INTRODUCTION

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THUS FAR, THE PHYSIOLOGICAL ROLE of the paranasal sinuses in mammals has not been clarified: olfaction, humidification and warming of inhaled air, resonation, and lightening of the skull, none of these is of importance (11, 12). Recently, Lundberg et al. (6) showed that large amounts of nitric oxide (NO) are produced in human paranasal sinuses. From the paranasal sinuses NO is continuously excreted into the upper airways during normal breathing and will follow the airstream to the lower airways and lungs with each inhalation. The autoinhalation of NO may contribute to the regulation of pulmonary blood flow (2, 6, 7) or may play a role in host defense (8). High exhaled or nasal NO levels were found in humans (6), elephants (5), and rhesus monkeys (13), all mammals with paranasal sinuses.

We hypothesize that the physiological role of nasal NO production from sinus cavities may be better understood through studies of comparative anatomy and physiology. Therefore, we measured exhaled and nasal NO from 17 healthy baboons (Papio hamadryas), which are the only mammals known to lack paranasal sinuses (11) to determine whether significant nasal NO concentrations could be found in the absence of paranasal sinuses.

	METHODS

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In the investigation, 17 healthy baboons (P. hamadryas) from the German Primate Center, Göttingen, Germany, were used. The 17 animals belonged to a colony kept for behavioral studies in an open-air enclosure. Once a year, the animals are rounded up and anesthetized with ketamine hydrochloride (10 mg/kg body wt im; Ketavet, Parke-Davis, Berlin, Germany) for veterinary examination, taking of blood samples, determination of weight and height, prophylactic inoculation against tuberculosis, and tattooing of an international animal number. We took advantage of this annual routine examination and measured the concentration of NO in the exhaled gas and nasal airways while the animals were anesthetized and lying in a supine position.

Radiological investigation of nasal airways. In the literature, only one reference exists on the absence of paranasal sinuses in baboons (11). For confirmation, we radiologically investigated two skulls of the species P. hamadryas (see Fig. 1).

View larger version (88K): [in this window] [in a new window]   View larger version (104K): [in this window] [in a new window]   Fig. 1.   A: frontal (top left), lateral (top right), and craniocaudal (bottom right) view of skull of adult baboon (Papio hamadryas). X-rays were performed at 65 kV with conventional film-foil system (Diagnost 76, Philips, Netherlands). X-rays clearly show that no paranasal sinuses are present. Large nasal cavity is recognizable. B: computerized tomography (CT) study of baboon skull. Shown are 6 coronal slices of 2-mm thickness. Large nasal cavity is clearly visible. There are no paranasal sinuses.

Measurement of NO in exhaled gas (mask technique). NO was measured in exhaled gas while the baboons were breathing spontaneously through an anesthesia face mask connected to a respirator (Siemens Servo 900 C ventilator, Elema, Sweden) that was set on the pressure-support mode at 8 cmH₂O, positive end-expiratory pressure at 3 cmH₂O, and trigger sensitivity at 0.5 cmH₂O. The face mask was tightly fitted to the animals by the investigator, and the animals' mouths were closed. The baboons breathed synthetic air (fraction of inspired oxygen 0.21) with a NO concentration of 0.12 parts/billion (ppb) via the inspiratory limb of the ventilator. Gas was continuously sampled for 60 s from the expiratory limb via a thin plastic tube with a constant flow rate of 1.35 l/min and analyzed online for NO concentration by chemiluminescence. The chemiluminescence analyzer (CLD 780 TR, ECO Physics, Duernten, Switzerland) was equipped with a prereaction chamber to minimize interference of hydrocarbons and NH₃ and had a lower detection limit of <50 parts/trillion. A dry-ice trap was included to remove water vapor from the detector sampling line. Calibration was performed before any analyses by using a tank containing 208 ppb NO in N₂. The chemiluminescence analyzer was preset to an integration time of 6 s. Expiratory flow was measured with a Wright spirometer positioned in the expiratory limb of the respiratory circuit before the NO suction.

Measurement of NO in the upper airways (nasal sampling). In the conducting of direct nasal sampling, a thin plastic tube was connected to a nasal olive that was gently introduced into the vestibulum of one nostril, avoiding contact with the nasal mucosa. The contralateral nostril was left open. Nasal gas was continuously aspirated via the tube for 60 s with a sample flow rate of 1.35 l/min and analyzed online for NO concentration by chemiluminescence (integration time 6 s). During the measurements the animals were spontaneously breathing ambient air. NO concentration in the ambient air was 2.52 ± 1.53 ppb during the measurements, representing the inspiratory NO concentration.

Statistical analysis. In the setting described above, the chemiluminescence analyzer emitted discrete readings of the NO concentrations every 6 s. The reported NO concentration for each animal is the mean value of the 10 NO concentrations measured during the whole 60-s registration period. In this study all measured NO concentrations were corrected for inspiratory (mask technique) or ambient (nasal sampling) NO concentrations according to the formula NO concentration (corrected value) = measured NO concentration  inspiratory or ambient NO concentration. Only NO concentrations that were corrected for inspiratory/ambient NO levels are reported in RESULTS. NO release was calculated according to the formula NO release (nl/min) = NO concentration (corrected value) (ppb) × flow rate (expiratory flow or sample flow rate, according to measurement method) (l/min). Results of the descriptive statistics are expressed as means ± SD.

	RESULTS

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Frontal and lateral radiographs and computerized tomography scans of the skulls of two baboons (species P. hamadryas) clearly showed the absence of any paranasal sinuses. The radiographs and computerized tomography scan of one of the baboon skulls are presented in Fig. 1.

A total of 17 baboons were investigated regarding exhaled and nasal NO concentrations, 12 females and 5 males. The animals ranged in age from 0.6 to 16 yr, and the mean age was 5.6 ± 6 yr (median 1.9 yr). Weight was 9.8 ± 6.3 kg, and height, measured from head to breech, was 48.5 ± 10.2 cm. During the measurements the animals breathed with a minute volume of 4.30 ± 1.27 l/min (spontaneous respiration via anesthesia face mask connected to a Servo 900 C respirator, 8 cmH₂O pressure support, positive end-expiratory pressure 3 cmH₂O). Minute volume ranged from 2.8 to 7.1 l/min, correlating with the baboons' age (r = 0.67, P = 0.003).

Sampling of exhaled gas via face mask in healthy baboons yielded NO concentrations of 1.00 ± 0.59 ppb. When gas was sampled directly from the nose of the animals, NO concentrations of 4.79 ± 2.08 ppb were recorded (Fig. 2A). Corresponding NO release was 4.28 ± 2.72 nl/min for the mask technique and 7.18 ± 3.13 nl/min for the nasal sampling technique (Fig. 2B). The scatterplots of NO concentrations and NO release from the 17 baboons are displayed in Fig. 2. The differences between mask and nasal NO levels were statistically significant. A statistically significant correlation was found between the NO concentrations measured with the mask technique and nasal sampling (r = 0.55, P = 0.024).

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Scatterplot of nitric oxide (NO) concentrations (A) and NO release (B) in exhaled gas and upper airways of 17 healthy baboons. Horizontal bars, means. Mask, NO concentrations and NO release were determined in exhaled gas while baboons were breathing continuously through a face mask; nasal, NO concentrations and NO release were determined in gas directly sampled from 1 nostril. ppb, Parts/billion. Differences between mask and nasal NO levels were statistically significant (Wilcoxon signed-rank test, 2-tailed).

No correlation was found between age and NO levels detected with the mask technique (NO concentration vs. age: r = 0.00, P = 0.992; NO release vs. age: r = 0.31, P = 0.225). Furthermore, no correlation was detected between age and NO levels sampled from the nose (NO concentration vs. age: r = 0.06, P = 0.828; NO release vs. age: r = 0.06, P = 0.827).

	DISCUSSION

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We report that baboons, the only known mammals to lack paranasal sinuses, have very low nasal NO concentrations and nearly undetectable NO levels from samples of exhaled gas. These findings are in striking contrast to past findings in humans (1, 4, 9) and other small and large mammals (3, 5, 13, 15) that have shown much higher NO levels.

Origin of exhaled NO. Knowledge concerning the origin of exhaled NO mainly derives from studies in humans. Lundberg et al. (6) showed that human paranasal sinuses contain high concentrations of NO that are excreted into the upper airways. The authors also identified the paranasal epithelium as a major production site of NO in humans, whereas the nasal epithelium produces only small amounts of NO. Kimberly et al. (4) reported that increasingly high NO concentrations can be recorded during breath holding via a NO-sampling catheter positioned in the nasal cavity, whereas samples from the pharynx and trachea contained only low NO concentrations. It can be deducted that the major portion of exhaled NO is produced in the paranasal sinuses and flows continuously from there into the nasal cavity.

Reason for the investigation of baboons. It has been demonstrated that exhaled and nasal NO can be found in humans, rats, rabbits, guinea pigs, minipigs, rhesus monkeys, horses, and elephants (3, 5, 10, 13, 15). There are some differences, however, in the exhaled NO levels among the species (see discussion of this issue in Comparison of our results with those in other species: exhaled NO levels). NO could not be detected in the exhaled gas of seals (14). Research procedures in humans indicate that nasal NO is mainly produced in the paranasal sinuses (6). The differences in NO exhalation among the species are possibly correlated with anatomic or functional specialities of the paranasal sinuses, the functional role of which has not yet become clear. An interesting observation is that all mammals that exhale NO have open paranasal sinuses. Findings in seals, which do not have open sinus air cavities (11) and do not exhale NO (14), support our observation. The data from seals, however, should be interpreted with caution because these animals have paranasal sinuses, but the sinuses are completely filled out with maxilloturbinal bodies. In addition, seals are not land-living mammals and were measured with a different technique under extreme climatic conditions. The functional role of the paranasal sinuses for nasal NO levels can be better understood and interpreted by having more knowledge of comparative physiology. Baboons were selected for this study of exhaled and nasal NO concentrations because this species has an exceptional characteristic. Baboons are the only mammals without paranasal sinuses, which makes them the ideal object for further investigation. Because we wanted representative data from a larger animal collective living under the utmost natural conditions to exclude effects of laboratory conditions and animal captivity, we studied baboons living in a colony that was composed similarly to those of free-living animals.

Comparison of our results with those in other species: methodological considerations. The first evidence that NO is exhaled by mammals was provided by studies in humans and some laboratory animals (rats, rabbits, guinea pigs) (3, 15). The laboratory animals were tracheotomized and mechanically ventilated while NO was detected in the expiratory limb of the ventilator. The applicable method sampled exhaled NO mainly from the trachea. Through studies in humans it is known that the site of sampling markedly influences the measured concentration of exhaled NO (4); thus it is not valid to compare a method that samples from the trachea with a method sampling from the nose or exhaled gas. Results obtained with comparable methods, that is, NO concentrations determined by either nasal sampling or the mask technique, have been published only with reference to humans, elephants, horses, seals, rhesus monkeys, and minipigs.

Comparison of our results with those in other species: exhaled NO levels. Our results show that baboons, when breathing spontaneously through a face mask, exhaled NO concentrations of only 1 ppb, the corresponding calculated NO release being 4.3 nl/min. With the animals' mouths closed during spontaneous ventilation via a face mask, the NO value can be attributed to nose breathing. Compared with the findings of other studies, the NO concentration in the exhaled gas of baboons is extremely low. Lundberg et al. (9) recorded exhaled NO concentrations of 22 ± 3 (SE) ppb in four nonsmoking healthy adults. The subjects breathed normal tidal volumes through their noses while exhaled gas was analyzed for NO concentrations. In elephants, NO concentrations of ~19-28 ppb were found in the exhaled gas while the animals were nose breathing (5). Alving et al. (1) measured NO concentrations of 23 ± 2 (SE) ppb in the exhaled gas of 12 healthy adults who were spontaneously nose breathing through a tight face mask. Using the same technique, Kimberly et al. (4) found NO concentrations of 30.7 ± 1.8 (SE) ppb in eight healthy adults, the corresponding NO release being 141 ± 17 nl · min¹ · m². Mills et al. (10) reported exhaled NO concentrations of 3.25 ± 0.75 ppb obtained by a mask technique in five horses at rest. The corresponding minute volume of the horses at rest was ~350 ml · min¹ · kg¹. From these data, the NO release can be calculated to be 3.25 ppb × 0.35 l · min¹ · kg¹ = 1.14 nl · min¹ · kg¹. Compared with our findings in baboons (NO release of 4.3 nl/min, i.e., 0.44 nl · min¹ · kg¹), the NO release in horses is 2.6-fold higher. Our findings reveal that the levels of exhaled NO in baboons are lower than those in humans, elephants, and horses.

Comparison of our results with those in other species: nasal NO levels. When gas was sampled directly from the baboons' noses, slightly higher NO concentrations of 4.8 ppb (corresponding NO release was 7.2 nl/min) were found than in the exhaled gas. Comparison of our data with the findings from other authors is limited to some studies in humans and only anecdotal data from those in rhesus monkeys and minipigs. In humans, Lundberg et al. (9) found 270 ± 37 (SE) ppb NO in the gas sampled directly from the nose of four healthy adults while they were mouth breathing. Kimberly et al. (4) reported 334 ± 52 (SE) ppb NO sampled via bronchoscope in the nasopharynx of five healthy adults during mouth breathing. Nasal NO release was determined to be 217 ± 19 nl · min¹ · m² during mouth breathing (n = 8). Nasopharyngeal NO levels decreased to 29 ± 7 (SE) ppb when the subjects breathed through their noses. Schedin et al. (13) measured nasal NO levels in three anesthetized, spontaneously breathing rhesus monkeys and in two minipigs that were intubated and mechanically ventilated. In rhesus monkeys they obtained plateau concentrations that averaged 237 ± 10 (SE) ppb. In the two minipigs, lower nasal NO concentrations of 18.1 ± 0.5 ppb were found. Unfortunately, no NO release rates were calculated in this study. The nasal NO concentrations in baboons are markedly lower than those in humans and rhesus monkeys. The NO levels measured in two minipigs must be interpreted cautiously when compared with the results obtained in baboons. It has to be kept in mind that only two animals had been studied (whether they were healthy was not stated), the animals were especially bred for laboratory experiments and kept caged, and the minipigs were intubated and mechanically ventilated during the nasal NO sampling, all of which could have influenced the measurement. Nevertheless, NO levels in minipigs are higher than those in baboons, especially considering that a portion of nasal NO is produced in the lower respiratory tract, which was excluded from the measurement due to intubation. Whether the species-specific differences in nasal NO concentrations can be confirmed in larger animal groups by using comparable measurement techniques, as well as their origin, are still unknown.

Comparison of our results with those in other species: NO levels in the lower airways. When comparing the NO concentrations found in baboons with the NO levels found in the lower airways (trachea, bronchi) of tracheotomized or intubated mammals with paranasal sinuses, including humans, we recognized more similarities. Stewart et al. (15) measured ~5 to 10 ppb NO in the exhaled gas of tracheotomized rats. Gustafsson et al. (3) reported NO levels of 15 ± 0.8 (SE) ppb in 18 rabbits, when exhaled gas from the tracheostoma was analyzed. In 10 intubated healthy adults, steady-state NO concentrations of 4-5 ppb were measured in the expiratory limb of the ventilator (2), and, in 4 healthy subjects breathing continuously through a tracheostomy, NO levels of 2 ± 0 (SE) ppb were found in the exhaled gas. We can assume that the NO found in the upper airways and exhaled gas in baboons stem mainly from the lower airways.

Age dependency of NO exhalation. In newborns, the paranasal sinuses are poorly developed. Lundberg et al. (6) observed that nasal NO concentrations are reduced. These authors also noticed an age-dependent increase in nasal NO levels from 4-20 ppb in newborns to ~300 ppb in adults that seemed to follow the development and pneumatization of the paranasal sinuses. Baboons do not show an age-dependent increase in nasal NO levels; the level stays at a value of ~5 ppb. Together with the low exhaled and nasal NO levels, this result suggests that the development of paranasal sinuses might be an anatomic requirement for significant NO production in the upper airways. Even a large nasal cavity, as found in baboons (see Fig. 1), cannot take over the functional role of the paranasal sinuses regarding NO production.

Conclusions. In the absence of paranasal sinuses, very low NO concentrations can be found in the nasal airways and exhaled gas of healthy baboons. Because NO concentrations in all mammals with open sinus air cavities studied so far are higher than those in baboons, we hypothesize that the paranasal sinuses might be an anatomic requirement for production of relevant nasal NO concentrations. The anatomic dead space in the paranasal sinuses might be of functional importance as a reservoir for NO produced by the epithelial paranasal cells because only in dead space is NO not absorbed or rapidly inactivated to any large extent. Other species-specific causes for the differences in exhaled and nasal NO levels, however, cannot be fully excluded. Rather, the theory of Schedin et al. (13) that high NO levels in monkeys and humans might be related to the upright body posture or relative longevity of these species can be questioned in view of our findings in baboons.