercise test results had
a normal distribution and were analyzed without transformation.
                                                                                higher geometric mean eNO levels (12.22 ppb) than did atopic
Comparisons between the groups were done using a one-way analysis
of variance (one-way ANOVA). Differences indicated by the ANO-
                                                                                children with negative exercise test results (5.69 ppb), non-
VAs were analyzed using the Tukey-HSD test. Statistical significance            atopic children with positive exercise test results (3.17 ppb), or
was set at the conventional 5% level (p 0.05).                                  nonatopic children with negative exercise test results (3.47
   The study had ethical approval from the local research ethics com-           ppb) (p 0.05, Tukey-HSD test) (Figure 2). Atopic children
mittee, and all the children and their parents or guardians gave in-            with positive exercise test results also tended to have higher
formed consent.                                                                 mean nNO levels (1,143 ppb) than did nonatopic children with
                                                                                positive exercise test results (631 ppb) and nonatopic children
One hundred fifty-eight children attended for clinical assess-
ment. Nitric oxide measurements were not possible in 61 as                                                          TABLE 2
                                                                                                ATOPY, ASTHMA, AND THEIR COMBINED
                                                                                                       EFFECT ON NITRIC OXIDE
                                      TABLE 1
                                                                                                             Geometric   95%    Mean Nasal    95%
    NITRIC OXIDE AND ITS RELATIONSHIP TO ASTHMA, ATOPY,                                             Subjects Mean NO Confidence    NO      Confidence
            AND BRONCHIAL CHALLENGE TO EXERCISE                                                       (n)      (ppb)   Interval   (ppb)     Interval
                             Geometric      95%       Mean Nasal      95%       Atopic probable
                  Subjects   Mean NO     Confidence      NO        Confidence     asthmatics           23         12.5       8.3–18.8       1,108       934–1,282
                    (n)        (ppb)      Interval      (ppb)       Interval    Nonatopic
                                                                                  asthmatics           10          3.2       2.3–4.6          752       535–969
  asthmatic         34          8.3       5.8–11.8      1,008      868–1,148
                                                                                Atopic 10–90%
                                                                                  chance of
  asthmatic         13          4.6       3.2–6.6         716      520–911
                                                                                  asthma*              12           8        4.4–14.8          896      639–1,153
  asthmatic         13          5.7       3.0–10.7        733      454–1,013
                                                                                  10–90% chance
Nonasthmatic        33          3.6       3.0–4.4         781      673–889
                                                                                  of asthma*           14          3.5       2.6–4.6          578       390–764
Atopic              51          7.8       5.9–10.3        996      889–1,103    Atopic
Nonatopic           40          3.4       3.0–3.9         658      563–754        nonasthmatics        16          3.8       2.7–5.5          910       750–1,069
Positive                                                                        Nonatopic
  exercise test     27          8.5       5.6–12.8      1,031      845–1,216      nonasthmatics        16          3.4       2.8–4.1          671       529–813
                                                                                  * Children in the possible and unlikely asthma groups were grouped together to in-
  exercise test     62          4.4       3.7–5.3         793      712–873      crease the numbers and then subdivided with relation to atopic status.
1034                                                  AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE                            VOL 158         1998

                Figure 1. Scatter plot of tidal nitric oxide with relation to atopy and likelihood of asthma.

with negative exercise test results (696 ppb) (p 0.05, Tukey-            DISCUSSION
HSD test) (Table 3).
                                                                         Our results show that eNO levels in children with probable
   There were no significant differences in eNO between male
                                                                         asthma are higher than those found in children less likely to
and female subjects or between different age groups.
                                                                         have the condition, in those with positive exercise test results
   The mean wheal size for the five allergens tested were
                                                                         compared with those with negative exercise test results, and in
added together and the combined mean wheal size was com-
                                                                         atopic children compared with nonatopic children. The higher
pared with the exhaled eNO levels using bivariate analysis.
                                                                         eNO levels in asthmatic children, however, were mainly due
Pearson’s correlation coefficient was calculated at 0.66 (p
                                                                         to associated atopy. These results suggest that atopic asth-
0.05), showing good correlation between mean wheal size and
                                                                         matic children produce greater amounts of eNO than do the
exhaled NO.
                                                                         nonatopic asthmatic children, perhaps because of different
                                                                         mechanisms operating in the two groups. This theory is sup-
                                                                         ported by Tang and colleagues (14) who found differences in
                                                                         cells in bronchoalveolar lavage samples taken from atopic
                                                                         asthmatics exposed to house dust mite compared with those
                                                                         found in nonatopic asthmatics. On the other hand, Humbert
                                                                         and colleagues (15) found some evidence that both atopic and
                                                                         nonatopic asthmatic patients had infiltration of the bronchial
                                                                         mucosa with cells expressing Th2 type cytokines, suggesting
                                                                         similarities in the immunopathogenesis of these clinically dis-
                                                                         tinct types of asthma.
                                                                            We found that atopic children had higher eNO than did

                                                                                                               TABLE 3
                                                                                  ATOPY, BRONCHIAL CHALLENGE TO EXERCISE, AND
                                                                                     THEIR COMBINED EFFECT ON NITRIC OXIDE

                                                                                                          Geometric   95%    Mean Nasal    95%
                                                                                                 Subjects Mean NO Confidence   NO*      Confidence
                                                                                                   (n)      (ppb)   Interval   (ppb)     Interval

                                                                         Atopic positive
                                                                           exercise test            19        12.22     7.59–19.65       1,143      930–1,357
                                                                         Nonatopic positive
                                                                           exercise test              7        3.17     1.99–5.04          631      313–949
                                                                         Atopic negative
                                                                           exercise test            30         5.69     4.07–7.96          907      786–1,028
                                                                         Nonatopic negative
                                                                           exercise test            31         3.47     2.97–4.05          696      596–796
Figure 2. Scatter plot of tidal nitric oxide with relation to atopy
and result of exercise test.                                               * The number of subjects in this category was 19, 6, 30, and 32, respectively.
Frank, Adisesh, Pickering, et al.: Exhaled Nitric Oxide and Childhood Asthma                                                                    1035

                                                                       tion made available to our consultants, therefore, is more
                                                                       likely to have been normal, and this may have reduced the
                                                                       chances of them diagnosing asthma.
                                                                          Although this study showed that there may be differences
                                                                       in the amounts of eNO produced by atopic and nonatopic
                                                                       asthmatics, further work is needed to confirm the finding in
                                                                       children who have not yet been started on inhaled steroid
                                                                       Acknowledgment : The writers thank the three consultant pediatricians, Dr.
                                                                       John Couriel, Dr. Warren Lenny, and Dr. Andrew Bradbury, who produced
                                                                       our expert consensus diagnosis. They also thank Brian Farragher, who pro-
                                                                       vided statistical support, Roseanne McNamee for her help with the sam-
                                                                       pling design method, and the staff of Bowland Medical Practice, Tregenna
                                                                       Group Practice, North West Lung Centre, and the Royal College of General
                                                                       Practitioners, Manchester Research Unit, for their help in conducting the

                                                                        1. Hamid, Q., D. R. Springall, V. Riveros-Moreno, P. Chanez, P. Howarth,
                                                                             A. Redington, J. Bousquet, P. Godard, S. Holgate, and J. M. Polak.
                                                                             1993. Induction of nitric oxide synthase in asthma. Lancet 342:1510–
                                                                        2. Alving, K., E. Weitzberg, and J. M. Lundberg. 1993. Increased amount
                                                                             of nitric oxide in exhaled air of asthmatics. Eur. Respir. J. 6:1368–1370.
                                                                        3. Kharitonov, S. A., D. Yates, R. A. Robbins, R. Logan-Sinclair, E. A.
                                                                             Shinebourne, and P. J. Barnes. 1994. Increased nitric oxide in exhaled
                                                                             air of asthmatic patients. Lancet 343:133–135.
Figure 3. Scatter plot of tidal nitric oxide levels in children with
                                                                        4. Massaro, A. F., B. Gaston, D. Kita, C. Fanta, J. S. Stamler, and J. M.
asthma in relation to their atopic status and the treatment they             Drazen. 1995. Expired nitric oxide levels during treatment of acute
were receiving.                                                              asthma. Am. J. Respir. Crit. Care Med. 152:800–803.
                                                                        5. Persson, M. G., O. Zetterstrom, V. Agrenius, E. Ihre, and L. E. Gustafs-
                                                                             son. 1994. Single breath nitric oxide measurements in asthmatics pa-
nonatopic children, but these differences were explained by                  tients and smokers. Lancet 343:146–147.
                                                                        6. Kharitonov, S. A., F. Chung, D. Evans, B. J. O’Connor, and P. J. Barnes.
each child’s asthma status since nonasthmatic children had                   1996. Increased exhaled nitric oxide in asthma is mainly derived from
similar eNO levels whether atopic or nonatopic.                              the lower respiratory tract. Am. J. Respir. Crit. Care Med. 153:1773–
   Children with positive exercise test results had higher eNO               1780.
than did those with negative exercise test results, but the im-         7. Dotsch, J., S. Demirakca, H. G. Terbrack, G. Huls, W. Rascher, and
portant cofactor was atopic status, with the highest values be-              P. G. Kuhl. 1996. Airway nitric oxide in asthmatic children and pa-
ing seen in atopic asthmatics.                                               tients with cystic fibrosis. Eur. Respir. J. 9:2537–2540.
                                                                        8. Nelson, B. V., S. Sears, J. Woods, C. L. Ling, J. Hunt, L. M. Clapper, and
   Children were not asked to stop their medication prior to                 B. Gaston. 1997. Expired nitric oxide as a marker for childhood
the review, and this could have affected our findings. If pre-               asthma. J. Pediatr. 130:423–427.
ventative asthma treatment lowered eNO levels, as suggested             9. Frank, P. I., S. Ferry, T. Moorhead, and P. Hannaford. 1996. Use of a
by other workers (16, 17), we might expect the differences be-               postal questionnaire to estimate the likely under-diagnosis of asthma-
tween asthmatic and nonasthmatic children to be less marked.                 like illness in adults. Br. J. Gen. Pract. May:295–297.
We found that atopic asthmatic children, whether receiving             10. Frank, P. I., S. Ferry, and P. Hannaford. 1996. The use of a postal ques-
                                                                             tionnaire to estimate the likely underdiagnosis of asthma in children
preventative asthma medication or not, had higher eNO levels                 living in South Manchester UK. Eur. Respir. J. 8(Suppl. 19):284s.
than did nonatopic asthmatic children (Figure 3) and that,             11. Wright, T., T. L. Frank, P. I. Frank, S. Hirsh, and P. Hannaford. 1996. A
overall, children with probable asthma receiving preventative                questionnaire to identify adult patients with possible asthma and to
treatment had higher eNO levels than did those who were not.                 estimate the rate of potential underdiagnosis. Eur. Respir. J. 9(Suppl.
This observation may reflect differences in the severity and di-             23):117s.
agnosis of asthma in the community, children with more se-             12. Keil, U., and S. Wieland. 1992. International asthma and allergy study.
                                                                             Lancet 340:46.
vere asthma perhaps being more likely to have their asthma             13. Adisesh, A., S. A. Kharitonov, D. H. Yates, D. C. Snashall, A. J. New-
diagnosed and therefore given preventative treatment than                    man Taylor, and P. J. Barnes. 1998. Exhaled and nasal nitric oxide is
those with less severe asthma. Alternatively, it may reflect in-             increased in laboratory animal allergy. Clin. Exp. Allergy (In press)
adequate compliance with the preventative treatment in the             14. Tang, C., L. M. Rolland, C. Ward, B. Quan, and E. H. Walters. 1997.
asthmatics studied in this population. There were no nonasth-                IL-5 production by bronchoalveolar lavage and peripheral blood
matic children receiving asthma medication and only one                      mononuclear cells in asthma and atopy. Eur. Respir. J. 10:624–632.
                                                                       15. Humbert, M., S. R. Durham, S. Ying, P. Kimmitt, J. Barkans, B. Assoufi,
atopic nonasthmatic child was receiving nasal steroids.                      R. Pfister, G. Menz, D. S. Robinson, A. B. Kay, and C. J. Corrigan.
   We used a consensus diagnosis of asthma based on three                    1996. IL-4 and IL-5 and protein in bronchial biopsies from patients
consultant pediatricians reviewing all the information, except               with atopic and nonatopic asthma: evidence against “intrinsic” asthma
the NO levels, available after a clinical review. By this ap-                being a distinct immunopathologic entity. Am. J. Respir. Crit. Care
proach we tried to replicate the type of diagnostic information              Med. 154:1497–1504.
that would be available to a consultant when seeing a child            16. Kharitonov, S. A., D. Yates, and P. J. Barnes. 1996. Inhaled glucocorti-
                                                                             coids decrease nitric oxide in exhaled air of asthmatic patients. Am. J.
referred by their general practitioner for diagnostic opinion.               Respir. Crit. Care Med. 153:454–457.
In the study, however, the children were more likely to be             17. Kharitonov, S. A., D. Yates, K. F. Chung, and P. J. Barnes. 1996.
asymptomatic at the time of their clinical assessment than if                Changes in the dose of inhaled steroid affect exhaled nitric oxide lev-
they were attending a new patient clinic. The clinical informa-              els in asthmatic patients. Eur. Respir. J. 9:196–201.
1036                                     AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE   VOL 158   1998

                                                 APPENDIX 1
                                         CHILDREN’S QUESTIONNAIRE

       (To be completed by the parent or Guardian. Please tick the appropriate box)
       What is your child’s date of birth?                              __/__/__
        1. Has your child had wheezing or whistling in the chest
           in the last 12 months?                                                     NO__    YES__

        2. How many attacks of wheezing has your child had
           in the last 12 months?
                                               i) None                                           __
                                               ii) 1 to 3                                        __
                                               iii) 4 to 12                                      __
                                               iv) More than 12                                  __

        3. In the last 12 months, how often, on average, has
           your child’s sleep been disturbed due to wheezing?
                                                   i) Never woken with wheezing                  __
                                                   ii) Less than one night per week              __
                                                   iii) One or two nights per week               __
                                                   iv) More than two nights per week             __

        4. In the last 12 months, has wheezing ever been severe
           enough to limit your child’s speech to only one or two
           words at a time between breaths?                                           NO__    YES__

        5. Has your child been woken by an attack of wheezing
           in the last 12 months?                                                     NO__    YES__

        6. In the last 12 months, has your child’s chest sounded
           wheezy during or after exercise?                                           NO__    YES__

        7. In the last 12 months, has your child had a dry cough at night,
           apart from a cough associated with a cold or chest infection?              NO__    YES__

        8. Has your child had more than 3 courses of antibiotics for respiratory
           infections (chest, ears or throat) in the last 12 months?                  NO__    YES__

        9. Is your child currently taking any medicine (including inhalers,
           aerosols or tablets) for asthma?                                           NO__    YES__

       10. Has your child had an attack of asthma in the last 12 months?              NO__    YES__

       11. Has your child had hay fever or eczema?                                    NO__    YES__

       12. Has anyone in your child’s family (parents, grandparents,
           sisters or brothers) had asthma?                                           NO__    YES__

                               THANK YOU FOR YOUR HELP

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