Exhaled breath condensate nitrite in premature infants with bronchopulmonary dysplasia

Abstract

BACKGROUND:

Tracheal aspirate is the conventional method to measure biomarkers of inflammation and oxidation from premature infants on mechanical ventilation at risk for bronchopulmonary dysplasia (BPD), but this method is invasive. Exhaled breath condensate (EBC) is a novel, non-invasive method that has been used in older populations. Nitrite, a stable metabolite of nitric oxide (NO), is elevated in inflammatory conditions. We aim to investigate the feasibility of EBC nitrite collection from ventilated premature infants and to quantify EBC nitrite in infants with and without BPD. We hypothesize that EBC nitrite correlates with TA nitrite, and that EBC nitrite in the first week of life is higher in infants who will develop BPD than those without BPD.

METHODS:

In a pilot prospective cohort study, TA and EBC were collected in the first week of life from mechanically ventilated premature infants. Nitrite levels were measured using chemiluminescence.

RESULTS:

EBC nitrite significantly correlated with TA nitrite (r = 0.45, p = 0.025). Of 40 infants, 33 (82.5%) developed BPD. EBC and TA nitrite levels collected in the first week of life had a higher trend in infants with BPD than those without BPD (p = 0.23 and 0.38 respectively).

CONCLUSIONS:

Higher trend of EBC nitrite in the first week of life was associated with the development of BPD. Correlation of nitrite level in EBC with that in TA (conventional method) highlights the utility of EBC as an alternative, non-invasive method to measure inflammation. Further refinement of conditions and timing may optimize the predictive value of EBC nitrite.

Keywords

Exhaled breath condensate tracheal aspirates nitrite inflammation oxidative stress bronchopulmonary dysplasia

1 Introduction

Bronchopulmonary dysplasia (BPD) was first described by Northway et al. in 1967 [1] based on the clinical, radiological, and pathophysiological changes in infants treated with mechanical ventilation. The most recent consensus workshop in 2000 by National Institute of Child Health and Human Development/National Heart, Lung, and Blood Institute Workshop (NICHD/NHLBI) defined BPD as any oxygen requirement at 36 weeks post-conceptional age, and further categorized it into severity-based criteria [2]. Despite the ongoing innovations and interventions surrounding prematurity and maternal care, BPD remains a major cause of mortality and morbidity in premature infants, especially in very low birth weight (VLBW) and extremely low birth weight (ELBW) infants. Stoll et al. [3] reported an increased rate of BPD in the last two decades, likely due to resuscitation involving younger gestational age and smaller infants.

Prolonged ventilation, mechanical injury, oxygen toxicity, airway infection, and fluid overload all contribute to inflammatory and oxidative processes that culminate in the pathophysiologic presentation of BPD. Thus early markers of inflammation may be useful in identifying at-risk patients and guiding therapy prior to the clinical presentation of BPD at 36 weeks post-conception. Samples obtained by tracheal aspirate (TA), bronchoalveolar lavage (BAL), and sputum induction (SI) have been used to monitor the progression of pulmonary diseases in adults and older pediatric patients [4]. In neonates, it has been shown that inflammatory mediators in TA samples from infants who develop BPD are elevated compared to controls [5, 6]. However, TA analyses are not routinely used because obtaining samples is invasive. Endotracheal tube (ETT) lavage and suction is associated with an increased risk of unintended extubation and alterations in blood pressure, possibly contributing to intraventricular hemorrhage [7, 8].

Alternatively, lung metabolites can be sampled by collection of exhaled breath condensate (EBC), a procedure that is not associated with any physiologic disturbance or potential interaction with underlying disease processes. EBC contains measurable concentrations of lung metabolites because shear forces generated with each breath create aerosols that contain volatile and non-volatile markers from the air-fluid interface [9, 10]. EBC has been shown to be non-invasive, quick, safe, easy, and feasible, [11] yielding reproducible results on the progression of oxidative and/or inflammatory lung conditions in various patient populations without disturbing underlying pathophysiology [12 –14]. However, its use in premature infants has been limited.

Nitric oxide (NO) is generated in large amounts by alveolar macrophages that are activated in lung inflammation, so tracheal NO and its metabolites may be a useful marker of pulmonary disease. NO is labile and readily reacts with oxygen radicals to produce nitrite, nitrate, nitrosothiols, and 3-nitrotyrosine. Nitrosothiols and 3-nitrotyrosine are significantly elevated in lung tissues of infants with BPD [15]. Nitrite and nitrate are particularly promising as biomarkers of inflammatory lung conditions because of their relative stability [16]. They can be detected and quantified in EBC using calorimetric, fluorometric, and chemiluminescent assays, or by ion, gas, and liquid chromatography [17, 18]. EBC nitrite may be more specific to pulmonary inflammation than EBC nitrate, which can be contaminated by saliva and influenced by circulating nitrate from dietary intake [19]. Nitrite has been shown to be elevated in inflammatory conditions and has been used to follow progression of cystic fibrosis and asthma [14 , 20–22].

In this study we tested the feasibility of collecting EBC nitrite from extremely premature and extremely low birth weight (ELBW) ventilated infants. The aim of this study was to evaluate the correlation between nitrite levels in TA and EBC, and to determine if infants with BPD have elevated EBC nitrite levels in the first week of life. Studies looking at markers associated with BPD commonly evaluated samples collected on days 1, 3, 7, 14, even up to 21 and 28 days [23 –25]. As an attempt to look at early inflammatory and oxidative injury markers, our study used samples collected on days of life (DOL) three and seven. We hypothesized that nitrite concentrations in EBC collected from ventilated preterm infants on DOL three and seven are higher in those who develop BPD than in those who do not develop BPD.

2 Methods

2.1 Subjects

In this pilot prospective cohort study, forty infants were enrolled within the first three days of life from the neonatal intensive care unit at Bristol-Myers Squibb Children’s Hospital at Robert Wood Johnson University Hospital from July 2013 to December 2015. Informed consent was obtained from mothers as approved by Institutional Review Board at Rutgers Robert Wood Johnson Medical School. Inclusion criteria were ≤28 weeks gestational age, ≤1500 grams birth weight, and on mechanical ventilation at the time of enrollment. Infants with known genetic disorders or congenital pulmonary, tracheal, or chest wall anomalies, or those who had been treated with inhaled NO at or before the time of enrollment were excluded. BPD was defined as any oxygen requirement at 36 weeks postmenstrual age.

2.2 Sample/data collection

EBC samples were collected from intubated infants on conventional mechanical ventilation on DOL three and again on DOL seven if they remained intubated and on conventional ventilation. Infants were not suctioned for at least 1 hour prior to collection of EBC in order to ensure that agitation not affect the baseline levels of exhaled nitrite. Following each EBC collection, a TA sample was also collected for comparison. For infants who received high frequency ventilation, only TA was collected. Clinical parameters were collected to document safety prior to, during, and at the completion of the EBC sample collection [ventilatory rate, positive inspiratory pressure (PIP), positive end expiratory pressure (PEEP), and fraction of inspired oxygen (FiO₂)], as well as infants’ clinical parameters [respiratory rate (RR), heart rate (HR), blood pressure (BP), oxygen saturation (SaO₂)].

2.3 Exhaled breath condensate (EBC) collection

EBC was collected from infants on conventional ventilation by connecting one end of an RTube^TM (Respiratory Research, Inc., Austin, TX) to the endotracheal tube and the other end to the exhalation limb of the ventilator circuit via a valve-less connector. In order to maintain constant –80°C temperature, the RTube^TM was encased in a dry ice during the entire collection time of 20 min. Subjects’ vital signs and ventilator parameters were recorded at the beginning and at the end of the sample collection, and were closely monitored throughout the collection period. The RTube^TM was disconnected from the ventilator circuit and immediately capped on both ends while maintained in the dry ice. RTubes^TM were transported to the laboratory in an adjacent building immediately on dry ice and stored at –80°C until analysis.

2.4 Tracheal aspirate (TA) collection

Tracheal aspirates were obtained by instilling 1 mL of sterile normal saline at room temperature into the ETT and suctioned using an inline suctioning catheter into a closed, sterile Lukens trap (Covidien, Mansfield, MA). Samples were brought to the laboratory in an adjacent building within 30 min of collection, transferred to 1.5 mL microtainer tubes, and centrifuged at 5000 rpm for ten min. Pellets and supernatants were then separately stored at –80°C until analysis.

2.5 Antenatal data collection

Data collected from the medical record included prenatal history such as the presence of pregnancy-related or pre-existing medical conditions in the mother, prenatal corticosteroid treatment, preterm premature rupture of membrane (PPROM), and presence of chorioamnionitis (defined as maternal fever ≥38°C, uterine tenderness, maternal or fetal tachycardia or malodorous amniotic fluid). Demographic variables such as gestational age, birth weight, gender, race, Apgar scores, details of resuscitation in the delivery room and delivery method, were collected.

2.6 Nitrite measurement

RTubes^TM and TA samples were transported on dry ice to Rutgers Environmental and Occupational Health Sciences Institute. Nitrite concentrations were measured using selective catalytic reduction and chemiluminescence detection (Sievers Instrument NOA 280i, GE Analytics, Boulder, CO). The detection limit approached 125 nM (5 picomoles of samples in 40 microliter injection volume) with precision of 21.4% (IQR 6.7, 27.1%) and 18.6% (IQR 6.1, 27.7%) for EBC and TA respectively. Nitrite was assayed after reduction in potassium iodide.

2.7 Data analysis

The data analysis for this paper was conducted using SAS software, Version 9.4 of the SAS System for Windows (Copyright © 2013 SAS Institute Inc.). For observations with two non-zero observed values of nitrite in TA and/or EBC, values were averaged to create a single observation. Analyses considered differences in characteristics between those who died or not and those with BPD versus those without, including gender, race, delivery method (vaginal versus cesarean section (CS)), gestational age, birth weight, Apgar scores at one and five minutes, EBC on DOL three and seven, and TA on DOL three and seven. Exact chi-square tests, appropriate for tables with small cell counts, were used to test for associations between either death or BPD and other categorical variables. Satterwaithe two-group t-tests to account for unequal variations were used to test for associations between either death or BPD and continuous characteristics (including EBC and TA). Additional analyses were performed that considered DOL three and seven as repeated measures in a mixed linear model, with random effect for subject. Because of the different standard errors between groups, the variances for each group were allowed to differ for this mixed model. F-tests examined whether day (timing of collection) modified the association between either BPD or death and EBC/TA nitrite levels. If no modification effect was found, F-tests examined the main effect of day on nitrite levels. If there was no significant main effect of day, then a repeated measures analysis with just BPD or death was conducted. Mixed linear models were used to examine the relationship between EBC and TA nitrite levels.

In particular, an interaction term examined whether day modified the relationship between the two. In addition, correlations were calculated between EBC and TA nitrite, with further analysis done separately for DOL three and seven. Confidence limits for the correlations were calculated using Fisher’s method. Sensitivity analyses included non-parametric tests for continuous variables. For EBC and TA, in particular, a few individuals had extreme values. For comparing univariate outcomes across either Death or BPD statuses, Wilcoxon rank sum test was applied and 2-tailed p values of <0.05 were used to determine significance. When comparison repeated measures (DOL three and seven for EBC or TA), the Wilcoxon rank sum test was applied to the average of the repeated measures. Correlation of EBC nitrite and TA nitrite was conducted using Spearman rank order correlation, accounting for non-parametric distribution of the data.

3 Results

3.1 Study population

Subject recruitment and sample collection are shown in Fig. 1. Of the 40 subjects enrolled, 22 received conventional ventilation (SIMV) and 18 received high frequency ventilation (HFJV, HFOV). All infants on SIMV and 12 infants on high frequency remained intubated at DOL7. Table 1 demonstrated the demographic data and outcomes of subjects in our study. As demonstrated, BPD was diagnosed at 36 weeks postmenstrual age in 33 infants, of whom 4 died. None of the infants developed pulmonary hemorrhage. Of those without BPD (n = 7), none died. The odds of male subjects having BPD were 6.4 times higher than for females (95% CI 0.63–310.11). Twelve infants had culture-proven bacteremia. In this group, none died, however, culture-proven bacteremia increased the odds of developing BPD by 25.80 times (95% CI 1.38, 491.17; OR and CI calculated adding 0.5 to all cells as small sample correction). Similarly, data suggested an association between delivery method and death, with the odds of death for those delivered by vaginal delivery being 5.00 times higher than those delivered by cesarean section (95% CI 0.58–42.80). The weight of infants who did not die was also 30% higher than those who did die. The odds of death for those with intestinal perforation/necrotizing enterocolitis were 4.60 times the odds of death for those without (95% CI 0.62, 34.55 with small sample correction). Finally prenatal steroids and postnatal steroids were associated with increased odds of BPD (OR: 5.10, 95% CI: 0.93, 28.28 and OR: 5.82, 95% CI: 0.31, 112.15, respectively).

Fig.1

Flow chart of patient recruitment. Of 44 subjects approached, 40 subjects enrolled in the study. On day 3, 22 subjects were on conventional mechanical ventilation and 18 subjects were on high frequency ventilation. On day 7, six subjects were extubated, leaving 22 subjects on mechanical ventilation and 12 subjects on high frequency ventilation. Exhaled breath condensate (EBC) followed by tracheal aspirate (TA) were collected from those on mechanical ventilation. Only TA was collected from those on high frequency ventilation.

Table 1

Subject characteristics and outcomes, by death and BPD status. P values use either exact chi-square (for categorical variables) or t-test (for continuous variable) to test for significant difference between groups (death = Yes vs. No or BPD = Yes vs. No). (Counts and percentages are presented for categorical variables; means and standard deviations for continuous variables)

Variables	Label	Overall	Death (Yes)	Death (No)	p value	BPD (Yes)	BPD (No)	p value
		n = 40	n = 4	n = 36		n = 33	n = 7
GA		25.2 (1.4)	24.7 (1.1)	25.2(1.4)	0.49	24.9 (1.4)	25.8 (1.7)	0.24
BW		688.8 (141.7)	535 (158.5)	695.1 (143.9)	0.13	670.0 (150.1)	722.1 (161.4)	0.45
Gender	Female	22 (55%)	2 (9%)	20 (91%)	1.00	16 (73%)	6 (27%)	0.10
	Male	18 (45%)	2 (11%)	16 (89%)		17 (94%)	1 (6%)
Race	Asian	9 (22.5%)	0 (0%)	9 (100%)	0.72	8 (89%)	1 (11%)	0.80
	Black	7 (17.5%)	1 (14%)	6 (86%)		5 (71%)	2 (29%)
	White	21 (52.5%)	3 (14%)	18 (86%)		17 (81%)	4 (19%)
	Other	3 (7.5%)	0 (0%)	3 (100%)		3 (100%)	0 (0%)
Delivery	CS	32 (80%)	2 (6%)	30 (94%)	0.17	26 (81%)	6 (85.7%)	1.00
	NSVD	8 (20%)	2 (25%)	6 (75%)		7 (87%)	1 (14.3%)
Prenatal Steroids	Yes	33 (85.5%)	1 (25%)	6 (16.7%)	0.55	29 (87.9%)	4 (57.1%)	0.09
PPROM	Yes	5 (12.5%)	4 (100%)	31 (86.1%)	1.00	4 (12.1%)	1 (14.3%)	1.00
Chorioamnionitis	Yes	2 (5%)	4 (100%)	34 (94.4%)	1.00	2 (6.1%)	0 (0%)	1.00
Postnatal Steroids	Yes	9 (22.5%)	4 (100%)	27 (75%)	0.56	9 (27.3%)	0 (0%)	0.17
Surfactant	Yes	38 (95%)	0 (0%)	2 (5.6%)	1.00	32 (97%)	6 (85.7%)	0.32
Indo/Ibu	Yes	15 (37.5%)	3 (75%)	12 (33.3%)	0.28	13 (39.4%)	2 (28.6%)	0.69
Positive culture	Yes	12 (30%)	0 (0%)	12 (33.3%)	1.00	12 (36.4%)	0 (0%)	0.08
Perforation/NEC	Yes	5 (12.5%)	2 (50%)	3 (8.3%)	0.07	5 (15.2%)	0 (0%)	0.56
Feeding Day 3	Yes	10 (25%)	4 (100%)	26 (72.2%)	0.56	8 (24.4%)	2 (28.6%)	1.00
Feeding Day 7	Yes	20 (50%)	2 (50%)	18 (50%)	1.00	4 (57.1%)	16 (48.5%)	1.00
Apgar 1		4 (2)	4 (3)	4 (2)	0.92	4 (2)	3 (2)	0.34
Apgar 5		6 (2)	6 (3)	6 (2)	0.91	7 (2)	6 (3)	0.63

GA = gestational age; BW = birth weight; CS = Cesarean section; NSVD = normal spontaneous vaginal delivery; PPROM = premature preterm rupture of membrane; Indo/Ibu = Indomethacin/Ibuprofen, NEC = necrotizing enterocolitis; Feeding Day 3 = presence of feeding by day of life 3; Feeding Day 7 = presence of feeding by day of life 7; Apgar 1 = Apgar at 1 minute; Apgar 5 = Apgar at 5 minutes.

3.2 Correlation of TA with EBC nitrite

TA and EBC nitrite measurements were significantly correlated (r = 0.45, 95% CI 0.06–0.71, p = 0.025). When further analyzed based on day of collection, the correlation between EBC and TA nitrite levels on DOL three was 0.30 (95% CI –0.17–0.65, p = 0.19) and on DOL seven was 0.34 (95% CI –0.13–0.68, p = 0.14) (Fig. 3b, c).

Fig.2

Nitrite levels based on BPD status (median with brackets indicating interquartile ranges. (a) Nitrite level in TA collected in the first week of life. (b) Nitrite level in EBC collected in the first week of life. (c) Nitrite level in TA collected on day of life (DOL) 3. (d) Nitrite level in TA collected on DOL 7. (e) Nitrite level in EBC collected on DOL 3. (f) Nitrite level in EBC collected on DOL 7.

Fig.3

Correlation between TA nitrite and EBC nitrite: (a) in the first week of life, (b) on DOL 3, and (c) on DOL 7.

3.3 Correlation of nitrite with BPD

TA nitrite concentrations obtained in the first week of life trended higher in infants with BPD compared to those without BPD (median 0.19μM±(IQR 0.05, 0.56μM), p = 0.23) (Fig. 2a). EBC nitrite levels also trended higher in infants with BPD (0.13μM (IQR 0.00, 0.26μM), p = 0.38) (Fig. 2b). When data were analyzed as repeated measures, there was a trend toward higher TA nitrite in infants with BPD on both DOL three (0.21μM (IQR 0.05, 0.82μM) vs. 0.01μM (IQR 0.00, 0.25μM), p = 0.11) and DOL seven (0.16μM (IQR 0.04, 0.37μM) vs. 0.14μM (IQR 0.06, 0.25μM)), p = 0.66) (Fig. 2c, e). EBC nitrite trended higher in infants with BPD on DOL seven only (0.25μM (IQR 0.13, 0.39μM) vs. 0.17μM (IQR 0.10, 0.23μM)), p = 0.28) (Fig. 2d, f). Neither TA nor EBC nitrite was significantly associated with death.

4 Discussion

We found statistically significant correlations between nitrite measurements obtained via TA and EBC. This important finding serves as a basis to support further research in EBC as an alternative to TA collection for the prediction and diagnosis of lung inflammatory pathology. Measureable quantities of both volatile and non-volatile compounds from the lung can be retrieved by EBC without directly impacting the airway milieu. There were no adverse changes in ventilator setting or clinical status during collection of EBC (data not shown), demonstrating the safety of this technique. This safety profile is consistent with a previous report by Muller et al. [4].

Furthermore, we found that similar to TA nitrite, EBC nitrite collected in infants with BPD had a higher trend compared to those without BPD. This difference was more notable on DOL seven than on DOL three. The larger difference on DOL seven compared to DOL three may reflect cumulative injury and inflammation. An early marker of risk for BPD could be useful to guide therapy for specific affected infants. These findings are consistent with prior studies in children and adults in which EBC nitrite was elevated in oxidative injury and inflammatory conditions [22].

These findings demonstrate that EBC nitrite measurements are feasible in ELBW infants. With a mean birth weight of 688.8 grams and average gestational age of 25 weeks, this cohort represents the smallest and youngest in which EBC collection has been reported. Prior studies in newborns had a mean age of 32 weeks gestation and a mean birth weight of 2300 grams [26, 27]. Other studies have reported primarily on the use of EBC in infants older than one month of age [28].

An additional strength of this study is that all EBC samples were collected from intubated infants, bypassing the upper airway to selectively sample the lower airways and lungs and minimize the potential for oral or gastric contamination [29]. A recent American Thoracic Society/European Respiratory Society (ATS/ERS) Task Force acknowledged this issue and recommended that EBC be collected only from patients with ETT or tracheostomies [11]. This study used the standard approach set forth by ATS/ERS on oral EBC collection. Although the ATS/ERS Task Force reported that there was no concentration difference found in EBC collected over 10, 15, or 20 minutes, [11] we used 20 minutes to optimize the volume of collection. The average EBC volume collected was 2672.50μL (IQR 2100, 3422μL), and the detection limit of nitrite in EBC and TA approached 125nM, similar to a previous report [30].

This study has several limitations. It had a relatively small sample size, and may have been underpowered to detect differences in EBC nitrite between BPD and non-BPD infants on DOL three. Additionally, the BPD/death rate in our study was 87.5%, much higher than previous reports [3, 31]. This high rate is likely related to the low birth weight and gestational age of the subjects enrolled. It could also reflect a center-based difference in practice and outcome, and as such, may limit the generalizability of the results. A larger sample size would allow us to appropriately control and match gender and gestational age of the infants. The results of this pilot study justify further research using larger samples. Measurement of EBC nitrite at multiple time points, and inclusion of total length of ventilation in the model, may improve its predictive value for BPD.

Despite its safety and non-invasiveness, methodological issues related to EBC collection remain. Uncertainty surrounding the origin of the condensate and the dilution effect can make interpretation of results difficult. EBC contains 99.9% water, with significant variability of the concentration of non-volatile compounds between individuals [10]. Multiple strategies to calculate for a dilution marker exist, including comparison of airway lining fluid osmolality to serum osmolality, [32, 33] as well as measurements of total cation and ionic contents in EBC samples and its conductivity, [33, 34] the use of urea, [34 –36] and the use of conductance [37]. None of these strategies has been proven effective. With the exception of a few publications, [38, 39] dilution factor calculations are not generally used for interpreting EBC [9]. A few studies proposed that ratios among interactive or biologically related biomarkers eliminate the need for dilution markers [30, 40]. As pointed out by Kononikhin et al. [27] a single biomarker lacks the sensitivity and specificity needed to diagnose and monitor complex conditions. Further study would benefit from using one of the above strategies to account for the dilution factor.

5 Conclusion

This pilot study demonstrated the feasibility of collecting EBC in ventilated premature infants and that EBC nitrite is correlated with TA nitrite, indicating that it is a promising alternative to tracheal aspiration for sampling airway contents. EBC nitrite may be elevated in the first week of life as a marker of oxidative stress preceding the development of BPD. EBC nitrite, and other markers of oxidative stress and inflammation, may prove useful to guide therapy and improve pulmonary outcomes in premature infants.

Funding

The study was funded by New Jersey Health Foundation and supported by Rutgers Pediatric Clinical Research Center (CRC).

Disclosure statements

None of the authors listed in this manuscript has any potential or actual conflict of interests to disclose.

Footnotes

Acknowledgments

Special thanks to the infants and families who participated in this study, as well as to Laura Shanahan, RRT and the other respiratory therapists in helping with sample collections, Clarimel Cepeda (Laumbach Lab) and Changjiang Guo (Gow Lab) in sample handling and analysis.

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