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Mixed exhaled nitric oxide and plasma nitrites and nitrates in newborn infants
Life Sciences 68 (2001) 2789–2797
Mixed exhaled nitric oxide and plasma nitrites and nitrates in newborn infants Paolo Biban*, Tiziana Zangardi, Eugenio Baraldi, Noemi Dussini, Lino Chiandetti, Franco Zacchello Department of Pediatrics, University of Padova, via Giustiniani 3, 35128 Padova, Italy Received 27 April 2000; accepted 27 October 2000
Abstract Plasma nitrite (NO22) and nitrate (NO32) are the stable end-products of endogenous nitric oxide (NO) metabolism. NO is present in the exhaled air of humans, but it is not clear if exhaled NO may be an indicator of the systemic endogenous NO production. The aims of the study were to determine the levels of exhaled NO and plasma NO22/NO32 in healthy term and preterm newborns, and to assess if exhaled NO correlates with plasma NO22/NO32 at birth. After the stabilization of the newborn, we measured by chemiluminescence the concentration of NO in the mixed expired breath of 133 healthy newborns. Measurement of exhaled NO was repeated after 24 and 48 hours. Plasma NO22/NO32 levels at birth were measured by the Griess reaction. NO concentrations were 8.9 (CI 8.1–9.8) parts per billion (ppb), 7.7 (CI 7.2–8.3) ppb and 9.0 (CI 8.4–9.6) ppb at birth, 24 and 48 hours, respectively. At birth, exhaled NO was inversely correlated with gestational age (p50.008) and birth weight (p,0.001). Plasma NO22/NO32 level was 27.30 (CI 24.26–30.34) mmol/L. There was no correlation between exhaled NO and plasma NO22/NO32 levels at birth (p50.88). We speculate that the inverse correlation between exhaled NO and gestational age and birth weight may reflect a role of NO in the postnatal adaptation of pulmonary circulation. At birth, exhaled NO does not correlate with plasma NO22/NO32 and does not seem to be an index of the systemic endogenous NO production. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Exhaled air; Nitric oxide; Nitrate; Newborn infant
Introduction Nitric oxide (NO) is an endogenous gas involved in many complex biological functions, such as the physiological regulation of the vasodilatory tone, the central and peripheral neurotransmission and the host defense [1]. Experimental and clinical studies suggest a role for * Corresponding author. Patologia Neonatale e Terapia Intensiva Pediatrica, Ospedale Maggiore, Azienda Ospedaliera di Verona, Piazzale Stefani 1, 37126, Verona, Italy. Tel.: 139045-8072377; fax: 139045-8073481. E-mail address: [email protected] (P. Biban) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 0 8 6 -4
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NO also under various pathophysiological conditions, such as sepsis [2,3] asthma [4] or persistent pulmonary hypertension of the newborn [5]. Nitric oxide has been detected in the exhaled breath of experimental animals and humans [6,7], but little data are available in the neonatal age [8]. There is strong evidence that high concentrations of NO are produced in the upper airways [7–12], but there is also measurable release of NO in the lower respiratory tract [9–10]. Notably, the endothelial constitutive NO synthase, one of the enzymes which forms NO from L-arginine, is expressed also in the alveolar walls [13] and metabolites of NO have been detected in broncho-alveolar lavage fluid [14]. Even though exhaled NO seems to reflect the origination of NO from the respiratory system, it is unclear if exhaled NO may be also related to the systemic endogenous NO production. Plasma nitrite (NO22) and nitrate (NO32) are the stable oxidative end products of NO and are considered as specific markers of the whole body NO formation [1]. Interestingly, some authors have observed an increase of endogenous NO synthesis [15] and of plasma NO22/NO32 concentrations in patients with sepsis syndrome [2,3,16]. In a recent study, the administration of L-arginine increased both exhaled NO and plasma nitrate levels [17]. This phenomenon might suggest a dynamic interrelationship between the whole body NO production, the end products of NO metabolism and the exhaled NO concentrations. The aims of this study were to measure the mixed exhaled NO and plasma nitrite/nitrate levels in healthy term and preterm newborns and to evaluate the possible effect of gender, gestational age (GA), birth weight (BW), mode of delivery, Apgar scores and other maternal factors on the exhaled NO concentration. Finally, we evaluated if exhaled NO concentrations were correlated with plasma NO22/NO32 levels, the specific markers of the systemic endogenous NO production. Methods This protocol was approved by the Ethical and Scientific Committee of the Department of Pediatrics, University of Padova. Informed consent was obtained from the patients’ parents before the study. All infants born at the Department of Obstetrics at the University of Padova were considered eligible for the study. Exclusion criteria were a GA less than 32 weeks or the need for resuscitation maneuvers in the delivery room. Between July and October 1997, we enrolled 133 newborn infants. Newborns were defined as premature if GA was less than 37 weeks. For each infant we recorded data about mother’s history, pregnancy, maternal drug use, parity, mode of delivery, GA, BW and Apgar score at 1 and 5 minutes of life. Measurement of exhaled NO Following the initial stabilization of the newborn in the delivery room and the calculation of the Apgar score, we collected non-invasively the total expired breath during 60 consecutive respiratory cycles, by means of a soft-edge face-mask. The mask was applied gently but firmly to the infant’s face, including the nose and the mouth. The mask was connected to a two-way valve that allowed inspiration of NO free gas (100% oxygen) from a 14 liter col-
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lapsible reservoir to ensure no contamination by ambient NO and expiration into a 2-liter polyvinylchloride collection bag (Braun, Milano, Italy). There was no gas leakage over the valve. The collection bag was connected to the expiratory side of the valve only after 10 breaths of NO free gas, in order to permit a wash-out of the lungs. The mixed exhaled NO samples were immediately analyzed by chemiluminescence. The expired gas was sampled from the collection bag at a constant flow of 0.7 L/min by the aspiration port of a chemiluminescence NO analyzer with 1-ppb resolution (CLD 700, ECO Physics, Dürnten, Switzerland). Such measurements were performed in each subject within 15 minutes after birth, at 24 and 48 hours of life. Before each study, the chemiluminescence analyzer was calibrated with a certified calibration mixture (300 ppb) of NO in nitrogen (SIAD, Bergamo, Italy) with guaranteed stability. The mixtures are prepared with a gravimetric method using NO which has a purity .99.9%.Total NOx within the aluminium cylinders did not exceed by more than 10 ppb the value of NO itself. During the analysis, NO concentration stabilized after few seconds and the steady state value was recorded. Measurements of exhaled NO was easily accomplished in all subjects and no complications were observed during sampling. Measurement of plasma nitrite and nitrate levels A blood sample (z2 ml) was obtained at birth from the umbilical cord. Samples were immediately centrifuged at 5000 g for 10 minutes, the supernatant aliquoted in microtubes, and stored at 280 C8 until analysis. Plasma samples were thawed at room temperature and ultrafiltered through a 10000 Mr cut-off Ultrafree-MC filter (Millipore Corp., Milford, MA, USA) for 60 min at 4000 g prior analysis. Each filter was washed with water thoroughly before use to remove nitrate left by the manufacturing process. Determination of the plasma concentrations of total nitrite and nitrate was performed using Cayman’s Nitrate/Nitrite Colorimetric Assay Kit (Alexis Corp., San Diego, CA, USA) according to the manufacturer’s directions. A 40 mL sample was used. The test tubes we used for blood samples contained litio-heparin. Before starting the study we checked twelve sample tubes filled with 2 ml of ultrafiltered pure water. No trace of nitrites/nitrates was found. In brief, the total nitrite/nitrate was determined after the reduction of nitrate to nitrite by nitrite reductase, followed by addition of the Griess reagents, which convert nitrite into an azo compound [18]. Standards and samples were assayed in duplicate. The absorbance was read at 540 nm on a detector Auto Reader III (Ortho Clinical Diagnostics, Milano, Italy). The calibration curve shows a linear relationship between the absorbances and total nitrite/nitrate concentrations (2.5, 5, 10, 20, 30, 35 mmol/L). The regression equation of curve was y50.0289x 1 0.0575, where y is the absorbance and x is the nitrite concentration. The standard error of the slope was 0.00043 and the correlation coefficient (r) was 0.999. Statistical analysis Data are presented as mean and the 95% confidence intervals (CI). Differences between groups were tested for significance with the Student’s t-test and the Mann-Whitney U test. Differences in frequencies of findings between groups were analyzed by Fisher’s exact test. The Pearson’s and Spearman correlation coefficients were used to evaluate the relation between
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Table 1 Demographic data, exhaled NO and plasma NO22/NO32 levels Total group (n5133)
Term infants (n5122)
Preterm infants (n511)
Statistical significance
Gestational age (weeks)
39.2 (39.0–39.4)
39.2 (39.0–39.4)
34.9 (34.1–35.9)
p,0.001*
Birth weight (grams)
3173 (3083–3263)
3261 (3184–3338)
2195 (1850–2540)
p,0.001*
Exhaled NO at birth (ppb)
8.9 (8.1–9.8)
8.7 (7.8–9.6)
12.2 (9.2–15.2)
p50.013*
Exhaled NO on day 1 (ppb)
7.7 (7.2–8.3)
7.8 (7.2–8.4)
7.3 (4.6–10.0)
p50.72*
Exhaled NO on day 2 (ppb)
9.0 (8.4–9.6)
9.0 (8.4–9.7)
8.7 (7.2–10.1)
p50.92*
27.30 (24.26–30.34)
27.62 (24.42–30.82)
23.47 (12.57–34.38)
p50.22*
Plasma NO22/NO32 (mmol/L)
Data are given as mean (95% confidence intervals). * Data are compared between term and preterm infants.
variables. Changes in mixed exhaled NO concentrations over time were analyzed with Friedman ANOVA. A p, 0.05 was considered significant. Data elaboration was performed by using a specific software program (Statistica, ©Statsoft Inc., Tulsa, OK, USA) Results Overall, we enrolled 133 newborns (61 males, 72 females). As expected, Apgar scores at 1 and 5 minutes were correlated with GA and BW (data not shown). Demographic characteristics and exhaled NO and plasma NO22/NO32 levels are summarized in Table 1. Exhaled NO concentrations at birth and at 48 hours were slightly higher compared with those at 24 hours (p50.024 and p50.007, respectively). At birth, the subgroup of term infants had lower exhaled NO levels (8.7 ppb, 95% CI 7.8–9.6) compared to the subgroup of preterm infants (12.2 ppb, 95% CI 9.2–15.2) (p50.013). Such difference was no longer present at 24 and 48 hours of life (Table I). In the whole group, an inverse correlation was found between BW and exhaled NO concentrations at birth (see figure 1) and at 24 hours (beta520.23, p50.007). Such inverse correlation was present regardless the mode of delivery (data not shown). Infants born from cesarean section tended to show higher NO concentrations at birth (9.8 ppb, 95% CI 8.6–11.1) when compared to infants born vaginally (8.0 ppb, 95% CI 6.9–9.1) (p50.032). Exhaled NO at birth was inversely correlated with GA (beta520.23, p50.007) and Apgar score at 1 minute (beta520.18, p50.033). Gender did not affect the exhaled NO concentrations except on day 2, when males showed slightly higher NO levels (9.7 ppb, 95% CI 8.8–10.6) compared with females (8.4 ppb, 95% CI 7.6–92; p50.037). Exhaled NO levels were not influenced by parity, maternal diseases or maternal drug assumption (data not shown).
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Fig. 1. Relationship between exhaled NO concentration and weight in 133 newborn infants. Data are presented as mean and 95% confidence intervals. Exhaled NO at birth was inversely correlated with birth weight (r52.3938, p, 0.001).
Plasma NO22/NO32 level was 27.30 (CI 24.26–30.34) mmol/L. There was no correlation between exhaled NO and plasma NO22/NO32 levels at birth (p50.88). Discussion Our study provides a base of normal mixed exhaled NO as well as plasma nitrite/nitrate levels in the healthy newborn and it shows that small amounts of NO are produced in the respiratory system since the first minutes of life. From our data, mixed exhaled NO at birth appear to be inversely correlated with BW and GA, while it does not seem to be influenced by gender and mode of delivery. Finally, mixed exhaled NO concentrations at birth do not correlate with plasma NO22/NO32, the stable end products of endogenous NO formation. To measure exhaled NO in newborns is quite a methodological challenge. A reliable method for measuring nasal NO has been described [8] but, hitherto, a practical method for measuring mixed exhaled NO in neonates has not been presented. Furthermore, newborns are almost exclusively nasal breathers, thus making measurement of orally exhaled NO very difficult at this age. Even though the exact source of NO production in the airway has not been determined, NO formation seems to occur in both upper and lower respiratory tract [7–12]. Yet, some authors have demonstrated in adults, children and neonates, that the majority of mixed exhaled NO emanates from the nose and the paranasal sinuses [8,12]. Recently, Zetterquist et al. have described a salivary contribution to exhaled NO due to nitrate intake in adults, with oral NO formation which may markedly affect the measurement of exhaled NO [19]. As of today, it is not known if this mechanism is present in newborns. Experimental studies suggest that exhaled NO originates, at least in part, also from the pulmonary endothelium [20]. The endogenous NO gas formed within the pulmonary endo-
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thelial cells mainly diffuses into underlying vascular smooth muscle, causing relaxation, and into the intravascular space, where it is avidly bound and inactivated by hemoglobin [1]. NO may also diffuse in the alveolar air space, thus contributing to the final expired NO concentration. Therefore, a complex dynamic exchange between the blood, the endothelial cells producing nitric oxide and the airspaces has been hypothesized [21]. In two recent studies, NO concentrations varied from few parts per billion to 4.6 parts per million in the nose and the pharynx of newborn infants shortly after birth [8,22]. The authors suggested that in newborns, as shown in adults [7,12], the major part of NO is formed in the upper airways, particularly in the nose, and can be reliably determined by sampling at constant nasal flow [8,22]. With various types of sampling techniques, other authors have reported rather different exhaled NO concentrations in healthy children and adults, varying from 2–3 to several hundreds part per billion of NO [6–12,21–24]. Technical factors and diverse methodology may substantially affect the measurement of exhaled NO. For instance, breathholding or variation of ventilation during tidal breathing, as well as different sampling site and sampling flow, can markedly influence the concentration of NO in the nasal air [22]. Thus, comparisons between different studies may be difficult unless standardized techniques are used. We used an offline tidal breathing method with variable flows. This non invasive method has the advantage to be extremely simple and feasible in newborns and infants, as recently indicated by other authors [25], but has some limitations related to technical aspects. One is that using a mask over the mouth and the nose we measured a mix of upper and lower NO production, making impossible to distinguish between these two compartments. This is particularly relevant in the newborn infant, who nearly exclusively breathe through the nose. In fact, even if the development of paranasal sinuses is limited in newborns and, theoretically, nasal NO contamination would be expected to be low, Schedin et al. showed marked nasal NO production also in newborns [8]. Another limitation of our technique is that we did not measure the expiratory flow, which has been shown to affect the exhaled NO concentration in adults and school aged children [26]. Nonetheless, the exhaled NO concentrations we found in our population were similar to those reported by other authors using similar sampling techniques [25,27]. van Erk and coworkers have found a mean exhaled NO of 12 ppb in 30 newborns spontaneously breathing in ambient air [25]. Artlich and coworkers observed a mean NO concentration of 3.57 ppb in six preterm infants, with a mean postnatal age of 21.7 days, during spontaneous respiration of NO-purified air [27]. In our study, exhaled NO levels were inversely correlated with BW and GA, and a small subgroup of preterm infants showed slightly higher exhaled NO concentrations when compared with term infants. Indeed, endogenous NO seems to play an important role during transition of pulmonary circulation from fetal to neonatal conditions at birth [28]. We speculate that higher exhaled NO concentrations may reflect a greater production of NO in premature airway favoring a rapid postnatal adaptation of pulmonary circulation. Alternatively, the observed correlation between exhaled NO and GA and BW could be simply due to lower expiratory flows which may be expected in this group, and not to any pulmonary adaptive changes. Exhaled NO at birth was inversely correlated with birth weight regardless the mode of delivery. The fact we observed slightly higher NO levels in the exhaled air of infants born from
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cesarean section may be partially related to the lower GA and BW seen in this group of patients. Indeed, Schedin et al. reported that neonates born after planned cesarean section had similar nasal NO concentrations as normally delivered newborns [ 8]. Exhaled NO concentrations tended to be slightly higher in males at 48 hours of age. In a recent study on adults, exhaled NO as well as plasma nitrate levels were higher in males compared to females, even when data were corrected for body weight [29]. The physiological implication of this phenomenon remains to be elucidated. In our population, mean plasma NO22/NO32 level was 27.3 mmol/L. This value is slightly lower when compared with the 34.9 mmol/L reported by Shi and co-workers in 30 newborns with a median age of 2.5 days [3]. Such little variation may be related to differences in age and food intake between the two populations at the time of sampling. Food intake from the mother may in fact be a source of NO22/NO32 potentially affecting the infant plasma NO22/ NO32 level at birth. Yet, we did not observe any correlation between NO22/NO32 levels measured in 32 infants and their respective mothers (unpublished data). Plasma NO22/NO32 measurements have been discussed as well as their potential as markers of the systemic NO synthesis in mammalians, for the only known endogenous source of NO22/NO32 production is through the conversion of L-arginine to NO [1]. However, dietary content can be several fold larger than the endogenous production, and microbes directly or through induction of endogenous production may also contribute and seriously compromise such studies. Several authors have found increased NO22/NO32 levels in patients with sepsis syndrome, a clinical condition which may be associated with elevated endogenous NO production [2,3,16]. Interestingly, an increase of exhaled NO has been demonstrated in a model of sepsis [30], in inflammatory lung conditions such as asthma, and during exercise [4,23,24,31,32]. Recently, Kharitonov and coworkers observed that the administration of L-arginine increased the exhaled NO concentrations in normal subjects, suggesting an increment in the activity of the endogenous NO synthase due to a larger substrate availability [17]. Such increase of exhaled NO concentrations was matched by an increase of plasma NO32 levels, which were indicative of an increased NO formation in the circulation [17]. We then hypothesized that mixed exhaled NO concentration, like plasma NO22/NO32levels, could be an indicator of the whole-body endogenous NO production. The fact we did not observe any correlation between exhaled NO and plasma nitrite/nitrate levels may indicate that expired NO, at least in healthy conditions, reflects local rather than systemic NO formation, as suggested by a previous experience in healthy volunteers [33]. Alternatively, mixed exhaled NO may be scarcely representative of the lung NO component, due to upper airway contamination. Notably, nasal and intratracheal NO measurements performed in preterm infants do suggest that more than 90% of the total NO production may occur in the upper airways [27]. Our study was not designed to clarify the site of origin and the relative contribution of exhaled NO in the various part of the respiratory system. Nonetheless, these data could be useful for future comparison with other populations, in health as well as in various pathological conditions in which an unbalanced endogenous NO production may be implicated. Further investigation is needed to evaluate whether mixed exhaled NO and plasma nitrite/nitrate levels may have relevance as diagnostic or therapeutic indicators in different clinical settings, such as for instance in newborns or older patients with sepsis.
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Mixed exhaled nitric oxide and plasma nitrites and nitrates in newborn infants Paolo Biban*, Tiziana Zangardi, Eugenio Baraldi, Noemi Dussini, Lino Chiandetti, Franco Zacchello Department of Pediatrics, University of Padova, via Giustiniani 3, 35128 Padova, Italy Received 27 April 2000; accepted 27 October 2000
Abstract Plasma nitrite (NO22) and nitrate (NO32) are the stable end-products of endogenous nitric oxide (NO) metabolism. NO is present in the exhaled air of humans, but it is not clear if exhaled NO may be an indicator of the systemic endogenous NO production. The aims of the study were to determine the levels of exhaled NO and plasma NO22/NO32 in healthy term and preterm newborns, and to assess if exhaled NO correlates with plasma NO22/NO32 at birth. After the stabilization of the newborn, we measured by chemiluminescence the concentration of NO in the mixed expired breath of 133 healthy newborns. Measurement of exhaled NO was repeated after 24 and 48 hours. Plasma NO22/NO32 levels at birth were measured by the Griess reaction. NO concentrations were 8.9 (CI 8.1–9.8) parts per billion (ppb), 7.7 (CI 7.2–8.3) ppb and 9.0 (CI 8.4–9.6) ppb at birth, 24 and 48 hours, respectively. At birth, exhaled NO was inversely correlated with gestational age (p50.008) and birth weight (p,0.001). Plasma NO22/NO32 level was 27.30 (CI 24.26–30.34) mmol/L. There was no correlation between exhaled NO and plasma NO22/NO32 levels at birth (p50.88). We speculate that the inverse correlation between exhaled NO and gestational age and birth weight may reflect a role of NO in the postnatal adaptation of pulmonary circulation. At birth, exhaled NO does not correlate with plasma NO22/NO32 and does not seem to be an index of the systemic endogenous NO production. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Exhaled air; Nitric oxide; Nitrate; Newborn infant
Introduction Nitric oxide (NO) is an endogenous gas involved in many complex biological functions, such as the physiological regulation of the vasodilatory tone, the central and peripheral neurotransmission and the host defense [1]. Experimental and clinical studies suggest a role for * Corresponding author. Patologia Neonatale e Terapia Intensiva Pediatrica, Ospedale Maggiore, Azienda Ospedaliera di Verona, Piazzale Stefani 1, 37126, Verona, Italy. Tel.: 139045-8072377; fax: 139045-8073481. E-mail address: [email protected] (P. Biban) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 0 8 6 -4
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NO also under various pathophysiological conditions, such as sepsis [2,3] asthma [4] or persistent pulmonary hypertension of the newborn [5]. Nitric oxide has been detected in the exhaled breath of experimental animals and humans [6,7], but little data are available in the neonatal age [8]. There is strong evidence that high concentrations of NO are produced in the upper airways [7–12], but there is also measurable release of NO in the lower respiratory tract [9–10]. Notably, the endothelial constitutive NO synthase, one of the enzymes which forms NO from L-arginine, is expressed also in the alveolar walls [13] and metabolites of NO have been detected in broncho-alveolar lavage fluid [14]. Even though exhaled NO seems to reflect the origination of NO from the respiratory system, it is unclear if exhaled NO may be also related to the systemic endogenous NO production. Plasma nitrite (NO22) and nitrate (NO32) are the stable oxidative end products of NO and are considered as specific markers of the whole body NO formation [1]. Interestingly, some authors have observed an increase of endogenous NO synthesis [15] and of plasma NO22/NO32 concentrations in patients with sepsis syndrome [2,3,16]. In a recent study, the administration of L-arginine increased both exhaled NO and plasma nitrate levels [17]. This phenomenon might suggest a dynamic interrelationship between the whole body NO production, the end products of NO metabolism and the exhaled NO concentrations. The aims of this study were to measure the mixed exhaled NO and plasma nitrite/nitrate levels in healthy term and preterm newborns and to evaluate the possible effect of gender, gestational age (GA), birth weight (BW), mode of delivery, Apgar scores and other maternal factors on the exhaled NO concentration. Finally, we evaluated if exhaled NO concentrations were correlated with plasma NO22/NO32 levels, the specific markers of the systemic endogenous NO production. Methods This protocol was approved by the Ethical and Scientific Committee of the Department of Pediatrics, University of Padova. Informed consent was obtained from the patients’ parents before the study. All infants born at the Department of Obstetrics at the University of Padova were considered eligible for the study. Exclusion criteria were a GA less than 32 weeks or the need for resuscitation maneuvers in the delivery room. Between July and October 1997, we enrolled 133 newborn infants. Newborns were defined as premature if GA was less than 37 weeks. For each infant we recorded data about mother’s history, pregnancy, maternal drug use, parity, mode of delivery, GA, BW and Apgar score at 1 and 5 minutes of life. Measurement of exhaled NO Following the initial stabilization of the newborn in the delivery room and the calculation of the Apgar score, we collected non-invasively the total expired breath during 60 consecutive respiratory cycles, by means of a soft-edge face-mask. The mask was applied gently but firmly to the infant’s face, including the nose and the mouth. The mask was connected to a two-way valve that allowed inspiration of NO free gas (100% oxygen) from a 14 liter col-
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lapsible reservoir to ensure no contamination by ambient NO and expiration into a 2-liter polyvinylchloride collection bag (Braun, Milano, Italy). There was no gas leakage over the valve. The collection bag was connected to the expiratory side of the valve only after 10 breaths of NO free gas, in order to permit a wash-out of the lungs. The mixed exhaled NO samples were immediately analyzed by chemiluminescence. The expired gas was sampled from the collection bag at a constant flow of 0.7 L/min by the aspiration port of a chemiluminescence NO analyzer with 1-ppb resolution (CLD 700, ECO Physics, Dürnten, Switzerland). Such measurements were performed in each subject within 15 minutes after birth, at 24 and 48 hours of life. Before each study, the chemiluminescence analyzer was calibrated with a certified calibration mixture (300 ppb) of NO in nitrogen (SIAD, Bergamo, Italy) with guaranteed stability. The mixtures are prepared with a gravimetric method using NO which has a purity .99.9%.Total NOx within the aluminium cylinders did not exceed by more than 10 ppb the value of NO itself. During the analysis, NO concentration stabilized after few seconds and the steady state value was recorded. Measurements of exhaled NO was easily accomplished in all subjects and no complications were observed during sampling. Measurement of plasma nitrite and nitrate levels A blood sample (z2 ml) was obtained at birth from the umbilical cord. Samples were immediately centrifuged at 5000 g for 10 minutes, the supernatant aliquoted in microtubes, and stored at 280 C8 until analysis. Plasma samples were thawed at room temperature and ultrafiltered through a 10000 Mr cut-off Ultrafree-MC filter (Millipore Corp., Milford, MA, USA) for 60 min at 4000 g prior analysis. Each filter was washed with water thoroughly before use to remove nitrate left by the manufacturing process. Determination of the plasma concentrations of total nitrite and nitrate was performed using Cayman’s Nitrate/Nitrite Colorimetric Assay Kit (Alexis Corp., San Diego, CA, USA) according to the manufacturer’s directions. A 40 mL sample was used. The test tubes we used for blood samples contained litio-heparin. Before starting the study we checked twelve sample tubes filled with 2 ml of ultrafiltered pure water. No trace of nitrites/nitrates was found. In brief, the total nitrite/nitrate was determined after the reduction of nitrate to nitrite by nitrite reductase, followed by addition of the Griess reagents, which convert nitrite into an azo compound [18]. Standards and samples were assayed in duplicate. The absorbance was read at 540 nm on a detector Auto Reader III (Ortho Clinical Diagnostics, Milano, Italy). The calibration curve shows a linear relationship between the absorbances and total nitrite/nitrate concentrations (2.5, 5, 10, 20, 30, 35 mmol/L). The regression equation of curve was y50.0289x 1 0.0575, where y is the absorbance and x is the nitrite concentration. The standard error of the slope was 0.00043 and the correlation coefficient (r) was 0.999. Statistical analysis Data are presented as mean and the 95% confidence intervals (CI). Differences between groups were tested for significance with the Student’s t-test and the Mann-Whitney U test. Differences in frequencies of findings between groups were analyzed by Fisher’s exact test. The Pearson’s and Spearman correlation coefficients were used to evaluate the relation between
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Table 1 Demographic data, exhaled NO and plasma NO22/NO32 levels Total group (n5133)
Term infants (n5122)
Preterm infants (n511)
Statistical significance
Gestational age (weeks)
39.2 (39.0–39.4)
39.2 (39.0–39.4)
34.9 (34.1–35.9)
p,0.001*
Birth weight (grams)
3173 (3083–3263)
3261 (3184–3338)
2195 (1850–2540)
p,0.001*
Exhaled NO at birth (ppb)
8.9 (8.1–9.8)
8.7 (7.8–9.6)
12.2 (9.2–15.2)
p50.013*
Exhaled NO on day 1 (ppb)
7.7 (7.2–8.3)
7.8 (7.2–8.4)
7.3 (4.6–10.0)
p50.72*
Exhaled NO on day 2 (ppb)
9.0 (8.4–9.6)
9.0 (8.4–9.7)
8.7 (7.2–10.1)
p50.92*
27.30 (24.26–30.34)
27.62 (24.42–30.82)
23.47 (12.57–34.38)
p50.22*
Plasma NO22/NO32 (mmol/L)
Data are given as mean (95% confidence intervals). * Data are compared between term and preterm infants.
variables. Changes in mixed exhaled NO concentrations over time were analyzed with Friedman ANOVA. A p, 0.05 was considered significant. Data elaboration was performed by using a specific software program (Statistica, ©Statsoft Inc., Tulsa, OK, USA) Results Overall, we enrolled 133 newborns (61 males, 72 females). As expected, Apgar scores at 1 and 5 minutes were correlated with GA and BW (data not shown). Demographic characteristics and exhaled NO and plasma NO22/NO32 levels are summarized in Table 1. Exhaled NO concentrations at birth and at 48 hours were slightly higher compared with those at 24 hours (p50.024 and p50.007, respectively). At birth, the subgroup of term infants had lower exhaled NO levels (8.7 ppb, 95% CI 7.8–9.6) compared to the subgroup of preterm infants (12.2 ppb, 95% CI 9.2–15.2) (p50.013). Such difference was no longer present at 24 and 48 hours of life (Table I). In the whole group, an inverse correlation was found between BW and exhaled NO concentrations at birth (see figure 1) and at 24 hours (beta520.23, p50.007). Such inverse correlation was present regardless the mode of delivery (data not shown). Infants born from cesarean section tended to show higher NO concentrations at birth (9.8 ppb, 95% CI 8.6–11.1) when compared to infants born vaginally (8.0 ppb, 95% CI 6.9–9.1) (p50.032). Exhaled NO at birth was inversely correlated with GA (beta520.23, p50.007) and Apgar score at 1 minute (beta520.18, p50.033). Gender did not affect the exhaled NO concentrations except on day 2, when males showed slightly higher NO levels (9.7 ppb, 95% CI 8.8–10.6) compared with females (8.4 ppb, 95% CI 7.6–92; p50.037). Exhaled NO levels were not influenced by parity, maternal diseases or maternal drug assumption (data not shown).
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Fig. 1. Relationship between exhaled NO concentration and weight in 133 newborn infants. Data are presented as mean and 95% confidence intervals. Exhaled NO at birth was inversely correlated with birth weight (r52.3938, p, 0.001).
Plasma NO22/NO32 level was 27.30 (CI 24.26–30.34) mmol/L. There was no correlation between exhaled NO and plasma NO22/NO32 levels at birth (p50.88). Discussion Our study provides a base of normal mixed exhaled NO as well as plasma nitrite/nitrate levels in the healthy newborn and it shows that small amounts of NO are produced in the respiratory system since the first minutes of life. From our data, mixed exhaled NO at birth appear to be inversely correlated with BW and GA, while it does not seem to be influenced by gender and mode of delivery. Finally, mixed exhaled NO concentrations at birth do not correlate with plasma NO22/NO32, the stable end products of endogenous NO formation. To measure exhaled NO in newborns is quite a methodological challenge. A reliable method for measuring nasal NO has been described [8] but, hitherto, a practical method for measuring mixed exhaled NO in neonates has not been presented. Furthermore, newborns are almost exclusively nasal breathers, thus making measurement of orally exhaled NO very difficult at this age. Even though the exact source of NO production in the airway has not been determined, NO formation seems to occur in both upper and lower respiratory tract [7–12]. Yet, some authors have demonstrated in adults, children and neonates, that the majority of mixed exhaled NO emanates from the nose and the paranasal sinuses [8,12]. Recently, Zetterquist et al. have described a salivary contribution to exhaled NO due to nitrate intake in adults, with oral NO formation which may markedly affect the measurement of exhaled NO [19]. As of today, it is not known if this mechanism is present in newborns. Experimental studies suggest that exhaled NO originates, at least in part, also from the pulmonary endothelium [20]. The endogenous NO gas formed within the pulmonary endo-
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thelial cells mainly diffuses into underlying vascular smooth muscle, causing relaxation, and into the intravascular space, where it is avidly bound and inactivated by hemoglobin [1]. NO may also diffuse in the alveolar air space, thus contributing to the final expired NO concentration. Therefore, a complex dynamic exchange between the blood, the endothelial cells producing nitric oxide and the airspaces has been hypothesized [21]. In two recent studies, NO concentrations varied from few parts per billion to 4.6 parts per million in the nose and the pharynx of newborn infants shortly after birth [8,22]. The authors suggested that in newborns, as shown in adults [7,12], the major part of NO is formed in the upper airways, particularly in the nose, and can be reliably determined by sampling at constant nasal flow [8,22]. With various types of sampling techniques, other authors have reported rather different exhaled NO concentrations in healthy children and adults, varying from 2–3 to several hundreds part per billion of NO [6–12,21–24]. Technical factors and diverse methodology may substantially affect the measurement of exhaled NO. For instance, breathholding or variation of ventilation during tidal breathing, as well as different sampling site and sampling flow, can markedly influence the concentration of NO in the nasal air [22]. Thus, comparisons between different studies may be difficult unless standardized techniques are used. We used an offline tidal breathing method with variable flows. This non invasive method has the advantage to be extremely simple and feasible in newborns and infants, as recently indicated by other authors [25], but has some limitations related to technical aspects. One is that using a mask over the mouth and the nose we measured a mix of upper and lower NO production, making impossible to distinguish between these two compartments. This is particularly relevant in the newborn infant, who nearly exclusively breathe through the nose. In fact, even if the development of paranasal sinuses is limited in newborns and, theoretically, nasal NO contamination would be expected to be low, Schedin et al. showed marked nasal NO production also in newborns [8]. Another limitation of our technique is that we did not measure the expiratory flow, which has been shown to affect the exhaled NO concentration in adults and school aged children [26]. Nonetheless, the exhaled NO concentrations we found in our population were similar to those reported by other authors using similar sampling techniques [25,27]. van Erk and coworkers have found a mean exhaled NO of 12 ppb in 30 newborns spontaneously breathing in ambient air [25]. Artlich and coworkers observed a mean NO concentration of 3.57 ppb in six preterm infants, with a mean postnatal age of 21.7 days, during spontaneous respiration of NO-purified air [27]. In our study, exhaled NO levels were inversely correlated with BW and GA, and a small subgroup of preterm infants showed slightly higher exhaled NO concentrations when compared with term infants. Indeed, endogenous NO seems to play an important role during transition of pulmonary circulation from fetal to neonatal conditions at birth [28]. We speculate that higher exhaled NO concentrations may reflect a greater production of NO in premature airway favoring a rapid postnatal adaptation of pulmonary circulation. Alternatively, the observed correlation between exhaled NO and GA and BW could be simply due to lower expiratory flows which may be expected in this group, and not to any pulmonary adaptive changes. Exhaled NO at birth was inversely correlated with birth weight regardless the mode of delivery. The fact we observed slightly higher NO levels in the exhaled air of infants born from
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cesarean section may be partially related to the lower GA and BW seen in this group of patients. Indeed, Schedin et al. reported that neonates born after planned cesarean section had similar nasal NO concentrations as normally delivered newborns [ 8]. Exhaled NO concentrations tended to be slightly higher in males at 48 hours of age. In a recent study on adults, exhaled NO as well as plasma nitrate levels were higher in males compared to females, even when data were corrected for body weight [29]. The physiological implication of this phenomenon remains to be elucidated. In our population, mean plasma NO22/NO32 level was 27.3 mmol/L. This value is slightly lower when compared with the 34.9 mmol/L reported by Shi and co-workers in 30 newborns with a median age of 2.5 days [3]. Such little variation may be related to differences in age and food intake between the two populations at the time of sampling. Food intake from the mother may in fact be a source of NO22/NO32 potentially affecting the infant plasma NO22/ NO32 level at birth. Yet, we did not observe any correlation between NO22/NO32 levels measured in 32 infants and their respective mothers (unpublished data). Plasma NO22/NO32 measurements have been discussed as well as their potential as markers of the systemic NO synthesis in mammalians, for the only known endogenous source of NO22/NO32 production is through the conversion of L-arginine to NO [1]. However, dietary content can be several fold larger than the endogenous production, and microbes directly or through induction of endogenous production may also contribute and seriously compromise such studies. Several authors have found increased NO22/NO32 levels in patients with sepsis syndrome, a clinical condition which may be associated with elevated endogenous NO production [2,3,16]. Interestingly, an increase of exhaled NO has been demonstrated in a model of sepsis [30], in inflammatory lung conditions such as asthma, and during exercise [4,23,24,31,32]. Recently, Kharitonov and coworkers observed that the administration of L-arginine increased the exhaled NO concentrations in normal subjects, suggesting an increment in the activity of the endogenous NO synthase due to a larger substrate availability [17]. Such increase of exhaled NO concentrations was matched by an increase of plasma NO32 levels, which were indicative of an increased NO formation in the circulation [17]. We then hypothesized that mixed exhaled NO concentration, like plasma NO22/NO32levels, could be an indicator of the whole-body endogenous NO production. The fact we did not observe any correlation between exhaled NO and plasma nitrite/nitrate levels may indicate that expired NO, at least in healthy conditions, reflects local rather than systemic NO formation, as suggested by a previous experience in healthy volunteers [33]. Alternatively, mixed exhaled NO may be scarcely representative of the lung NO component, due to upper airway contamination. Notably, nasal and intratracheal NO measurements performed in preterm infants do suggest that more than 90% of the total NO production may occur in the upper airways [27]. Our study was not designed to clarify the site of origin and the relative contribution of exhaled NO in the various part of the respiratory system. Nonetheless, these data could be useful for future comparison with other populations, in health as well as in various pathological conditions in which an unbalanced endogenous NO production may be implicated. Further investigation is needed to evaluate whether mixed exhaled NO and plasma nitrite/nitrate levels may have relevance as diagnostic or therapeutic indicators in different clinical settings, such as for instance in newborns or older patients with sepsis.
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