sign in sign up
<<
Home , Exhaled nitric oxide

View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector

RESPIRATORY (2001) 95, 153–158 doi:10.1053/rmed.2000.1010, available online at http://www.idealibrary.com on

Increased in exhaled air after intake of a -rich meal

{ { A.-C. OLIN*,A.ALDENBRATT*,A.EKMAN*,G.LJUNGKVIST*,L.JUNGERSTEN ,K.ALVING AND } K. TORE´ N*

*Department of Occupational and Environmental Medicine, Sahlgrenska University Hospital, Go¨teborg, {Department of Clinical Physiology, Sahlgrenska University Hospital, Go¨teborg, {Department of Physiology and Pharmacology, Karolinska Institute, Stockholm and }Section of Respiratory Medicine and Allergology, Sahlgrenska University Hospital, Go¨teborg, Sweden

Exhaled and nasal NO (ENO, NNO) have been suggested as markers for inflammation in lower and upper respectively. It is still unknown how a number of factors, apart from airway inflammation, can influence NO levels. The aim of this study was to determine the effect of a nitrate-rich meal on ENO and NNO. Sixteen healthy subjects were observed during 1 week on normal diet before a nitrate-restricted diet was introduced in the next. On day 3 of the second week they were made to ingest a nitrate rich meal. ENO, NNO, plasma nitrate and plasma L- were followed before the meal and afterwards for 3 h. ENO and NNO as well as plasma nitrate and plasma L-arginine were significantly elevated after the nitrate-rich meal. The median maximal increase of ENO and NNO was 47% and 13% respectively. We found a moderate but significant correlation between the rise in plasma nitrate and ENO (rs¼0?57, P¼0?027) but none between plasma nitrate and NNO (rs¼70?02, P¼0?95). As nitrate in the diet seems to substantially influence the levels of ENO it is important either to restrict or register the intake of nitrate-rich food prior to measuring ENO.

Key words: nitric oxide; nasal nitric oxide; nitrate; ; L-arginine; breath analysis.

RESPIR.MED. (2001) 95, 153–158 # 2001 HARCOURT PUBLISHERS LTD

Introduction inflammation, having been shown to increase in patients with allergic rhinitis (5, 6) and during upper respiratory Nitric oxide in exhaled air (ENO) has been suggested as a tract infections (7). In the upper airways, very high levels of marker for airway inflammation (1,2), mainly on the basis nitric oxide are produced in the paranasal sinuses (8). of the observation that steroid-naive asthmatic patients However, the increased NO levels found in patients with have high levels of ENO, which readily decreases to normal allergic rhinitis are believed to originate mainly in the nasal levels after steroid treatment (3). If so, measurements of mucosa (6). ENO would offer a fast, easy and non-invasive procedure The method of choice for measurement of ENO has for instantaneous detection of airway inflammation, at least been developed over the past few years. In 1997 an in steroid-naive non-smoking patients. European Respiratory Society (ERS) task force (9) ENO seems to be representative for NO produced in the published guidelines for the performance of measure- airways (4). NO in blood entering alveolar space binds very ments, and recently guidelines for the standardization have quickly to haem, and will only contribute in a minor way to been published by the American Thoracic Society (ATS) the exhaled NO levels. (10). Knowledge of a number of factors, for instance diet, Although the knowledge of nasal NO (NNO) still is which could influence the ENO levels, is however still limited, NNO has been suggested as a marker for nasal lacking. Nitric oxide is formed enzymatically from L-arginine in Received 13 October 2000 and accepted 8 November 2000. the presence of NO synthase, which exists in at least three Correspondence should be addressed to: Dr Anna-Carin Olin, different isoforms. Nitric oxide is moreover also formed Department of Occupational and Environmental Medicine, Sahl- non-enzymatically, in the oral cavity (11) and in the gastric grenska University Hospital, St Sigfridsgatan 85 B, S-412 66 ventricle (12), by reduction of nitrite ðNO2 Þ, a reaction Go¨ teborg, Sweden. E-mail: [email protected] which is enhanced by an acidic environment. NO might,

0954-6111/01/020153+06 $35?00/0 # 2001 HARCOURT PUBLISHERS LTD 154 A.-C. OLIN ET AL. too, be formed non-enzymatically during oxidative stress, TABLE 1. Basic data on the subjects as proposed by Nagase et al. (13). Although it is well known that the intake of food with a Females (n=7) Males (n=9) high nitrate ðNO3 Þ content to a large extent influences blood nitrate levels, it is not clear how variations in plasma Age (years) 27 25 nitrate influence the exhaled NO levels. VC (% pred) 101 100 In the gut, the nitrate in the diet is absorbed into FEV (% pred) 101 102 the blood and actively transported to the saliva by 1 Weight (kg) 54 79 the salivary glands (14). In the oral cavity it is converted Phadiatop* 0/7 1/9 to nitrite by bacteria, mainly in the deep clefts on the rear part of the tongue. The balance that exist between NO and nitrite is regulated by the pH, i.e. the more *0=negative, 1=positive. acidic environment the more NO will be formed. Salivary nitrate and nitrite have been shown to increase with increasing doses of ingested nitrate (15), although The local committee of ethics approved the study and there has been shown to be a wide inter- informed consent was obtained from all volunteers prior to individual variability of conversion of nitrate to nitrite their inclusion in the study. (16). Zetterquist et al. (17) have investigated the effect of an intake of 400 mg potassium nitrate solution on ENO levels, which they found to increase by a maximum of 150% 2 h EXHALED AND NASAL NO after the ingestion. Concomitantly, they found an increase of nitrite in saliva following the pattern of its increase in Exhaled NO was measured during a slow single-exhalation plasma. Mouthwash with an anti-bacterial agent reduced for 20 sec against an oral pressure of 5 cm H2Oata NO release by almost 50%. They did not find any increase constant flow rate of 50 ml sec71, in accordance with ATS in nasal NO. recommendations (10). The measurements were performed Studies have not been made of the way L-arginine in triplicate at each occasion and the mean NO concentra- in the diet can influence exhaled or nasal NO levels. After tion (ppb) and mean output (nl min71) during the plateau 71 intake of L-arginine capsules in a dose of 0?05 g kg ,no phase (12–18 sec of the exhalation) were registered. Nasal increase of exhaled NO was shown among asthmatic NO was measured by letting the analyser sample nasal air patients (18), but Kharitonov et al. (19) found an increase directly from one nostril at a constant sample rate of 71 71 in healthy subjects after ingestion of 0?1g L-arginine kg . 8 ml sec , while the subject held their breath for 20 sec and Nasal NO has been shown to increase after infusion of L- closed the velum voluntarily. The nasal NO concentration arginine (20). during the plateau phase was registered. One measurement The aim of the present study was to investigate whether was performed in each nostril and the mean NO the intake of a nitrate-rich meal would influence ENO and concentration was calculated. NNO levels. We analysed ENO and NNO after a normal A analyser was used to measure nitric diet, a nitrate-restricted diet and after intake of a nitrate- oxide, (CLD 700 AL; Ecophysics, Du¨ rnten, Switzerland). A rich meal in 16 healthy volunteers. The levels of plasma special computerized biofeedback system was used to TM nitrate and plasma L-arginine levels were simultaneously regulate exhalation flow rate (Aerocrine NO System , recorded. Aerocrine AB, Stockholm, Sweden). A two-point calibra- tion of the analyser was performed daily with a certified NO calibration gas. The exhaled NO output, i.e. ENO elimination rate [NO concentration (ppb ¼ nl l71)6flow rate (l min71)] is presented because these values are more Subjects and methods accurate, the NO concentration being strongly influenced by small changes in the exhalation flow. To make SUBJECTS comparisons possible with other studies where only NO concentrations are presented, the individual concentrations Twenty healthy (10 males/10 females), non-smoking of exhaled NO are also presented. The intra-individual volunteers with no history of obstructive lung disease variability expressed as coefficient of variation (CV) for or respiratory tract infection during the previous 3 weeks ENO, NNO and plasma nitrate were estimated through were recruited for the study. Three females had measurements taken on 3 days at a normal diet, with an int- to be excluded because of an infection of the respiratory erval of at least 2 days between each, on all included subjects. tract caught during the study period as did one male subject who developed symptoms of allergic rhinitis. ENO and NNO as well as plasma nitrate and plasma L-arginine NITRATE levels were measured in 16 subjects. All subjects passed an examination including spirometry (21) and a Phadia- Nitrate in plasma was analysed by the gas chromatography/ TM top test (22). Basic data on the subjects are presented in mass spectrometry (GC/MS) method described by Tesch et Table 1. al. (23) and further developed by Wennmalm et al. (24). NO IN EXHALED AIR AFTER A NITRATE-RICH MEAL 155

L-ARGININE Spearman’s rank correlation coefficient (rs) have been calculated to analyse any correlation between both maximal L-arginine was measured with a fully automatic ion- and total change in ENO/NNO and nitrate/L-arginine. exchange-chromatography system, as previously described (25). Results We found significant increases of ENO and NNO, plasma DIET nitrate and plasma L-arginine after intake of the nitrate-rich During the first week the subjects were kept on normal diet meal [see Fig. 1(A–D) (mean + SEM)]. The individual NO without any restrictions. During week 2 a low-nitrate diet concentrations before the nitrate-rich meal, the maximal increase of exhaled NO, irrespective of time-point (range 15 was introduced (26), but with a normal L-arginine content. After 48 h of a low-nitrate diet the subjects were given a min–2 h 45 min), and the levels 3 h after the meal are meal rich in nitrate: 100 g lettuce, 75 g spinach, 100 g presented in Fig. 2. The median of maximal ENO increase ham, 75 g cucumber, three to five potatoes and a tomato. after the meal was 47% (range 7–89%) and the median of maximal NNO increase was 13% (range 0?1–36%). The nitrate content was equivalent to 1 g nitrate, and the L- arginine content to 1?4 g. ENO and NNO and plasma The maximal increase in ENO and plasma nitrate nitrate were then analysed every 15 min for 3 h and occurred 2 h after the meal and subsequently decreased, although both ENO and nitrate were found to be thereafter daily for 2 days. Plasma L-arginine was analysed before the meal and hourly during the first 3 h afterwards. significantly elevated throughout the measurement period. The nitrate rich meal was served at 09.00 hours for half L-arginine was only examined before the meal and 1, 2 and of the subjects and for the other half at 12.00 hours in a 3 h afterwards. Here, too, the maximal increase occurred 2 h randomized way. The ENO and NNO measurements taken after the meal. on days of normal diet, and on the days following the nitrate-rich meal, were performed at the same time of the day as on the meal was served.

LEAN BODY MASS (LBM)

The increase in plasma nitrate and plasma L-arginine was considered to be dependent on plasma volume. A lean body mass index has been calculated for each individual as this is the best available index for plasma volume (27). The lean body mass index has been used in analysing the correlation between the changes in ENO, NNO, plasma nitrate and plasma L-arginine.

STATISTICS

Statistical analyses were carried out with SAS, version 6.12 (SAS Statistical package, Cary, NC, U.S.A.). The results have been given as medians of change in ENO output (nl min71), NNO concentration (ppb), nitrate 71 concentration (mmol l ) and L-arginine concentration (mmol l71) in plasma, unless otherwise stated. The indivi- dual changes from baseline (i.e. the value before the meal was served) of exhaled and nasal NO, plasma nitrate and plasma L-arginine after the nitrate-rich meal were calculated as [value at respectively time point] 7 [baseline value]. For each individual the maximal change from baseline in ENO concentration was also noted. Significance testing was based on Student’s t-test using logarithm- transformed values. The area under the curve (AUC), representing the total increase from baseline during the follow-up period, was calculated for each parameter. Since the observations were equally spaced in time, the mean change for each parameter can be used, representing the FIG. 1. (A) Change in exhaled NO, mean+SEM. (B) area under the curve (28). As this method does not allow for Change in nasal NO, mean+SEM. (C) Change in plasma any missing values, the mean of the two adjacent values has nitrate, mean+SEM. (D) Change in plasma L-arginine, been used to represent the missing value. mean+SEM. 156 A.-C. OLIN ET AL.

apart, when the subjects were on normal diet, was 19% for ENO, 9% for NNO and 22% for plasma nitrate.

Discussion Both ENO and NNO were significantly increased after the intake of a meal rich in nitrate. The increase in ENO was dose-dependent, i.e. the subjects with the highest dose per LBM (an index for plasma volume) showed the greatest increase. The median increase of ENO for all subjects was 43% occurring 2 h after the meal. These findings are in agreement with Zetterquist et al., reporting a maximal increase of ENO 2 h after ingestion of 400 mg potassium nitrate solution. However the ENO increase was very much lower (43%) after intake of nitrate-rich food intake compared with the intake of potassium nitrate solution (150%), even though the total nitrate content of the meal was higher than the nitrate content of the solution. One possible explanation may be the different bio-availability of the nitrate in food and in potassium nitrate solution, FIG. 2. Maximal change in exhaled NO concentration enabling the latter to be more rapidly and completely (ppb). absorbed than the nitrate in the food. Contrary to Zetterquist et al., we found an increase of We presumed that the increase of plasma nitrate and NNO. One explanation for this might be that the meal also plasma L-arginine would depend on the subjects’ plasma contained L-arginine. The production of NNO may be volume. As all the subjects were given the same amount of limited by the availability of the substrate L-arginine (20). food, the intake of nitrate and L-arginine in relation to The L-arginine content of the meal, due to its content in the plasma volume differed markedly. We had chosen to use ham, was equivalent to 1?4gL-arginine; we also found a lean body mass (LBM) as an index for plasma volume. We significant increase of plasma L-arginine after the meal. found an inverse correlation between maximal increase in Lundberg et al. (20) have shown that an L-arginine infusion ENO and LBM, (rs¼70?78, P¼0?0003) and between of 16 mg per kg body weight, increases NNO from 420 ppb maximal increase in plasma nitrate and LBM (rs¼70?78, to 475 ppb (13%) as against the ingestion of 15 mg L- P¼ 0?0006). The maximal rise in plasma L-arginine was not arginine per kg body weight in this study, also resulting in dependent on LBM (rs¼70?0044, P¼0?98). The maximal increased NNO by 13%. increase in NNO did not correlate to LBM (rs¼ 0?11, Nevertheless, we found no significant decreases of either P¼0?68). ENO or NNO after 3 days with a nitrate-restricted diet. As a measure of the total change of ENO, NNO, plasma The plasma nitrate levels were, however, significantly lower nitrate and plasma L-arginine the area under the curve with a nitrate-restricted diet. (AUC) for each factor was calculated, and used in the This might indicate that the plasma nitrate levels are less subsequent analysis. We found a significant correlation important than the other factors determining ENO levels, between the total change of ENO and plasma nitrate at least when there are only minor changes of plasma (rs¼0?57, P¼0?027), but no significant correlation between nitrate. The median decrease in plasma nitrate, on a nitrate- 71 ENO and plasma L-arginine (rs¼0?19, P¼0?47). NNO was restricted diet, was 5?9 mmol l compared with the median not significantly correlated with change of either plasma maximal increase of 81?5 mmol l71 after the nitrate-rich nitrate (rs¼70?018, P¼0?95) or plasma L-arginine meal. Alternatively, since ENO is influenced by salivary (rs¼70?094, P¼0?73). nitrate/nitrite concentrations (18) the result may indicate We did not find any significant change of ENO or NNO that nitrate uptake in the salivary glands is biologically when the subjects were kept on a nitrate-restricted as resulted to keep a minimum of nitrate in the saliva for host compared with a normal diet (median 35?1 vs.34?6 defence purposes (12). nl min71 and 587 vs. 534 ppb respectively). In contrast, One limitation of the study is that the diet also contained the plasma nitrate levels were significantly lower with a L-arginine, which makes it difficult to separate the effects of 71 nitrate-restricted diet (median 36?9 vs.45?6 mmol l , increased plasma nitrate from that of L-arginine, at the P50?02). For these analyses we compared the median of ENO and NNO levels. The time courses for the increases of three measurements on different occasions during week 1 nitrate and L-arginine were also similar. However, in with the median value before the meal on day 3 of the previous investigations the ingestion of L-arginine in second week. amounts equivalent to the contents in the nitrate-rich meal, The intra-individual coefficient of variation (CV), for has not been shown to induce any increase in ENO levels in repeated measurements on three occasions at least 2 days normal subjects (19). NO IN EXHALED AIR AFTER A NITRATE-RICH MEAL 157

The area under the curve (AUC) was used as an index of Council for Worklife Research, Torsten och Ragnar the total change of ENO, NNO, plasma nitrate and L- So¨ derberghs Foundation and the Gothenburg Medical arginine during the follow-up period of 3 h. Although one Society. could speculate that changes of plasma nitrate and plasma L-arginine would precede the changes in ENO and NNO, the kinetics for these processes are not known, making for ambiguity in any analysis based on lag-time. References Another limitation of the study is that we did not compare the changes in ENO after a nitrate-rich meal with 1. Alving K, Weitzberg E, Lundberg JM. Increased variations in ENO during a normal day. There has been amount of nitric oxide in exhaled air of asthmatics. contradicting data concerning diurnal variation of ENO. Eur Respir J 1993; 6: 1368–1370. Ten Hacken et al. (29) have not been able to demonstrate 2. Kharitonov SA, Yates D, Robbins RA, Logan-Sinclair any such variation in asthmatic patients with nocturnal R, Shinebourne EA, Barnes PJ. Increased nitric oxide . On the other hand Massaro et al. (30) have in exhaled air of asthmatic patients. Lancet 1994; 343: presented an abstract indicating great diurnal variation, 133–135. although with a maximal increase of ENO between 04.00 3. Massaro AF, Gaston B, Kita D, Fanta C, Stamler JS, hours and 08.00 hours and a maximal decrease at 20.00 Drazen JM. Expired nitric oxide levels during treat- hours in healthy individuals. In our study half of the ment of acute asthma. Am J Respir Crit Care Med subjects had their meal at 09.00 hours and the other half at 1995; 152: 800–803. noon. If there were a diurnal variation of ENO in the 4. Byrnes CA, Dinarevic S, Busst C, Bush A, Shinebourne pattern suggested by Massaro et al., the ENO values would EA. Is nitric oxide in exhaled air produced at airway or tend to decrease during the morning hours when we alveolar level? Eur Respir J 1997; 10: 1021–1025. followed half of the group, as well as among the subjects 5. Martin U, Bryden K, Devoy M, Howarth P. Increased receiving their meal at noon. levels of exhaled nitric oxide during nasal and oral The ENO levels in our study started to return to baseline breathing in subjects with seasonal rhinitis. J within 3 h, indicating that the changes were related to the Clin Immunol 1996; 97: 768–772. food intake and not to diurnal variation. 6. Kharitonov SA, Rajakulasingam K, O’Connor B, The finding that ENO could increase up to 89% after a Durham SR, Barnes PJ. Nasal nitric oxide is increased nitrate-rich meal, indicates that it is important to register or in patients with asthma and allergic rhinitis and may be cease the intake of nitrate-rich food the same day as ENO modulated by nasal . J Allergy Clin measurements take place. Although a normal diet rarely Immunol 1997; 99: 58–64. consists of, for example, 100 g of salad and 75 g spinach, the 7. Olin A-C, Tore´ n K, Ljungkvist G, Nielsen J, Granung effect on ENO might be substantial even with lower nitrate G. Measurement of nasal nitric oxide. Eur Respir J contents in recently ingested food. 1998; 12(Suppl. 28): 249S. How long subjects should refrain from nitrate-rich food 8. Lundberg JO, Farkas-Szallasi T, Weitzberg E, et al. may vary according to the need for accuracy and this will High nitric oxide production in human paranasal also have to be considered in relation to other sources of sinuses. Nat Med 1995; 1: 370–373. measurement errors. We followed the ENO levels 3 h after 9. Kharitonov S, Alving K, Barnes PJ. Exhaled and the meal, and at that time-point half of the subjects still had nasal nitric oxide measurements: recommendations. higher ENO levels than before the meal. Eur Respir J 1997; 10: 1683–1693. It seems, however, of no value to introduce a nitrate 10. Recommendations for standardized procedures for the restricted diet for a longer period of time in order to avoid online and offline measurement of exhaled lower dietary influences on ENO levels. respiratory nitric oxide and nasal nitric oxide in adults Another way of reducing the influence of exogenous and children—1999. Am J Respir Crit Care Med 1999; nitrate on ENO levels might be to reduce the nitrate 160: 2104–2117. reducing bacteria in the oral cavity and/or increase the pH 11. Duncan C, Dougall H, Johnston P, et al. Chemical on the oral cavity before the measurements are performed. generation of nitric oxide in the mouth from the Before making any such recommendations further studies enterosalivary circulation of dietary nitrate. Nat Med are called for. 1995; 1: 546–551. In conclusion we found that ENO increases significantly 12. Lundberg JO, Weitzberg E, Lundberg JM, Alving K. after ingestion of a nitrate-rich meal. It seems therefore Intragastric nitric oxide production in humans: mea- important to restrict the intake of food rich in nitrate prior surements in expelled air. Gut 1994; 35: 1543–1546. to measuring nitric oxide 13. Nagase S, Takemura K, Ueda A, et al. A novel nonenzymatic pathway for the generation of nitric oxide by the reaction of hydrogen peroxide and D-orL- Acknowledgments arginine. Biochem Biophys Res Commun 1997; 233: 150–153. The authors acknowledge the valuable technical assistance 14. Tannenbaum SR, Weisman M, Fett D. The effect of of G. Granung and K. Wass. The work has been supported nitrate intake on nitrite formation in human saliva. by the Swedish Heart-Lung Foundation, the Swedish Food Cosmet Toxicol 1976; 14: 549–552. 158 A.-C. OLIN ET AL.

15. Eisenbrand G, Spiegelhalder B, Preussmann R. Nitrate 23. Tesch JW, Rehg WR, Sievers RE. Microdetermination and nitrite in saliva. Oncology 1980; 37: 227–231. of and in saliva, blood, water, and 16. Granli T, Dahl R, Brodin P, Bockman OC. Nitrate and suspended particulates in air by gas chromatography. J nitrite concentrations in human saliva: variations Chromatogr 1976; 126: 743–755. with salivary flow-rate. Food Chem Toxicol 1989; 27: 24. Wennmalm A, Benthin G, Edlund A, et al. Metabolism 675–680. and excretion of nitric oxide in humans. An experi- 17. Zetterquist W, Pedroletti C, Lundberg JO, Alving K. mental and clinical study. Circ Res 1993; 73: 1121– Salivary contribution to exhaled nitric oxide. Eur 1127. Respir J 1999; 13: 327–333. 25. Spackman DH SW, Moore S. Automatic recording 18. de Gouw HW, Verbruggen MB, Twiss IM, Sterk PJ. apparatus for use in the chromatography of amino Effect of oral L-arginine on airway hyperrespon- acids. Anal Chem 1958; 30: 1190–1206. siveness to histamine in asthma. Thorax 1999; 54: 26. Jungersten L, Edlund A, Petersson AS, Wennmalm A. 1033–1035. Plasma nitrate as an index of nitric oxide formation in 19. Kharitonov SA, Lubec G, Lubec B, Hjelm M, man: analyses of kinetics and confounding factors. Clin Barnes PJ. L-arginine increases exhaled nitric oxide Physiol 1996; 16: 369–379. in normal human subjects. Clin Sci 1995; 88: 27. Boer P. Estimated lean body mass as an index for 135–139. normalization of body fluid volumes in humans. Am J 20. Lundberg JO, Weitzberg E, Rinder J, et al. Calcium- Physiol 1984; 247: F632–F636. independent and steroid-resistant 28. Altman DG. Practical statistics for medical research. activity in human paranasal sinus mucosa. Eur Respir J 1st ed. London: Chapman and Hall, 1991. 1996; 9: 1344–1347. 29. ten Hacken NH, van der Vaart H, van der Mark TW, 21. Berglund E, Birath G, Bjure J, et al. Spirometric studies Koeter GH, Postma DS. Exhaled nitric oxide is in normal subjects. Acta Med Scand 1963; 173: higher both at day and night in subjects with nocturnal 185–191. asthma. Am J Respir Crit Care Med 1998; 158: 22. Matricardi PM, Nisini R, Pizzolo JG, D’Amelio R. The 902–907. use of Phadiatop in mass-screening programmes of 30. Massaro A, Coulston E, Foster J, et al. Diurnal inhalant : advantages and limitations. Clin Exp variation in expired nitric oxide (NO) in asthmatics. Allergy 1990; 20: 151–155. Am J Respir Crit Care Med 1999; 159: A860.