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Time-Dependent Effect of Nitrate-Rich Meals on Exhaled Nitric Oxide in Healthy Subjects
Time-Dependent Effect of Nitrate-Rich Meals on Exhaled Nitric Oxide in Healthy Subjects* Anne-Marie Vints, MD; Ellie Oostveen, PhD; Guy Eeckhaut; Mieke Smolders; and Wilfried A. De Backer, MD
Background: Exhaled nitric oxide (eNO) is a convenient noninvasive marker for airway inflammation in several pulmonary diseases. However, external factors such as nitrate-rich nutrition can affect the levels of eNO and thus compromise its diagnostic value. Study objectives: The objective of this investigation was to have a better understanding of the time-dependent effect of nitrate-rich meals on eNO in healthy subjects. Study design: Forty-two healthy, nonsmoking volunteers (age range, 25 to 62 years) were recruited for the study. They had no recent respiratory tract infections and were free of pulmonary history, rhinitis, and atopic disorders. eNO was measured before, and 0.5, 2, 4, 12, 15, and 20 h after the intake of a nitrate-rich meal equivalent to 230 mg of nitrate. Results: The intake of a nitrate-rich meal increased eNO by 60% 2 h after the meal. Even after 15 h, the mean eNO value was still 22% higher than the baseline value. Only after 20 h did eNO return to the normal baseline level. Conclusion: This finding stresses the importance of advising patients to avoid nitrate-rich nutrition at least 20 h before a scheduled measurement of eNO. (CHEST 2005; 128:2465–2470) Key words: exhaled nitric oxide; nitrate-rich meals; time dependence Abbreviations: eNO ⫽ exhaled nitric oxide; NO ⫽ nitric oxide; ppb ⫽ parts per billion
the past decade, the research of noninvasive I nmarkers to monitor airway inflammation has ex-
panded enormously. Of all noninvasive markers, exhaled nitric oxide (eNO) has been studied most extensively, especially in asthmatic patients.1 eNO has also been researched in other lung diseases involving bronchial inflammation such as COPD2 and bronchiectasis,3 and in some interstitial lung diseases such as cryptogenic fibrosing alveolitis.4 eNO also proved its use as an important inflammation parameter in the follow-up of patients after lung transplantation and more specific as an early marker of pulmonary graft dysfunction.5 Verleden et al6 showed that eNO can even be valuable in guiding the treatment of chronic rejection after a lung transplantation. The reason for this broad application of eNO is that eNO is easy to measure, it is highly reproducible in *From the Department of Respiratory Medicine, University Hospital Antwerp, Belgium. Manuscript received October 26, 2004; revision accepted March 7, 2005. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (www.chestjournal. org/misc/reprints.shtml). Correspondence to: Anne-Marie Vints, MD, Department of Respiratory Medicine, University Hospital Antwerp, Wilrijkstraat, 10, Edegem 2650, Belgium; e-mail: [email protected] www.chestjournal.org
normal subjects,7 and it shows no diurnal variation.8 However, some external factors can influence eNO. If eNO is used to monitor inflammation, it is important to know the influence of these external factors. One of these disturbing factors is nitrate-rich food, already studied by Olin and coworkers,9 who measured eNO in healthy subjects up to 3 h after the intake of a nitrate-rich meal and showed a median increase in eNO of 43% while eNO levels were not yet returned to baseline 3 h after the meal.9 A complete view of the long-lasting time effect of nitrate-rich meals is not known, so diet restrictions are not yet included in the guidelines for eNO measurements. The aim of this work was to examine in closer detail the influence, as function of time, of a nitrate-rich meal on the level of eNO in healthy adults. Our results imply that guidelines for eNO measurements should indeed include restrictions on the intake of nitrate-rich meals at least 20 h prior to the measurement. Materials and Methods Study Subjects The test persons were 42 healthy, nonsmoking volunteers (24 women and 18 men; age range, 24 to 61 years). They were free CHEST / 128 / 4 / OCTOBER, 2005
2465
of pulmonary history, rhinitis, and atopic disorders, and were without recent respiratory tract infections. The Medical Ethics Committee of the University Hospital of Antwerp approved the study. Study Design eNO levels of the test persons were measured before, and 0.5, 2, 4, 12, 15, and 20 h after the intake of a nitrate-rich meal. The “standard” nitrate-rich meal given to the subjects consisted of 100 g of lettuce and 50 g of radishes equivalent to approximately 230 mg of nitrate. All volunteers were instructed to avoid nitrate-rich nutrients 12 h prior to the baseline eNO measurement, performed in the morning. At approximately noon, 28 volunteers consumed our standard nitrate-rich meal and their eNO levels were measured 0.5, 2, 4, and 20 h after that meal. Twenty subjects had the standard nitrate-rich meal at approximately 8:00 pm, and their eNO values were recorded 12 h and 15 h later the next day. Under this schedule, eNO measurements during the night could be avoided. For practical reasons related to the daily professional activities of the volunteers, it was not possible to record eNO values at all the defined time lags for every subject. The sampled eNO values of all the volunteers were subclassified in seven subsets. The first subset, hereafter named as the reference set, contained the eNO values of all the 42 volunteers before they had eaten the nitrate-rich meal. The next six subsets contained eNO data of a variable number of subjects recorded at 0.5 h (n ⫽ 26), 2 h (n ⫽ 23), 4 h (n ⫽ 28), 12 h (n ⫽ 20), 15 h (n ⫽ 17), and 20 h (n ⫽ 18) after the consumption of the nitrate-rich meal.
Methods The eNO concentration was measured on-line (Niox Nitric Oxide Analyser; Aerocrine; Stockholm, Sweden) according to American Thoracic Society recommendations.10 The subjects were measured seated. After full exhalation, the subjects inhaled nitric oxide (NO)-free air from the apparatus to total lung capacity and then immediately exhaled slowly at a constant flow rate of 50 mL/s. The exhalation breathing maneuver lasted for 10 s, and the measurement was accepted when there was a NO plateau during the last 3 s of the measurement. The exhalation was performed against a fixed external resistance that ensured a positive mouth pressure of 5 to 20 cm H2O so that the soft palate was closed to prevent contamination with nasal NO. A visual biofeedback helped the subjects to achieve the desired expiratory flow of 50 mL/s (⫾ 10%). The maneuver was repeated with 30 s of relaxed breathing between the measurements until three reproducible NO values were obtained.10 The mean NO value of these three maneuvers was used for further analysis. Statistical Analysis The eNO values of the volunteers were log-normally distributed as already published by other authors,11 so the analysis of the eNO data was carried out on log10-transformed data. The distribution of the log10 (eNO) values in the seven subsets were tested for normality using the Shapiro-Wilk test as shown in Figure 1. The log10-transformed eNO data of all the subsets were normally distributed except the data set recorded after 15 h. However, an unpaired, two-tailed t test can tolerate a modest skewness in the population distribution.12 The averages of the
Figure 1. Normal plots of the log10 (eNO) values in the seven different subsets. According to the Shapiro-Wilk test, all subsets are normally distributed except the subset of the eNO data recorded after 15 h. 2466
Clinical Investigations
log10 (eNO) values of each of the seven subsets were compared with the average of the reference log10 (eNO) values by an unpaired, two tailed, t test at a 95% confidence level or a p level ⱕ 0.05.
Results The eNO levels of the men and women in the study group are given in Table 1. Mean log10 (eNO) levels were 1.24 for men (n ⫽ 18) and 1.21 for women (n ⫽ 24), corresponding to average eNO values of, respectively, 17.4 parts per billion (ppb) and 16.2 ppb eNO. The results of the unpaired, two-tailed, t test comparisons of the 0.5-h, 2-h, 4-h, 12-h, 15-h, and 20-h data with the reference set are summarized in Table 2. A statistically significant increase in eNO was found at 0.5 h, 2 h, 4 h, 12 h, and 15 h after the intake of a nitrate-rich meal. The relative increases of mean eNO values compared to the reference set were 45% (after 0.5 h), 62% (after 2 h), 47% (after 4 h), 30% (after 12 h), and 22% (after 15 h). The initial increase and the following decay of eNO are illustrated in Figure 2. Twenty hours after the meal, eNO levels were again back to normal. Discussion Our study clearly illustrates that the intake of a nitrate-rich meal may disturb eNO measurements for almost 20 h. The standardized procedures for the measurements of exhaled NO given by the European Respiratory Society13 and the American Thoracic Society10 do not include dietary restrictions, and they may well be adapted based on our findings. eNO was for the first time described in the air of healthy subjects in 1991 by Gustafsson et al.14 Two
Table 1—Baseline Data of eNO Levels in Healthy Men and Women
Variables Men (n ⫽ 18) Mean 95% confidence interval SD Women (n ⫽ 24) Mean 95% confidence interval SD Men and women (n ⫽ 42) Mean 95% confidence interval SD
www.chestjournal.org
Log10Transformed eNO, ppb
BackTransformed eNO, ppb
1.24 1.17–1.31 0.138
17.4 14.8–20.4
1.21 1.15–1.28 0.148
16.2 14.3–19.1
1.22 1.18–1.27 0.145
16.6 15.1–18.6
years later, in 1993, Alving et al15 described that NO was increased in the air exhaled by patients with asthma. Now, 14 years later, eNO is generally used as a marker of several inflammatory airway disorders, in particular asthma. Many studies have shown the importance of eNO in the follow-up of asthma patients and confirm that the results of eNO measurements are closely related to indirect indexes of asthma control such as symptom scores within the past 2 weeks, dyspnea score, and daily use of rescue medication.16 Although these indexes reflect the clinical control of asthma in the patients, they are all indirect indexes of airway inflammation. Direct indexes of airway inflammation are also related with eNO. eNO correlates with the quantity of eosinophils in bronchial biopsies in patients with atopic asthma17 and with an increased number of eosinophils in the induced sputum of asthma patients.18 In the follow-up of asthma patients, eNO reflects the evolution of the airway inflammation when starting, reducing, or stopping a treatment. When a treatment with inhaled glucocorticoids is started, eNO is reduced.19 However, when inhaled glucocorticoids were withdrawn in patients with mild-tomoderate asthma, eNO measurements were highly predictive (80 to 90%) for loss of their asthma control.20 The increase of eNO during an asthma exacerbation due to reduction in steroids may be detected even before a decrease in the pulmonary function tests occurs.21 Studies have also shown that the measurement of eNO in patients with undiagnosed chronic respiratory symptoms can be predictive for a diagnosis of asthma22 and that eNO correlates with asthma severity. eNO was significantly higher in patients with severe asthma compared with patients with mild asthma.23 A limitation in the use of eNO as inflammation marker in asthma is that it is a good marker in steroid-naı¨ve asthma patients but probably not so useful in asthma patients who are already treated with inhaled steroids.24 Beside asthma, eNO has also diagnostic value in the follow-up of patients after lung transplantation. In a number of other lung diseases, eNO is researched as a parameter of inflammation. eNO is a noninvasive and easy-to-perform test that can be regularly repeated. Earlier investigation has also proved that there is no evidence for a diurnal variation of eNO and that eNO is reproducible in normal subjects.8,25 Apart of all these advantages, there are also confounding factors in the use of eNO as a parameter of inflammation in lung diseases. The confounding factors in the interpretation of eNO can be classified in two groups. The first group consists of other CHEST / 128 / 4 / OCTOBER, 2005
2467
Table 2—Log10-Transformed and Back-Transformed eNO Data Measured Before and at Different Time Lags After a Nitrate-Rich Meal Together With the Results of the Unpaired, Two-Tailed t Test on the Log10-Transformed Data Time of eNO Measurement Baseline After 0.5 h After 2 h After 4 h After 12 h After 15 h After 20 h
Log10-Transformed eNO, ppb
Back-Transformed eNO, ppb
Average
SD
No.
p Value*
Average
⫾ 2 SD
Relative Increase, %
1.224 1.386 1.434 1.390 1.336 1.312 1.237
0.145 0.188 0.134 0.148 0.126 0.137 0.156
42 26 23 28 20 18 18
0.00017 ⬍ 0.000001 0.00016 0.005 0.033 0.77
16.6 24.3† 27.2† 24.6† 21.7† 20.5† 17.3
8.6–32.6 10.2–57.8 1.7–50.4 1.7–48.6 1.6–38.7 1.6–38.6 1.6–35.3
⫹ 45 ⫹ 62 ⫹ 47 ⫹ 30 ⫹ 22 ⫹3
*Significance of the change compared to the baseline set from an unpaired, two-tailed t test; p values ⱕ 0.01 are commonly considered as indicating a statistically significant difference between the test set and the reference set. †Statistically significant deviations from the baseline eNO value.
inflammatory diseases of the upper or lower airways, which can increase eNO like allergic rhinitis,26 viral airway infections,27 and pneumonia.28 The second group of confounding factors consists of external factors affecting the level of eNO. Factors that reduce the level of eNO are the consumption of caffeine in healthy persons29 (however, a recent study30 showed no effect on eNO in patients with asthma), smoking,31 a sputum induction procedure,32
and consumption of alcohol by asthma patients.33 An external factor that increases the level of eNO is the consumption of a nitrate-rich meal.9 The influence of this particular disturbing factor on the interpretation of eNO measurements was the main subject of this study. Vegetables are important sources of nitrate in our daily Western diet and are regularly used by our patients. Some examples of nitrate-rich food are lettuce, radishes, spinach, parsley, green cab-
Figure 2. The distribution characteristics of the log10 (eNO) values at different time lags after a nitrate-rich meal. Thirty minutes after the meal, the log10 (eNO) value is elevated, and the increase in log10 (eNO) is maximal after 2 h. After this, the effect begins to decay. After 20 h, the log10 (eNO) value is back to normal. The small square in the plot represents the mean (central tendency) log10 eNO value, while the dispersion (variability) is represented by ⫾ 1 times the SE (large box) and ⫾ 1 times the SD about the mean (“whiskers”). 2468
Clinical Investigations
bage, and rhubarb. The concentration of nitrate in different sorts of vegetables (eg, in lettuce and in spinach) fluctuates significantly. The differences are mostly related to the season and weather conditions in which the vegetables are grown but also the fact whether the vegetables are protected or outdoor grown influences their nitrate concentration.34 Our study examined in closer detail the longer time scale over which a nitrate rich-meal influences the level of eNO in healthy adults. It is known that 2 h after a nitrate-rich meal, the increase of eNO is maximal and that the effect still persists 3 h after the meal.9 The remaining practical question of how long this influence persists was not yet answered. By knowing the exact period over which nitrate-rich meals influence eNO, better advice can be given on restricting the intake of nitrate-rich food prior to eNO measurements. The statistical analysis of the eNO measurements confirms that the intake of a nitrate-rich meal, equivalent with 230-mg nitrate, definitely increases eNO values. The NO increase is maximal, ⬎ 60%, 2 h after the meal. This finding is in agreement with Olin et al,9 who also reported a maximal increase of eNO 2 h after ingestion of nitrate-rich nutrients, although via a different measurement technique. The effect lasts for at least 15 h. In conclusion, besides confirming the short-term effect of nitrate-rich food on eNO, the longer time effect is now mapped out clearly. Nitrate-rich meals induce a maximal increase of eNO after 2 h, but after 15 h the increase is still significant. Only after 20 h is the eNO value definitely back to normal. This finding has consequences for routine clinical practice and for the design and interpretation of clinical studies in which eNO is used as an inflammation parameter. It stresses the importance of informing patients to refrain from nitrate-rich meals at least 20 h prior to a scheduled measurement of eNO.
References 1 Dupont LJ, Demedts MG, Verleden GM. Prospective evaluation of the validity of exhaled nitric oxide for the diagnosis of asthma. Chest 2003; 123:751–756 2 Sterk PJ, De Gouw HW, Ricciardolo FL, et al. Exhaled nitric oxide in COPD: glancing through a smoke screen. Thorax 1999; 54:565–567 3 Kharitonov SA, Wells AU, O’Connor BJ, et al. Elevated levels of exhaled nitric oxide in bronchiectasis. Am J Respir Crit Care Med 1995; 151:1889 –1893 4 Paredi P, Kharitonov SA, Loukides S, et al. Exhaled nitric oxide is increased in active fibrosing alveolitis. Chest 1999; 115:1352–1356 5 Fisher AJ, Gabbay E, Small T, et al. Cross sectional study of exhaled nitric oxide levels following lung transplantation. Thorax 1998; 53:454 – 458 www.chestjournal.org
6 Verleden GM, Dupont LJ, Van Raemdonck D, et al. Effect of switching from cyclosporine to tacrolimus on exhaled nitric oxide and pulmonary function in patients with chronic rejection after lung transplantation. J Heart Lung Transplant 2003; 22:908 –913 7 Purokivi M, Randell J, Hirvonen MR, et al. Reproducibility of measurements of exhaled NO, and cell count and cytokine concentrations in induced sputum. Eur Respir J 2000; 16: 242–246 8 Kharitonov SA, Gonio F, Kelly C, et al. Reproducibility of exhaled nitric oxide measurements in healthy and asthmatic adults and children. Eur Respir J 2003; 21:433– 438 9 Olin AC, Aldenbratt A, Ekman A, et al. Increased nitric oxide in exhaled air after intake of a nitrate-rich meal. Respir Med 2001; 95:153–158 10 Recommendations for standardized procedures for the online and off-line measurement of exhaled lower respiratory nitric oxide and nasal nitric oxide in adults and children-1999. This official statement of the American Thoracic Society was adopted by the ATS Board of Directors, July 1999. Am J Respir Crit Care Med 1999; 160:2104 –2117 11 van der Lee I, van den Bosch JM, Zanen P. Reduction of variability of exhaled nitric oxide in healthy volunteers. Respir Med 2002; 96:1014 –1020 12 Ott RL. An introduction to statistical methods and data analysis. North Scituate, MA: Duxbury Press, 1988; 174 13 Kharitonov S, Alving K, Barnes PJ. Exhaled and nasal nitric oxide measurements: recommendations. The European Respiratory Society Task Force. Eur Respir J 1997; 10:1683– 1693 14 Gustafsson LE, Leone AM, Persson MG, et al. Endogenous nitric oxide is present in the exhaled air of rabbits, guinea pigs and humans. Biochem Biophys Res Commun 1991; 181:852– 857 15 Alving K, Weitzberg E, Lundberg JM. Increased amount of nitric oxide in exhaled air of asthmatics. Eur Respir J 1993; 6:1368 –1370 16 Sippel JM, Holden WE, Tilles SA, et al. Exhaled nitric oxide levels correlate with measures of disease control in asthma. J Allergy Clin Immunol 2000; 106:645– 650 17 van den Toorn LM, Overbeek SE, de Jongste JC, et al. Airway inflammation is present during clinical remission of atopic asthma. Am J Respir Crit Care Med 2001; 164:2107–2113 18 Jones SL, Herbison P, Cowan JO, et al. Exhaled NO and assessment of anti-inflammatory effects of inhaled steroid: dose-response relationship. Eur Respir J 2002; 20:601– 608 19 Kharitonov SA, Yates DH, Barnes PJ. Inhaled glucocorticoids decrease nitric oxide in exhaled air of asthmatic patients. Am J Respir Crit Care Med 1996; 153:454 – 457 20 Jones SL, Kittelson J, Cowan JO, et al. The predictive value of exhaled nitric oxide measurements in assessing changes in asthma control. Am J Respir Crit Care Med 2001; 164:738 – 743 21 Kharitonov SA, Yates DH, Chung KF, et al. Changes in the dose of inhaled steroid affect exhaled nitric oxide levels in asthmatic patients. Eur Respir J 1996; 9:196 –201 22 Smith AD, Cowan JO, Filsell S, et al. Diagnosing asthma: comparisons between exhaled nitric oxide measurements and conventional tests. Am J Respir Crit Care Med 2004; 169: 473– 478 23 Delgado-Corcoran C, Kissoon N, Murphy SP, et al. Exhaled nitric oxide reflects asthma severity and asthma control. Pediatr Crit Care Med 2004; 5:48 –52 24 Tsujino I, Nishimura M, Kamachi A, et al. Exhaled nitric oxide: is it really a good marker of airway inflammation in bronchial asthma? Respiration 2000; 67:645– 651 25 Ekroos H, Tuominen J, Sovijarvi AR. Exhaled nitric oxide and CHEST / 128 / 4 / OCTOBER, 2005
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26
27 28 29 30
its long-term variation in healthy non-smoking subjects. Clin Physiol 2000; 20:434 – 439 Henriksen AH, Sue-Chu M, Lingaas HT, et al. Exhaled and nasal NO levels in allergic rhinitis: relation to sensitization, pollen season and bronchial hyperresponsiveness. Eur Respir J 1999; 13:301–306 Murphy AW, Platts-Mills TA, Lobo M, et al. Respiratory nitric oxide levels in experimental human influenza. Chest 1998; 114:452– 456 Adrie C, Monchi M, Dinh-Xuan AT, et al. Exhaled and nasal nitric oxide as a marker of pneumonia in ventilated patients. Am J Respir Crit Care Med 2001; 163:1143–1149 Bruce C, Yates DH, Thomas PS. Caffeine decreases exhaled nitric oxide. Thorax 2002; 57:361–363 Taylor ES, Smith AD, Cowan JO, et al. Effect of caffeine
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31 32 33 34
ingestion on exhaled nitric oxide measurements in patients with asthma. Am J Respir Crit Care Med 2004; 169:1019 – 1021 Kharitonov SA, Robbins RA, Yates D, et al. Acute and chronic effects of cigarette smoking on exhaled nitric oxide. Am J Respir Crit Care Med 1995; 152:609 – 612 Piacentini GL, Bodini A, Costella S, et al. Exhaled nitric oxide is reduced after sputum induction in asthmatic children. Pediatr Pulmonol 2000; 29:430 – 433 Yates DH, Kharitonov SA, Robbins RA, et al. The effect of alcohol ingestion on exhaled nitric oxide. Eur Respir J 1996; 9:1130 –1133 Ysart G, Clifford R, Harrison N. Monitoring for nitrate in UK-grown lettuce and spinach. Food Addit Contam 1999; 16:301–306
Clinical Investigations
Background: Exhaled nitric oxide (eNO) is a convenient noninvasive marker for airway inflammation in several pulmonary diseases. However, external factors such as nitrate-rich nutrition can affect the levels of eNO and thus compromise its diagnostic value. Study objectives: The objective of this investigation was to have a better understanding of the time-dependent effect of nitrate-rich meals on eNO in healthy subjects. Study design: Forty-two healthy, nonsmoking volunteers (age range, 25 to 62 years) were recruited for the study. They had no recent respiratory tract infections and were free of pulmonary history, rhinitis, and atopic disorders. eNO was measured before, and 0.5, 2, 4, 12, 15, and 20 h after the intake of a nitrate-rich meal equivalent to 230 mg of nitrate. Results: The intake of a nitrate-rich meal increased eNO by 60% 2 h after the meal. Even after 15 h, the mean eNO value was still 22% higher than the baseline value. Only after 20 h did eNO return to the normal baseline level. Conclusion: This finding stresses the importance of advising patients to avoid nitrate-rich nutrition at least 20 h before a scheduled measurement of eNO. (CHEST 2005; 128:2465–2470) Key words: exhaled nitric oxide; nitrate-rich meals; time dependence Abbreviations: eNO ⫽ exhaled nitric oxide; NO ⫽ nitric oxide; ppb ⫽ parts per billion
the past decade, the research of noninvasive I nmarkers to monitor airway inflammation has ex-
panded enormously. Of all noninvasive markers, exhaled nitric oxide (eNO) has been studied most extensively, especially in asthmatic patients.1 eNO has also been researched in other lung diseases involving bronchial inflammation such as COPD2 and bronchiectasis,3 and in some interstitial lung diseases such as cryptogenic fibrosing alveolitis.4 eNO also proved its use as an important inflammation parameter in the follow-up of patients after lung transplantation and more specific as an early marker of pulmonary graft dysfunction.5 Verleden et al6 showed that eNO can even be valuable in guiding the treatment of chronic rejection after a lung transplantation. The reason for this broad application of eNO is that eNO is easy to measure, it is highly reproducible in *From the Department of Respiratory Medicine, University Hospital Antwerp, Belgium. Manuscript received October 26, 2004; revision accepted March 7, 2005. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (www.chestjournal. org/misc/reprints.shtml). Correspondence to: Anne-Marie Vints, MD, Department of Respiratory Medicine, University Hospital Antwerp, Wilrijkstraat, 10, Edegem 2650, Belgium; e-mail: [email protected] www.chestjournal.org
normal subjects,7 and it shows no diurnal variation.8 However, some external factors can influence eNO. If eNO is used to monitor inflammation, it is important to know the influence of these external factors. One of these disturbing factors is nitrate-rich food, already studied by Olin and coworkers,9 who measured eNO in healthy subjects up to 3 h after the intake of a nitrate-rich meal and showed a median increase in eNO of 43% while eNO levels were not yet returned to baseline 3 h after the meal.9 A complete view of the long-lasting time effect of nitrate-rich meals is not known, so diet restrictions are not yet included in the guidelines for eNO measurements. The aim of this work was to examine in closer detail the influence, as function of time, of a nitrate-rich meal on the level of eNO in healthy adults. Our results imply that guidelines for eNO measurements should indeed include restrictions on the intake of nitrate-rich meals at least 20 h prior to the measurement. Materials and Methods Study Subjects The test persons were 42 healthy, nonsmoking volunteers (24 women and 18 men; age range, 24 to 61 years). They were free CHEST / 128 / 4 / OCTOBER, 2005
2465
of pulmonary history, rhinitis, and atopic disorders, and were without recent respiratory tract infections. The Medical Ethics Committee of the University Hospital of Antwerp approved the study. Study Design eNO levels of the test persons were measured before, and 0.5, 2, 4, 12, 15, and 20 h after the intake of a nitrate-rich meal. The “standard” nitrate-rich meal given to the subjects consisted of 100 g of lettuce and 50 g of radishes equivalent to approximately 230 mg of nitrate. All volunteers were instructed to avoid nitrate-rich nutrients 12 h prior to the baseline eNO measurement, performed in the morning. At approximately noon, 28 volunteers consumed our standard nitrate-rich meal and their eNO levels were measured 0.5, 2, 4, and 20 h after that meal. Twenty subjects had the standard nitrate-rich meal at approximately 8:00 pm, and their eNO values were recorded 12 h and 15 h later the next day. Under this schedule, eNO measurements during the night could be avoided. For practical reasons related to the daily professional activities of the volunteers, it was not possible to record eNO values at all the defined time lags for every subject. The sampled eNO values of all the volunteers were subclassified in seven subsets. The first subset, hereafter named as the reference set, contained the eNO values of all the 42 volunteers before they had eaten the nitrate-rich meal. The next six subsets contained eNO data of a variable number of subjects recorded at 0.5 h (n ⫽ 26), 2 h (n ⫽ 23), 4 h (n ⫽ 28), 12 h (n ⫽ 20), 15 h (n ⫽ 17), and 20 h (n ⫽ 18) after the consumption of the nitrate-rich meal.
Methods The eNO concentration was measured on-line (Niox Nitric Oxide Analyser; Aerocrine; Stockholm, Sweden) according to American Thoracic Society recommendations.10 The subjects were measured seated. After full exhalation, the subjects inhaled nitric oxide (NO)-free air from the apparatus to total lung capacity and then immediately exhaled slowly at a constant flow rate of 50 mL/s. The exhalation breathing maneuver lasted for 10 s, and the measurement was accepted when there was a NO plateau during the last 3 s of the measurement. The exhalation was performed against a fixed external resistance that ensured a positive mouth pressure of 5 to 20 cm H2O so that the soft palate was closed to prevent contamination with nasal NO. A visual biofeedback helped the subjects to achieve the desired expiratory flow of 50 mL/s (⫾ 10%). The maneuver was repeated with 30 s of relaxed breathing between the measurements until three reproducible NO values were obtained.10 The mean NO value of these three maneuvers was used for further analysis. Statistical Analysis The eNO values of the volunteers were log-normally distributed as already published by other authors,11 so the analysis of the eNO data was carried out on log10-transformed data. The distribution of the log10 (eNO) values in the seven subsets were tested for normality using the Shapiro-Wilk test as shown in Figure 1. The log10-transformed eNO data of all the subsets were normally distributed except the data set recorded after 15 h. However, an unpaired, two-tailed t test can tolerate a modest skewness in the population distribution.12 The averages of the
Figure 1. Normal plots of the log10 (eNO) values in the seven different subsets. According to the Shapiro-Wilk test, all subsets are normally distributed except the subset of the eNO data recorded after 15 h. 2466
Clinical Investigations
log10 (eNO) values of each of the seven subsets were compared with the average of the reference log10 (eNO) values by an unpaired, two tailed, t test at a 95% confidence level or a p level ⱕ 0.05.
Results The eNO levels of the men and women in the study group are given in Table 1. Mean log10 (eNO) levels were 1.24 for men (n ⫽ 18) and 1.21 for women (n ⫽ 24), corresponding to average eNO values of, respectively, 17.4 parts per billion (ppb) and 16.2 ppb eNO. The results of the unpaired, two-tailed, t test comparisons of the 0.5-h, 2-h, 4-h, 12-h, 15-h, and 20-h data with the reference set are summarized in Table 2. A statistically significant increase in eNO was found at 0.5 h, 2 h, 4 h, 12 h, and 15 h after the intake of a nitrate-rich meal. The relative increases of mean eNO values compared to the reference set were 45% (after 0.5 h), 62% (after 2 h), 47% (after 4 h), 30% (after 12 h), and 22% (after 15 h). The initial increase and the following decay of eNO are illustrated in Figure 2. Twenty hours after the meal, eNO levels were again back to normal. Discussion Our study clearly illustrates that the intake of a nitrate-rich meal may disturb eNO measurements for almost 20 h. The standardized procedures for the measurements of exhaled NO given by the European Respiratory Society13 and the American Thoracic Society10 do not include dietary restrictions, and they may well be adapted based on our findings. eNO was for the first time described in the air of healthy subjects in 1991 by Gustafsson et al.14 Two
Table 1—Baseline Data of eNO Levels in Healthy Men and Women
Variables Men (n ⫽ 18) Mean 95% confidence interval SD Women (n ⫽ 24) Mean 95% confidence interval SD Men and women (n ⫽ 42) Mean 95% confidence interval SD
www.chestjournal.org
Log10Transformed eNO, ppb
BackTransformed eNO, ppb
1.24 1.17–1.31 0.138
17.4 14.8–20.4
1.21 1.15–1.28 0.148
16.2 14.3–19.1
1.22 1.18–1.27 0.145
16.6 15.1–18.6
years later, in 1993, Alving et al15 described that NO was increased in the air exhaled by patients with asthma. Now, 14 years later, eNO is generally used as a marker of several inflammatory airway disorders, in particular asthma. Many studies have shown the importance of eNO in the follow-up of asthma patients and confirm that the results of eNO measurements are closely related to indirect indexes of asthma control such as symptom scores within the past 2 weeks, dyspnea score, and daily use of rescue medication.16 Although these indexes reflect the clinical control of asthma in the patients, they are all indirect indexes of airway inflammation. Direct indexes of airway inflammation are also related with eNO. eNO correlates with the quantity of eosinophils in bronchial biopsies in patients with atopic asthma17 and with an increased number of eosinophils in the induced sputum of asthma patients.18 In the follow-up of asthma patients, eNO reflects the evolution of the airway inflammation when starting, reducing, or stopping a treatment. When a treatment with inhaled glucocorticoids is started, eNO is reduced.19 However, when inhaled glucocorticoids were withdrawn in patients with mild-tomoderate asthma, eNO measurements were highly predictive (80 to 90%) for loss of their asthma control.20 The increase of eNO during an asthma exacerbation due to reduction in steroids may be detected even before a decrease in the pulmonary function tests occurs.21 Studies have also shown that the measurement of eNO in patients with undiagnosed chronic respiratory symptoms can be predictive for a diagnosis of asthma22 and that eNO correlates with asthma severity. eNO was significantly higher in patients with severe asthma compared with patients with mild asthma.23 A limitation in the use of eNO as inflammation marker in asthma is that it is a good marker in steroid-naı¨ve asthma patients but probably not so useful in asthma patients who are already treated with inhaled steroids.24 Beside asthma, eNO has also diagnostic value in the follow-up of patients after lung transplantation. In a number of other lung diseases, eNO is researched as a parameter of inflammation. eNO is a noninvasive and easy-to-perform test that can be regularly repeated. Earlier investigation has also proved that there is no evidence for a diurnal variation of eNO and that eNO is reproducible in normal subjects.8,25 Apart of all these advantages, there are also confounding factors in the use of eNO as a parameter of inflammation in lung diseases. The confounding factors in the interpretation of eNO can be classified in two groups. The first group consists of other CHEST / 128 / 4 / OCTOBER, 2005
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Table 2—Log10-Transformed and Back-Transformed eNO Data Measured Before and at Different Time Lags After a Nitrate-Rich Meal Together With the Results of the Unpaired, Two-Tailed t Test on the Log10-Transformed Data Time of eNO Measurement Baseline After 0.5 h After 2 h After 4 h After 12 h After 15 h After 20 h
Log10-Transformed eNO, ppb
Back-Transformed eNO, ppb
Average
SD
No.
p Value*
Average
⫾ 2 SD
Relative Increase, %
1.224 1.386 1.434 1.390 1.336 1.312 1.237
0.145 0.188 0.134 0.148 0.126 0.137 0.156
42 26 23 28 20 18 18
0.00017 ⬍ 0.000001 0.00016 0.005 0.033 0.77
16.6 24.3† 27.2† 24.6† 21.7† 20.5† 17.3
8.6–32.6 10.2–57.8 1.7–50.4 1.7–48.6 1.6–38.7 1.6–38.6 1.6–35.3
⫹ 45 ⫹ 62 ⫹ 47 ⫹ 30 ⫹ 22 ⫹3
*Significance of the change compared to the baseline set from an unpaired, two-tailed t test; p values ⱕ 0.01 are commonly considered as indicating a statistically significant difference between the test set and the reference set. †Statistically significant deviations from the baseline eNO value.
inflammatory diseases of the upper or lower airways, which can increase eNO like allergic rhinitis,26 viral airway infections,27 and pneumonia.28 The second group of confounding factors consists of external factors affecting the level of eNO. Factors that reduce the level of eNO are the consumption of caffeine in healthy persons29 (however, a recent study30 showed no effect on eNO in patients with asthma), smoking,31 a sputum induction procedure,32
and consumption of alcohol by asthma patients.33 An external factor that increases the level of eNO is the consumption of a nitrate-rich meal.9 The influence of this particular disturbing factor on the interpretation of eNO measurements was the main subject of this study. Vegetables are important sources of nitrate in our daily Western diet and are regularly used by our patients. Some examples of nitrate-rich food are lettuce, radishes, spinach, parsley, green cab-
Figure 2. The distribution characteristics of the log10 (eNO) values at different time lags after a nitrate-rich meal. Thirty minutes after the meal, the log10 (eNO) value is elevated, and the increase in log10 (eNO) is maximal after 2 h. After this, the effect begins to decay. After 20 h, the log10 (eNO) value is back to normal. The small square in the plot represents the mean (central tendency) log10 eNO value, while the dispersion (variability) is represented by ⫾ 1 times the SE (large box) and ⫾ 1 times the SD about the mean (“whiskers”). 2468
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bage, and rhubarb. The concentration of nitrate in different sorts of vegetables (eg, in lettuce and in spinach) fluctuates significantly. The differences are mostly related to the season and weather conditions in which the vegetables are grown but also the fact whether the vegetables are protected or outdoor grown influences their nitrate concentration.34 Our study examined in closer detail the longer time scale over which a nitrate rich-meal influences the level of eNO in healthy adults. It is known that 2 h after a nitrate-rich meal, the increase of eNO is maximal and that the effect still persists 3 h after the meal.9 The remaining practical question of how long this influence persists was not yet answered. By knowing the exact period over which nitrate-rich meals influence eNO, better advice can be given on restricting the intake of nitrate-rich food prior to eNO measurements. The statistical analysis of the eNO measurements confirms that the intake of a nitrate-rich meal, equivalent with 230-mg nitrate, definitely increases eNO values. The NO increase is maximal, ⬎ 60%, 2 h after the meal. This finding is in agreement with Olin et al,9 who also reported a maximal increase of eNO 2 h after ingestion of nitrate-rich nutrients, although via a different measurement technique. The effect lasts for at least 15 h. In conclusion, besides confirming the short-term effect of nitrate-rich food on eNO, the longer time effect is now mapped out clearly. Nitrate-rich meals induce a maximal increase of eNO after 2 h, but after 15 h the increase is still significant. Only after 20 h is the eNO value definitely back to normal. This finding has consequences for routine clinical practice and for the design and interpretation of clinical studies in which eNO is used as an inflammation parameter. It stresses the importance of informing patients to refrain from nitrate-rich meals at least 20 h prior to a scheduled measurement of eNO.
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