The use of iNO in premature infants has only recently been investigated in a series of randomized, controlled clinical trials.4, 5, 6, 16
Most trial protocols for the use of iNO to prevent BPD have used <14 days of treatment at
p.p.m. iNO. The NO CLD trial was unique in its use of decreasing concentrations of iNO starting at 20
p.p.m. and in continuing treatment for 24 days by nasal cannulae when an infant was extubated. This protocol provided the opportunity to monitor NOx
at different iNO doses over an extended period. We report a dose-related effect of iNO on levels of NOx
in both TA and plasma, indicating effective delivery of NO to the injured lung and systemic uptake of exogenous NO and/or its metabolites. To our knowledge, this is the first description of metabolites related to use of iNO in the infant. In addition, we provide new information on endogenous NO levels, as reflected by metabolite concentrations, in premature infants. At study entry during the second or third postnatal week, before receiving study gas, total metabolite levels were not associated with degree of prematurity or severity of lung disease. However, a sub-population of the study infants with lower metabolite levels seemed to have a better clinical response to iNO, consistent with the concept of iNO as replacement therapy for a developmental deficiency of endogenous NO production.3, 17
In the premature baboon model of BPD, iNO at 5
p.p.m. for 2 weeks beginning at birth improved respiratory function and lung structure.3
The level of total NOx
in TA fluid did not change, consistent with our observations in the human infant at this dose.18
levels were increased ~twofold in treated animals throughout the time of iNO administration, which is similar to the findings at 5 to 10
p.p.m. iNO in this study of human infants.
As NO cannot be directly measured in stored biological samples, levels of nitrite and total NO oxidative metabolites were used as surrogate markers for NO. Nitrite represented ~14% of NOx
in TA fluid, indicating that nitrate and potentially other NO metabolites constitute most of NO species present. Although it is clear from both this study and the baboon model that iNO can lead to an increase in plasma-borne NOx
, it is uncertain whether NO and/or its metabolites diffuse across the lung. Studies in mice and rabbits showed that within 10
min after intratracheal instillation of nitrite, the levels in lung and plasma were equal.19
Nitrite represents a portion of the bioactive pool of NO metabolites and has been shown to provide protection after ischemia–reperfusion injury in a variety of tissues, including the brain, in animal studies.9
In this regard, it is noteworthy that Kinsella et al.5
showed on head ultrasound an overall decrease in the combined outcome of grade 3 or 4 intraventricular hemorrhage, periventricular leukomalacia, and ventriculomegaly in their iNO-treated group. Furthermore, Mestan et al.20
described an improvement in neurodevelopmental outcome, specifically in the cognitive portion of the Bayley Scale of Infant Development, in the iNO-treated infants compared with control in the Schreiber trial,4
which persisted after adjustment for the decrease in severe intraventricular hemorrhage and periventricular leukomalacia. The observations of elevated NOx
support the possibility for extrapulmonary effects of iNO in infants that may be independent of improved pulmonary status.
There were no associations observed between TA or plasma NOx
concentrations at study entry and measures of prematurity or severity of lung disease. Infant baboons delivered prematurely (at the end of the second trimester) are deficient in endogenous NO production, and NO synthase activity remains suppressed after birth.17
Our observations suggest that endogenous NO production in sick preterm human infants does not increase appreciably between 24 and 30 weeks, or, alternatively, that developmental changes are not observed within the postnatal timeframe of this study. The concentration of plasma NOx
in control infants (36.4
μ) is somewhat lower than the level reported for healthy full-term infants on day 5 (55.2
μ), perhaps reflecting an effect of gestational age.21
Infants with lower TA NOx
levels at entry seemed to have a better clinical response to iNO therapy (survival without BPD) than infants with higher entry NOx
, consistent with the concept that insufficiency of endogenous NO contributes to BPD. An alternative hypothesis is that the infants in the lowest quartile have a lesser degree of inflammation or oxidative stress at study entry. To support this hypothesis, we examined the 8-epi PGF 2α levels in these infants and found them to be lower (86
protein) than what we have earlier reported in a larger group of iNO responders,12
but there was wide variability in this measurement (0.35 to 534
protein). These results should be interpreted with caution and need confirmation because of the relatively small number of infants in each quartile of NOx
Conversely, we did not find an association with NOx
levels and adverse outcome at any dose in either the iNO-treated or control infants in this study. Specific NO metabolites have been described before in relation to BPD and response to therapy. Banks et al.
showed that a specific NO metabolite, 3-nitrotyrosine, is elevated in plasma in the first month of life in infants who develop BPD. Furthermore, increased levels of 3-nitrotyrosine after the first month of life correlated with the fraction of inspired oxygen the infant was receiving.22
Lorch et al.
described the plasma levels of 3-nitrotyrosine in infants with BPD who were receiving iNO. A significant decrease within the first 72
h after starting therapy was observed in those infants who were weaned off mechanical ventilation compared with those who remained on mechanical ventilation at discharge or who expired.23
These data suggest that markers of nitrative and oxidative stress are associated with both BPD and response to therapy. We have previously reported from data in the NO CLD trial that 3-nitrotyrosine did not change in infants with and without BPD, whether they were receiving iNO or placebo.13
This discrepancy may be explained by differences in study population, severity of illness, progression of clinical course, when samples were taken, or differences in assay techniques. However, given that 3-nitrotyrosine is a small component of total NOx
metabolites, it is not surprising that we did not see changes in this study.
There are several limitations to this study. First, TA samples could not be obtained after infants were extubated, which reduced the number of samples available at 5 and 2
p.p.m. and accordingly reduced the power of our study at these points. In general, variability in the TA data was greater than in plasma values, which reflects in part the necessity to use an imperfect denominator (total protein) to correct for dilution of epithelial lining fluid by the lavage saline. Second, infants from whom samples were received were from a subset of NO CLD trial sites enrolling patients. Although their demographics were the same as infants in the parent trial, and, therefore, representative of the entire cohort, this remains a limitation. Third, although infants in this study had similar nutritional management strategies, there was no specific control over or measurement of nitrate intake, which may influence the measurement of NOx
within plasma. Lastly, as NO cannot be directly measured within these samples, one must rely on measurements of its most abundant metabolites as markers of NO content.
In conclusion, we have shown that iNO therapy for premature infants results in dose-related increases in NOx in both lung fluid and the circulation. These findings indicate the efficiency and quantitative nature of NO delivery to infants with lung disease and raise the possibility of increased delivery of NO to other tissues with iNO therapy. As iNO treatment is not associated with increased incidence of co-morbidities of prematurity, and may offer neuroprotection, uptake of NO and metabolites into the circulation may represent an additional benefit of iNO for the at-risk premature infant.