INTRODUCTION

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Keywords: breathing control; mass spectrometry; peripheral chemoreceptors

Peripheral chemoreceptor function protects the body against changes in arterial PO₂. Peripheral chemoreceptors are active in utero (1), although in mammals their PO₂ threshold is reset to a higher level after birth (2, 3). The peripheral chemoreceptor component of ventilatory drive is thought to play a major role during the development of breathing control (4). Defective peripheral chemoreceptor function may contribute to sudden infant death syndrome (4). Therefore, testing peripheral chemoreceptor function may be clinically useful in infants. The first test described in the literature was the hyperoxic test (HT) (5). The alternate breath test (ABT) was developed more recently (2, 6).

The HT examines peripheral chemoreceptor function by measuring the decrease in minute ventilation (E) caused by hyperoxia as compared with normoxia (7). In an earlier study (13), we defined the HT response time as the time from hyperoxia onset to the first significant E drop during a 30-s HT and found that calculating the ventilatory response at the response time provided a reproducible assessment of peripheral chemoreceptor function. The E drop was associated with a significant decrease in tidal volume (VT), but not in respiratory frequency (13).

The ABT delivers rapid, alternating hypoxic stimuli to the peripheral chemoreceptors through breath-by-breath alternations in fractional inspired O₂ between normoxia and hypoxia (2). The ventilatory response is assessed by looking for significant alternations in E and in all its components, including VT, flows, and timing variables. Bilateral section of the carotid sinus nerve significantly reduces the ventilatory response to ABT in lambs, indicating that the respiratory variable alternations are produced mainly by a reflex arising from the peripheral chemoreceptors (14). ABT is a reproducible test of peripheral chemoreceptor function under standardized conditions in infants (15).

No previous studies have compared the ventilatory responses to both HT and ABT in the same infants or newborn animals. The present study compared the ventilatory responses to both tests in a group of 18 infants. Because both tests are believed to reflect the strength of the peripheral chemoreceptor drive, we hypothesized that the E drop during HT would be significantly related to E alternations during ABT. We examined the changes in respiratory variables underlying E changes during HT and ABT. Furthermore, we compared the reproducibility of HT and ABT by performing both tests twice during the same session in the study infants.

	METHODS

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Subjects

Eighteen infants were studied (mean gestational age, 36.6 ± 3.3 wk [range, 30 to 41]; mean postnatal age, 21 ± 14 d [range, 2 to 61]; mean body weight at time of study, 3.1 ± 0.9 kg [range, 1.9 to 5.0]). They were not healthy control infants. Of the 12 full-term infants, three had intrauterine growth retardation, two were born to diabetic mothers, and seven had neonatal infection. The remaining six infants were preterm; three had respiratory distress syndrome, which was managed by oxygen during the first 3 d, without mechanical ventilation. All 18 infants were free from neurologic, cardiac, and respiratory symptoms at testing. The parents gave their informed consent to participation of their infants in the study.

Protocol

A standardized protocol was used (13, 15). Tests were done during quiet sleep in unsedated infants (16). A face mask attached to a pneumotachograph was used (17). Each study infant underwent two successive HTs and two successive ABTs at intervals of 2 min, in random order. Each HT lasted 1 min and consisted of a 30-s normoxic control period, during which infants breathed room air, followed by a 30-s hyperoxic period, during which infants inspired 100% of O₂ from a bag and expired to room air (13). Each ABT lasted 4 min and included a 2-min control run (CR), during which ventilation alternated from a breath to a breath between ambient air and ambient air, followed by a 2-min test run (TR), during which breath-by-breath alternations were between ambient air and 15% O₂ (15).

Equipment

The experimental setup allowed measurement of respiratory flow, determination of O₂ and CO₂ fractional concentrations, and use of a fast respiratory valve to deliver gas mixtures during the HTs and ABTs (13, 15). Oxygen saturation was monitored throughout the tests.

Data Analysis

Respiratory flow was integrated for breath-by-breath measurements of the following respiratory variables: VT, inspiratory time (TI), expiratory time (TE), total respiratory cycle duration (Ttot), respiratory frequency (f), duty cycle (TI/Ttot), E, and mean inspiratory flow (VT/TI). Inspired (FI_O2, FI_CO2) and end-tidal (FET_O2, FET_CO2) fractions of O₂ and CO₂ were detected breath-by-breath. Numerical criteria were used to discard nonrepresentative breaths from HT and ABT calculations (13, 15). Ventilatory responses to HT (13) and ABT (3, 15) were determined as previously described.

Comparison of the Ventilatory Responses to HT and ABT

Correlation.In each infant and for each respiratory variable, we averaged the changes determined during the two HTs and the percentage alternation coefficients during the two ABTs. Only those respiratory variables that showed significant changes during both HTs and ABTs were used to compute the correlation coefficients and linear relationships between HT and ABT.

Principal components analysis (PCA).Only respiratory variables that showed significant changes during both HT and ABT were used for PCA (18).

Reproducibility of the ventilatory response.Interindividual and intraindividual coefficients of variation (CV) of the mean percentage alternation coefficients were computed during the two ABTs and of the changes in respiratory variables during the two HTs.

	RESULTS

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Average Sa_O2 was 97 ± 1% at baseline and 95 ± 2% during ABT, a nonsignificant difference. Sa_O2 remained above 92% during ABT in all infants. Minute ventilation and inspired and end-tidal gas fractions of O₂ and CO₂ in normoxia are given in Table 1 for the two HTs and the two ABTs. No significant differences were observed between normoxic conditions. Mean percentage of nonrepresentative breaths was 1.9% ± 0.6% and 1.7% ± 0.9% during ABTs and HTs, respectively.

Ventilatory Response to HT

During both HTs, all infants showed significant decreases in E, VT, and VT/TI. When the data were pooled over infants, E, VT, TI, and VT/TI exhibited significant percentage changes during both HT1 and HT2 (Table 2). When HT1 and HT2 were compared, no significant differences were found among changes in these respiratory variables. Neither were any significant changes seen during HTs for TE, Ttot, TI/Ttot, or f (Table 2).

Ventilatory Response to ABT

In all infants, mean percentage alternation coefficients in TR were significantly different from those of CR during both ABTs. For pooled data over infants, Table 2 shows mean percentage alternation coefficients of all respiratory variables in the TRs of ABT1 and ABT2. No significant differences were found between ABT1 and ABT2.

Comparison of HT and ABT

Correlation. The mean E decrease over the two HTs was linearly related to the corresponding mean percentage alternation coefficient over the two ABTs (r = 0.69, p < 0.01) (Figure 1A). A significant relationship was also found for VT/TI (r = 0.70, p < 0.01) (Figure 1B). No significant correlations were found for changes in VT and TI during HT and ABT.

View larger version (16K): [in this window] [in a new window]   Figure 1.   (A) Linear relationship between mean % decrease in E over the two HTs and mean % alternation coefficient of E over the two ABTs. (B) Linear relationship between mean % decrease in VT/TI over the two HTs and mean % alternation coefficient of VT/TI over the two ABTs.

Principal component analysis. PCA was applied to the respiratory variables that changed significantly during both HT and ABT. Figure 2 shows individual location of respiratory variables in a principal component space. VT/TI (HT) (factor loading = 0.87) and VT (HT) (factor loading = 0.85) determined axis 1. VT (ABT) (factor loading = 0.88) and TI (ABT) (factor loading = 0.86) determined axis 2. Thus, respiratory variables that collected much of the information were VT/TI and VT in HT and VT and TI in ABT.

View larger version (10K): [in this window] [in a new window]   Figure 2.   Individual location of respiratory variables in a principal component space. Solid circles indicate respiratory variables with a loading factor > 0.75 that contributed significantly to axis determination. Open circles refer to respiratory variables with a loading factor < 0.75. Axis 1 was determined by VT/TI (HT) and VT (HT) and axis 2 by VT (ABT) and TI (ABT). The units on those axes are arbitrary. The figure shows that the respiratory variables that collected much information were VT/TI and VT in HT and VT and TI in ABT.

Reproducibility. Mean intraindividual and interindividual CVs for changes in E, VT, TI, and VT/TI during HT and ABT are shown in Table 3. Intraindividual CVs for E, VT, and VT/TI were significantly lower for HT than for ABT (p < 0.01). Interindividual CVs of changes in E and VT showed large and similar values for both HT and ABT. Interindividual CVs of changes in TI and VT/TI were greater during HT than during ABT.

	DISCUSSION

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The aim of the present study was to compare ventilatory responses to HT and ABT in infants. Because both tests are believed to reflect peripheral chemoreceptor function, we hypothesized that the ventilatory responses they elicit should be related to each other. We found that E and VT/TI decreases during HT were significantly related to the corresponding percentage alternation coefficients during ABT. PCA showed that the E decrease during HT was mainly explained by the fall in VT and that the E alternations during ABT were explained by alternations in both VT and TI. Intraindividual CV of E changes were significantly lower during HT than during ABT.

Subjects and Measurement Conditions

Our study infants were not healthy control infants. Therefore, our study does not provide normative data on ventilatory responses to HT and ABT. However, because all study infants exhibited a significant response to HT and ABT, our data allowed us to compare ventilatory responses to both tests. The study objective was to compare ventilatory responses to HT and ABT performed in random order in the same infants. Therefore, we used an experimental setup involving breath-by-breath quantitative measurement of respiratory variables and gas fractions. This required application of a face mask attached to a pneumotachograph. Face-mask application disrupts the breathing pattern for 10 to 40 s (19). Consequently, we started HTs and ABTs at least 1 min after face-mask application. FI_CO2 remained below 0.3%, indicating that there was probably no sustained elevation in E caused by CO₂ rebreathing (Table 1). That ABT is safe has been established in earlier studies (6, 15, 20) and was confirmed in our study. A risk of retinopathy associated with exposure to high concentrations of oxygen has been reported in preterm infants weighing less than 1,200 g (21). All our study infants weighed far more than 1,200 g.

Hyperoxic Test

HTs in infants have been performed using various durations of O₂ inhalation, ranging from one or two breaths (7, 8) to 30 s (9). However, one or two breaths of O₂ inhalation may not be sufficient to ensure complete elimination of peripheral chemoreceptor input (22). We used O₂ inhalation for 30 s. Furthermore, we measured the ventilatory response to HT using breath-by-breath analysis at the response time, defined as the time from HT onset to the first significant E change. In an earlier study (13), we showed that the ventilatory response at the response time was significantly greater than the mean percentage E change over a 30-s HT (13). Ventilatory response at the response time is thought to be dependent only on suppression of the peripheral chemoreceptor drive, whereas the mean percent decrease over 30 s reflects both the initial drop caused by physiologic chemodenervation and the early phase of the subsequent increase in ventilation related to the metabolic response to hyperoxia in infants (23).

Alternate Breath Test

In previous studies in infants, breath-by-breath alternations in fractional inspired O₂ were delivered through a nasal catheter, and respiratory responses were measured noninvasively using an uncalibrated inductive plethysmograph, a setup that does not allow measurement of inspired and end-tidal gases (6, 20, 24). With our experimental setup, in contrast, we were able to measure inspired and end-tidal O₂ and CO₂ gas fractions. The constancy of FET_CO2 during ABT in our study shows that normocapnia was maintained. We verified that FI_O2 remained within a narrow range during each alternate hypoxic breath, and we removed from the raw data all TR breaths that had an FI_O2 greater than 0.17 because of defective computer detection of the end of expiration. The FET_O2 recordings showed stable FET_O2 oscillations reflecting the alternating peripheral chemoreceptor stimulation. The ABT repeatedly delivers a hypoxic stimulus to the peripheral chemoreceptors over a large number of breaths, allowing averaging of the response. Our study infants exhibited significant ventilatory responses to ABT, including alternations in VT, mean inspiratory flow, and timing variables, as previously reported in a smaller group of infants (15).

Comparison of the HT and the ABT

The experimental setup involving breath-by-breath quantitative measurement of respiratory variables and gas fraction measurements allowed us to compare ventilatory responses to HT and ABT performed in random order in the same infants. The results showed significant correlations between HT and ABT for E and VT/TI changes. These data support our hypothesis that two tests believed to reflect peripheral chemoreceptor function should elicit significantly related ventilatory responses despite opposite effects on peripheral chemoreceptor output to inspiratory drive. PCA corroborated our previous data by showing that the E and VT/TI decreases seen during hyperoxia were due mainly to a VT decrease (13). Hyperoxia had no effect on f and a slight but significant effect on TI. In contrast, ABT is known to induce significant alternations in all timing variables, as well as in VT and flows (15). In the present study, PCA showed that the alternations in E and VT/TI were ascribable to alternations in both VT and TI. The hypoxic stimulus delivered during ABT modifies both VT and the duration of inspiration. The effect of hypoxia on respiratory timing has been shown to be mediated in large part by the peripheral chemoreceptors (25).

Our study is the first to compare the reproducibility of ventilatory responses to HT and ABT during a single session in the same infants. In keeping with previous studies on different populations (13, 15), the present study showed that intraindividual variability of the ventilatory responses was lower during HT than during ABT (13, 15). Intraindividual CV of the E drop during HT was half the CV of E alternations during ABT in the study infants. This finding suggests that the HT may be the best test in clinical practice, especially to follow infants with deficient peripheral chemoreceptor function (26). However, interindividual CV showed large values for the E drop during HT and the E alternations during ABT. The interindividual variability in ABT in our study population was within the range previously reported in healthy infants, although these were exposed to a broader range of inspired O₂ fraction alternations than the infants in the present study (27).

It is unlikely that the large interindividual variability was related to measurements conditions because we used standardized protocols (13, 15). Rather, differences in peripheral chemosensitivity may contribute to the substantial interindividual variability of the E change during both tests (28). This variability may be a limitation for testing peripheral chemoreceptor function. Nevertheless, both tests have been found effective in demonstrating deficient peripheral chemoreceptor function in infants with bronchopulmonary dysplasia as compared with control preterm infants (12, 24). However, it may be difficult to test peripheral chemoreceptor function in infants with abnormal lungs. During ABT, poor alveolar gas mixing may prolong the time interval between the onset of inspired gas alternation and the occurrence of stable FET_O2 oscillations, thereby resulting in nonsignificant ventilatory alternations during ABT. Poor alveolar gas mixing would also be expected to limit the usefulness of HTs. Breath-by-breath analysis during a 30-s HT has the advantage of allowing determination of the response time even in infants with abnormal lung function (12).

In conclusion, we showed for the first time that ventilatory responses to HT and ABT as assessed based on E and VT/TI changes are significantly correlated with each other, although the mechanisms underlying the E changes during the two tests are different. However, the E change during HT had only half the variability of the E change during ABT, suggesting that HT may be more useful in clinical practice. Chemoreceptor dysfunction has been suggested as a risk factor for sudden infant death syndrome (4). Environmental factors such as perinatal hypoxia (2) have been reported to impair peripheral chemoreceptor function. Consequently, a reproducible test of chemoreceptor function may be useful in investigating the control of breathing in high-risk infants (29).