Exhaled breath condensate in intubated neonates- a window into the lung’s glutathione status

Intubated newborns were enrolled from the NICU at Children’s Healthcare of Atlanta at Egleston (CHOA), a level IV intensive care unit within the Emory Division of Neonatology System, in the city of Atlanta from January 2011 through October 2012. Informed consent was obtained from guardians using procedures approved by the Institutional Review Board at Emory University School of Medicine (Gauthier, IRB00038093). All intubated babies were eligible for enrollment. Exclusion criteria included patients deemed too unstable by the attending neonatologist or those on high frequency ventilation, inhaled nitric oxide, or extracorporeal membrane oxygenation (ECMO). Clinical information was ascertained from the electronic medical record.

Liquid condensate was collected from the exhaled breath of mechanically ventilated newborns for 30 minutes. The EBC collection device consisted of an RTUBE™ (Respiratory Research, Inc. Austin, TX) encased in an aluminum cooling sleeve that was cooled to -80°C for at least 2 hours prior to use. A modified collection device was used to collect EBC off the ventilator circuit. The RTUBE™ and cooling sleeve, covered by a blue insulating cover, were connected to the ETT and to the expiratory limb of the ventilator circuit via valve-less connectors provided in most standard ventilator circuits (Figure 1A). Figure 1B depicts a simulated collection from an intubated neonatal mannequin. Immediately after collection, the condensate tubes were capped, placed on wet ice and transported to the laboratory for analyses. The process of EBC collection was monitored at the bedside by the investigators (MR, SR, ET) and was tolerated well by all subjects, without significant changes in heart rate, oxygen saturation or blood pressure.

TA samples where obtained within one hour of EBC collection. For the suctioning procedure, bacteriostatic saline (~1 ml) was instilled into the trachea. After several ventilator breaths, an in-line suction catheter (Ballard, Kimberly-Clark, Irving, TX) was inserted just past the endotracheal tube and the sample was then retrieved into a closed, sterile Lukens trap (Covidien, Mansfield, MA). Additional bacteriostatic saline was used if necessary to clear the sample from the suction catheter. Samples were immediately placed on wet ice and transported to the laboratory for analyses.

Patients and samples were de-identified with a study number to ensure confidentiality. All samples were transported to the laboratory on wet ice within an hour of collection. In previous studies, we determined that the rate of oxidation was ~5% per hour if the sample was maintained in a sterile Lukens trap on wet ice [21]. TA samples were centrifuged for 10 minutes to remove the cell pellet. EBC was extracted from the RTUBE with a plunger apparatus. To prevent auto-oxidation before analysis, 240 μL of the EBC was aliquoted into pre-made tubes containing 21 μL of a diluted preservative with a final concentration of 5% perchloric acid, 0.1 M boric acid, 6.7 mM iodoacetic acid (IAA), and 5 nM of the internal standard γ-glutamyl-glutamate as we have previously described [22]. For TA, 250 μL of sample was added to pre-made tubes containing 250 μL of preservative for a final concentration of 10% perchloric acid, 0.2 M boric acid, 13.4 mM IAA, and 10 μMγ-glutamyl-glutamate. All samples were stored at -80°C until batch analysis by HPLC, also previously described by this laboratory [21, 23]. GSH and GSSG fractions were treated with dansyl chloride and the dansylated derivatives separated using an amino μBondaPak column (Waters, Milford MA) with fluorescent detection. The lower limits of detection were 15 fmol/μl for rGSH and were consistent with studies by Komatsu and Obata [24] which used similar methods and reported similar lower limits of detection. All of the samples in this study were above the noted limits of detection. To minimize operational variability, all samples from this study were analyzed within the same HPLC run. Inter-assay coefficient of variability was determined by performing HPLC analysis on a mixture of standards including cysteine, cystine, rGSH, and GSSG. This mixture was analyzed immediately before, in the middle and at the end of sample set analysis. Based on this cocktail of standards, the inter-assay coefficient of variability was calculated at 1%. Given the limited sample volume obtained, we were unable to calculate intra-assay variability.

The subject’s blood urea nitrogen level (BUN, mg/dl) was noted in the medical record on the day samples were obtained. Sample urea was measured using the Bethleot/ Colorimetric assay (Pointe Scientific, Canton, MI - sensitivity range of 0.05 to 150 mg/dl). Sample concentrations of rGSH, GSSG and total GSH (defined as: GSH + (2 X GSSG)) are presented as both raw values and urea corrected values. Samples were corrected for dilution by multiplication with the urea dilution factor (f) defined as f = Patient BUN/ sample urea used was as previously described for EBC samples [21].

TA samples where centrifuged as noted above to obtain cells (300 g, 10 minutes). The supernatant was processed as above and the cell pellets where re-suspended in RPMI 1640 media with 10% fetal calf serum (FCS) and plated under sterile conditions for 1 hour. The media was removed and the adhered cells washed three times with phosphate buffered saline (PBS). Cells where then fixed with 4% paraformaldehyde solution for 30 minutes, washed with PBS and stored at 4°C. At the time of analysis non-specific binding was blocked with 10% bovine serum albumin (BSA, 1 hour) and the cells were stained for protein-glutathione adducts (primary mouse monoclonal IgG GSH, 2 hours) (Virogen, Watertown, MA). After washing, a secondary Alexa fluorescence 488 green anti-mouse IgG was added for 2 hours (Life Technologies, Carlsbad, CA). The cells were identified as AM by cell morphology and then analyzed for fluorescence via confocal fluorescent microscopy (Olympus FluoView FV1000). The cellular fluorescence was quantified as relative fluorescent units (RFU) /cell as we previously described [25].

All data were analyzed with SPSS® for Windows software (Version 18.0, SPSS Inc., Chicago, IL) and SAS 9.3 (Cary, NC). Prior to analysis, data were assessed for normality using histograms, normal probability plots, and the Kolmogorov-Smirnov test of normality. For concentrations that were non-normally distributed, a log₁₀ normalizing transformation was applied. Pearson’s correlation coefficient and associated 95% confidence intervals were used to quantify the association between TA and EBC concentrations. Scatter plots were used to assess whether the relationship between two concentrations appeared linear and to confirm that the Pearson’s correlation coefficient was an appropriate measure of association. Statistical significance was defined as p ≤ 0.05 using two-tailed tests. The sample size was determined in order to detect a minimum correlation coefficient of 0.5, with 0.8 power and an alpha of 0.05.

Twenty-six (26) intubated newborns on mechanical ventilation were enrolled (Table 1). Enrolled subjects had a median birthweight of 1564 grams (interquartile range (IQR) 698, 2914 grams) and a median gestational age of 32.5 weeks (IQR: 24, 38 weeks). At the time of EBC and TA collection, the median weight was 2280 grams (IQR: 1500, 3254 grams) with a corrected gestational age of 35 weeks (IQR: 34, 39.5 weeks). Subjects were ventilated for a mean of 21 days (IQR: 7, 59.5 days) at the time of sample collection. As noted in Table 1, the majority of patients were admitted for surgical procedures.

The median volume recovered from the TA was 0.75 ml (IQR: 0.6, 1.75 ml) while the median EBC volume recovered after 30 minutes of sampling was 2.2 ml (IQR: 1.5, 2.5 ml). GSH, GSSG and total GSH were detected by HPLC in all samples (100%) in the nM range. The urea dilution factors, raw concentrations, and urea-corrected concentrations are presented in Table 2.

We postulated that rGSH and GSSG values measured in the TA would correlate with those measured via a non-invasive EBC. As seen in Figure 2, urea-corrected rGSH in the TA demonstrated a moderate positive correlation with urea-corrected rGSH in the EBC. Similarly, concentrations of TA rGSH not normalized to urea moderately correlated with EBC rGSH concentrations not normalized to urea (r = 0.574, p = 0.002, 95% CI: (0.230-0.782), R² = 0.329). Interestingly, the duration of mechanical ventilation demonstrated a moderate negative correlation with urea-corrected rGSH in the EBC (r = - 0.574, p = 0.003, 95% CI: (-0.779 – (-0.209), R² = 0.329) but did not correlate with urea-corrected rGSH in the TA (p = NS). Figure 3 demonstrates a strong positive correlation between urea-corrected GSSG in the TA and urea-corrected GSSG in the EBC. A similarly strong positive correlation existed between TA GSSG concentrations not normalized by urea and EBC GSSG concentrations not normalized to urea (r = 0.708, p < 0.001, 95% CI: (0.431 – 0.856), R² = 0.501). Although the fraction of oxidized GSH (i.e. the % GSSG) was approximately two times higher in the EBC than in the TA (EBC: 44.8 ± 4.6% vs TA: 21.8 ± 3%, p < 0.001), there was no significant correlation between the TA % GSSG and the EBC % GSSG (p = NS). When total GSH was compared between the two sampling sites, a moderate positive correlation was demonstrated in both urea-corrected values (Figure 4) and in non-urea normalized values (r = 0.649, p < 0.001, 95% CI: (0.338 – 0.824), R² = 0.421).

Since we hypothesized that the GSH in the TA or EBC sample may be reflective of cellular GSH in the isolated AM, we evaluated the correlation of total GSH measured in TA and EBC with AM GSH staining determined via immunofluorescence. Fluorescent staining for protein-GSH adducts in the AM demonstrated a moderate positive correlation with the urea-corrected concentration of total GSH in the TA and the EBC (Figure 5A and B). Furthermore, AM demonstrated a moderate positive correlation with urea-corrected concentrations of GSSG in the TA (Figure 6A) and in the EBC (Figure 6B). While we did not observe a correlation between AM GSH and urea-corrected concentrations of rGSH in the TA (p = NS), a moderate positive correlation was found with urea-corrected concentrations of rGSH in the EBC (r = 0.571, p = 0.004, 95% CI: (0.195 – 0.791), R² = 0.326).