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Exhaled nasal nitric oxide output is reduced in humans at night during the sleep period
Respiratory Physiology & Neurobiology 156 (2007) 94–101
Exhaled nasal nitric oxide output is reduced in humans at night during the sleep period Daniel J. O’Hearn ∗ , George D. Giraud, Jeffrey M. Sippel, Chad Edwards, Benjamin Chan, William E. Holden Medical Service, Portland Veterans Administration Medical Center and Oregon Health and Science University, Portland, OR, USA Accepted 9 August 2006
Abstract The physiologic function of nasal nitric oxide (NO) release is unknown. In prior experiments, topical NG -nitro-l-arginine methyl ester (l-NAME) on nasal mucosa reduced exhaled nasal NO output and caused daytime sleepiness. We hypothesized that nasal NO output is reduced at night during the sleep period. We measured exhaled nasal NO concentration and minute ventilation and calculated nasal NO output in humans over 24 h. Daytime awake NO output was greater than NO output at night during sleep or transient wakefulness. Exhaled NO concentration decreased during sleep along with minute ventilation. A daytime voluntary reduction in minute ventilation also decreased nasal NO output but exhaled NO concentration increased. Nasal NO output was not changed by body position. We conclude that exhaled nasal NO output is decreased at night due to decreased mass flow of NO into nasal air in addition to decreased minute ventilation. Our findings suggest a role of nasal NO in sleep or in the physiologic processes accompanying sleep. © 2006 Elsevier B.V. All rights reserved. Keywords: Sleep; Somnolence; Nitric oxide; Nose
1. Introduction Nitric oxide (NO) is present in the exhaled air of humans. The majority of exhaled NO is released into the nasal passages and paranasal sinuses (Lundberg et al., 1994; Kimberly et al., 1996). The function of NO in the upper airway and factors that modulate its release into nasal air are poorly understood. In prior experiments we have shown that an inhibitor of nitric oxide synthase, NG -nitro-l-arginine methyl ester (l-NAME), delivered by aerosol into the nasal cavity, decreased exhaled NO and altered temperature conditioning and humidity conditioning of inhaled and exhaled air in humans (Holden et al., 1999). Unexpectedly, the subjects complained of sleepiness during and following these experiments. This observation prompted us to study the effects of l-NAME on daytime sleepiness (Sippel et al., 1999). We found that l-NAME reduced exhaled NO and shortened sleep onset latency, indicating increased daytime sleepiness. These
∗ Correspondence to: Pulmonary and Critical Care Medicine, Portland VA Medical Center, P3-PULM, 3710 SW U.S. Veterans Hospital Road, Portland, OR 97201, USA. Tel.: +1 503 220 8262x56625; fax: +1 503 721 7852. E-mail address: [email protected] (D.J. O’Hearn).
1569-9048/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2006.08.002
findings suggested a potential relationship between decreased nasal exhaled NO output and increased sleepiness or sleep in humans, but the mechanism by which the topical l-NAME induced sleepiness remains unknown. Other researchers have shown that inhibition of NO synthase reduces wakefulness in rats (Dzoljic and De Vries, 1994). In addition, rat brain cortical NO may be reduced during sleep (Burlet and Cespuglio, 1997; Faradji et al., 2000). This observation led us to speculate that nasal NO output might be naturally deceased at night when humans are somnolent or asleep. We therefore hypothesized that exhaled nasal NO output is reduced at night. To test this hypothesis, we measured exhaled nasal nitric oxide output in adult humans during daytime wakefulness and nighttime sleep and wakefulness. Nighttime sleep was documented using polysomnography. Since minute ventilation is reduced in sleep compared to the awake state, and since NO output could be a function of minute ventilation alone, we also measured exhaled nasal NO output during daytime voluntary hypoventilation in a range of minute ventilation values similar to those measured during the night. Finally, in order to evaluate the potential variation in NO output due to the supine posture typical of sleep, we compared nasal NO output between the upright and supine positions.
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2. Methods 2.1. Subjects The Investigational Review Board of the Portland VA Medical Center approved the research protocol and subjects gave their written consent before participation. Eight healthy subjects (seven males and one female, mean age 38 years, range 21–61) participated in the daytime awake, nighttime sleep portion of the study. Eleven healthy subjects (five males and six females, mean age 42 years, range 22–62) participated in the voluntary reduction in minute ventilation experiments. Nine healthy subjects (seven males, two females, mean age 48 years, range 29–63) participated in the upright versus supine position experiments. 2.2. Measurement of nasal NO output, O2 consumption and CO2 output Subjects, with mouth closed, breathed into a nasal continuous positive airway pressure mask (Respironics, Inc., Murrysville, PA, USA) connected to a two-way valve (Hans–Rudolph, Inc., Kansas City, KS, USA) that allowed for inhalation of room air through one port and collection of exhaled air through the other port. The exhalation port of the valve was attached to a Mylar bag known to be impermeable to and non-reactive with NO. We measured NO concentration (ppb) using a chemiluminescence analyzer (Sievers Instruments, Inc., model 270B, Boulder, CO, USA) calibrated using precise dilutions of a certified gas mixture (45 ppm NO in air, Sievers Instruments, Inc.) in NO-free air using a calibrated two-liter syringe. With each exhaled gas measurement, concurrent ambient levels of NO were also measured. In general, ambient NO levels are negligible in Portland, OR, at night, and were consistently found to be ≤5 ppb during the daytime experiments reported herein. To determine minute ventilation, the volume of each timed collection of exhaled air was measured in a Tissot gasometer. Nasal NO output (nL/min) was calculated as the product of NO concentration in the collection bag and minute ventilation: NO output (nL/ min) = [NO] (ppb or nL/L) × minute ventilation (L/ min) Because we have previously noted a direct relationship between body mass and exhaled nasal NO output (unpublished observation), the results are expressed per m2 of body surface area. Oxygen consumption and carbon dioxide output were measured to document during the awake and asleep states during the day and at night. The percentage of O2 and of CO2 in exhaled air was measured using a calibrated (certified gas mixtures of O2 and CO2 in nitrogen) mass spectrometer (MGA 100, Perkin-Elmer, Inc., Pomona, CA, USA) or metabolic cart (Sensormedics, Inc., Yorba Linda, CA, USA). 2.3. Nasal gas collections over a 24-h period Hourly daytime measurements were made with the subjects seated and resting quietly for 2 min before exhaled nasal gas col-
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lections were collected over 2 min. Similar nighttime exhaled gas collections were made every 20 min while subjects were supine in bed during the sleep period (approximately from 11 p.m. to 6 a.m.). Sleep stages were monitored at night using surface electroencephalographic (EEG) activity of the central and occipital regions and submental electromyography. The sleep state (wake versus sleep) was determined from the EEG record using standard criteria (Rechtschaffen and Kales, 1968). While the subjects were supine in bed, a chinstrap was used during the nocturnal recordings to ensure mouth closure and nasal breathing. Subjects were monitored for oral breathing during the night by observation via video camera and oral air breathing was not observed during the nocturnal recordings. 2.4. Measurements of exhaled nasal NO output during voluntary changes in minute ventilation Minute ventilation is reduced during sleep compared with wakefulness, primarily due to decreased tidal volume and mean inhaled airflow (Phillipson and Bowes, 1986). In a previous study we observed that decreases in airflow in a closed nasal circuit decreased nasal NO output slightly over the range of airflow that occurs with breathing (Giraud et al., 1998). To determine if changes in minute ventilation could account for the differences in nasal NO output between daytime wakefulness and nighttime sleep in the 24-h measurements, we measured exhaled nasal NO output during the day in 11 awake, seated subjects during resting normal ventilation and voluntary hypoventilation. The subjects voluntarily controlled their minute ventilation by altering respiratory rate and/or tidal volume in whatever way they found most comfortable over a range of 5–10 L/min (the range of minute ventilation observed in our 24-h sleep wake study). No attempt was made to selectively decrease tidal volume, respiratory rate or airflow rate during voluntary hypoventilation over the 2-min gas collection. 2.5. Measurements of exhaled nasal NO output with change in position The supine posture of sleep typically differs from the upright status characteristic of daytime wakefulness. To determine the potential contribution of body position on the difference in nasal NO output between wakefulness and sleep, we measured nasal NO output during the day in nine awake subjects after a 30-min period of rest in both the seated and supine positions. 2.6. Statistical analysis All numeric data in the text is expressed as mean with standard deviation and in the illustrations as mean with standard error of the mean. The unit of analysis was each exhaled gas collection. Linear mixed models were used to fit the data where sleep state was the fixed effect and study subject was the random effect (Neter et al., 1990). Tukey-Kramer adjustments were used for multiple comparisons with an alpha level of 0.05. Paired Student’s t-tests were used in the analyses of ventilation state and body position data with statistical significance set at p < 0.05.
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Fig. 1. Exhaled nasal NO concentration (upper panel), minute ventilation (middle panel) and exhaled nasal NO output (lower panel) during the daytime with subjects awake and sitting (open bars) and during the night with subjects supine and in bed (solid bars) in eight subjects. * p < 0.05 comparing daytime awake to night values. During the night, exhaled NO concentration and minute ventilation were reduced, leading to a decrease in calculated nasal NO output.
3. Results 3.1. Nasal NO output over a 24-h period The major finding of this study was that exhaled nasal NO output was reduced when subjects were supine in bed at night compared with the daytime awake state (Fig. 1, lower panel). Reduced nasal NO output was due to reductions in both exhaled nasal NO concentration and minute ventilation (Fig. 1, upper and middle panels) during the night. Exhaled nasal nitric oxide concentration decreased from 28.0 ppb (S.D. 10.5) in the daytime awake state to 22.2 ppb (S.D. 10.5) at night (p < 0.001). Minute ventilation decreased from 8.8 L/min (S.D. 0.8) in the daytime awake state to 6.1 L/min (S.D. 0.8) at night (p < 0.001). These reductions in NO concentration and minute ventilation led to a 46% reduction of exhaled nasal
Fig. 2. Exhaled nasal NO output in two subjects over 24 h. Measurements were made hourly during the day with the subjects awake and sitting (open circles) and every 20 min during the night when the subjects were supine in bed. During the night, the awake (shaded circles) vs. asleep (solid circles) state was confirmed by EEG analysis. The shaded portion of the figure indicates the supine position in bed in the dark. Values of NO output were less at night when the subjects were supine and in bed, in both the awake and asleep state. Note that NO output decreased promptly when the subjects assumed the supine position (and before falling asleep) and increased promptly in the morning when the subjects rose from bed.
NO output from 136.3 nL/min/m2 (S.D. 48.8) in the daytime awake state to 77.4 nL/min/m2 (S.D. 48.8) during the night (p < 0.001). The second major finding of this study was that exhaled nasal NO output was reduced at night during periods of brief wakefulness as well as during EEG-documented sleep (Figs. 2 and 3). Fig. 2 illustrates the relationship between nasal nitric oxide output in the sitting daytime awake, supine nighttime awake and nighttime asleep states in two representative subjects. Interestingly, exhaled nasal NO output decreased shortly after the subjects assumed the supine position at bedtime while the subjects were still awake, and then continued to be decreased (compared with daytime awake values) until the final awakening in the morning. When the subjects awoke in the middle of the night, exhaled nasal NO output remained reduced compared to the daytime awake level. Fig. 3 shows group values of exhaled NO concentration (upper panel), minute ventilation (middle panel) and NO output (lower panel) for eight subjects. Similar to the findings of Fig. 1, exhaled NO concentration, minute ventilation
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3.2. Nasal NO output and voluntary hypoventilation Since calculated exhaled nasal NO output is the product of minute ventilation and the NO concentration of exhaled air, the finding of reduced exhaled nasal NO output at night could potentially be due to decreased NO concentration, decreased minute ventilation, or reductions in both. We observed that both minute ventilation and NO concentration were reduced at night compared with daytime values. In an additional experiment we examined the effect of daytime awake voluntary hypoventilation on exhaled nasal NO output (Fig. 4). Voluntary hypoventilation during the day reduced minute ventilation to a level (Fig. 4
Fig. 3. Exhaled nasal NO concentration (upper panel), minute ventilation (middle panel) and NO output (lower panel) during the sitting daytime awake (open bars), EEG-confirmed nighttime awake (diagonal bars) and nighttime asleep (solid bars) condition in eight subjects. * p < 0.05 comparing nighttime awake or asleep to daytime awake, # p < 0.05 comparing nighttime asleep to nighttime awake. Similar to the results of Fig. 1, during EEG-verified sleep, exhaled NO, minute ventilation and exhaled NO output decreased in comparison with the daytime awake condition. During the nighttime awake condition, NO output and minute ventilation were also reduced compared with the daytime awake state, but NO concentration was not changed.
and exhaled nasal NO output during EEG-documented sleep were reduced compared with the daytime awake state. However, the findings during nighttime awake periods suggested an intermediary state between daytime wakefulness and nighttime sleep, with decreased minute ventilation and NO output but unchanged exhaled NO concentration compared with the daytime awake state. As expected, oxygen uptake and carbon dioxide output during daytime hours (0.35 L/min, S.D. 0.07, and 0.30 L/min, S.D. 0.05, respectively) were significantly greater relative to the nighttime awake values (0.24 L/min, S.D. 0.08, and 0.21 L/min, S.D. 0.06, each p < 0.0001) and nighttime asleep values (0.21 L/min, S.D. 0.07, and 0.17 L/min, S.D. 0.05, each p < 0.0001).
Fig. 4. Exhaled nasal NO concentration (upper panel), minute ventilation (middle panel) and NO output (lower panel) in the daytime control sitting and resting condition (open bars) and during daytime voluntary hypoventilation (solid bars) in 11 subjects. * p < 0.05 comparing control normal resting ventilation to hypoventilation. The value of minute ventilation during voluntary hypoventilation was comparable to values of minute ventilation during the night in the studies of Figs. 1 and 3. During hypoventilation minute ventilation was reduced (p < 0.0001) but exhaled NO concentration increased (p < 0.01). Calculated nasal NO output also decreased (p < 0.006), but the reduction was less marked than that which occurred during sleep or the nighttime awake state.
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upper panel, 5.3 L/min, S.D. 1.3) that was comparable to nighttime minute ventilation in subjects either asleep (6.0 L/min, S.D. 1.1) or awake (6.8 L/min, S.D. 1.4) (values from Fig. 3, middle panel). Exhaled nasal NO output was reduced by approximately 14% (p < 0.05) during voluntary hypoventilation from 127.2 nL/min/m2 (S.D. 47.8) to 109.9 nL/min/m2 (S.D. 34.6) (Fig. 4, lower panel). Hence, at least some of the observed decrease in exhaled nasal NO output at night can be explained by a reduction in minute ventilation that occurs during nocturnal wakefulness and sleep. However, it is important to note that exhaled NO concentration increased significantly during daytime voluntary hypoventilation (Fig. 4, middle panel), whereas either no significant change or a decrease in exhaled NO concentration was observed at night in the awake or sleeping state respectively (Fig. 3, upper panel). This indicates that the mass output of NO into nasal air is reduced at night, causing a greater reduction in NO output than can be explained by reduced minute ventilation alone.
Fig. 5. Exhaled nasal NO concentration (upper panel), minute ventilation (middle panel) and NO output (lower panel) in the seated position (open bars) and in the supine position (solid bars) in nine subjects. No significant differences were noted between the seated and supine positioning.
3.3. The effect of posture on nasal NO output In order to assess the potential contribution of the typical difference in posture between sleep and wakefulness, we measured exhaled nasal NO output in both the seated and supine positions during the day after each of the subjects had rested in that position for 30 min while awake (Fig. 5). Exhaled NO output, NO concentration or minute ventilation did not differ in any appreciable manner in our subjects between the seated and supine positions. 4. Discussion The significant finding of this study was that exhaled nasal NO was reduced at night when humans are generally sleepy or asleep. Exhaled nasal NO output was reduced both during sleep and during periods of brief awakening compared to daytime awake levels. The decrease in exhaled nasal NO output during sleep was due to decreases in both of the factors which determine exhaled nasal NO output: minute ventilation and NO concentration in the exhaled nasal air. Daytime voluntary hypoventilation also decreased exhaled nasal NO output, but the decrease was less than the decrease seen during either sleep or wakefulness at night despite comparable changes in minute ventilation. In addition, voluntary hypoventilation was associated with an increase in exhaled nasal NO concentration, unlike during the nighttime in which exhaled NO concentration decreased. The observation that exhaled nasal NO concentration decreased at night along with decreased exhaled minute ventilation indicates that the mass flow of NO from the nasal mucosa into nasal air decreased at night. This finding also suggests that either sleep or sleepiness (or both) is associated with changes in NO output into nasal air, and raises the possibility that NO is involved in some way with the physiologic mechanisms of sleep or sleepiness, or with the physiologic changes that occur in the nose during nighttime sleep. An interesting observation is that exhaled nasal NO output often decreased following lights out for the study, and before the subjects entered sleep as measured by polysomnography. NO output increased in the morning promptly with the subject’s awakening, and usually prior to rising from bed. There are some methodological issues requiring consideration in our study. First, measurement of exhaled nasal NO output does not represent total NO output into nasal air. Nasal air NO is in equilibrium with NO in the nasal mucosa and NO is both excreted and quickly absorbed by the nasal mucosa (Dubois et al., 1998) as well as the lower airways (DuBois et al., 1999). Our group (Kimberly et al., 1996) and others (Tornberg et al., 2002) have shown that NO is continuously released into the nasal passages during respiration. During nose breathing with the mouth closed, nasal NO is inhaled into the lower respiratory tract and largely taken up by the lungs. Exhaled air from the lungs contains decreased concentrations of NO relative to nasal exhaled air and entrains additional nasal NO in the exhalant. Also, there is evidence that nasal NO output during inspiration is greater than during expiration in awake subjects (Tornberg et al., 2002). Hence, total nasal NO output is likely underestimated by our measurements of exhaled nasal NO output in this study,
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although the precise proportion of inhaled and exhaled nasal NO output under conditions of sleep is unknown. Measurement of total nasal NO output during sleep would require the relatively invasive placement of catheters in the posterior nasopharynx or oropharynx with potential impact on the quantity and quality of sleep. The use of a fixed flow rate and fixed expiratory resistance as suggested by the 2005 ATS/ERS Nitric Oxide Joint Consensus Statement (ATS/ERS, 2005) was not possible in our tidal breathing sleeping subjects. Therefore, we chose to measure NO output only during exhalation rather than throughout the respiratory cycle. Measurement of exhaled NO output during single breath exhalations is a reproducible and convenient method for clinical measurements in humans (Palm et al., 2000). A second methodological issue in our study is that accurate collection of exhaled nasal NO output requires nasal breathing with the mouth closed. Humans prefer nasal breathing during sleep (Fitzpatrick et al., 2003), and we assisted mouth closure during sleep with use of a chinstrap. Subjects were also monitored closely by video camera, especially during exhaled nasal air collections, to assess whether mouth breathing occurred. Nevertheless, if transient mouth breathing went undetected, it would be expected to lower the measured minute ventilation. A third methodological issue is that we did not independently measure respiratory airflow or tidal volumes in our study. It is thus unclear if the pattern of breathing leading to decreased minute ventilation at night was equivalent to that used by subjects who voluntarily decreased their minute ventilation during the day over a 2-min period. If differing patterns of breathing occurred – i.e., different inhalation or exhalation flows with the potential for variance in the mucosal shear stress between the two conditions – NO output measurements might be affected. Other groups (Dubois et al., 1998; Djupesland et al., 1999; Chambers et al., 2001) and we (Giraud et al., 1998) have shown a direct relationship between airflow through the nasal passages and nasal NO output. This effect is typically small over the range of airflow during tidal breathing (Giraud et al., 1998). In addition, when nasal airflow is decreased in awake subjects, exhaled NO concentration typically increases (Dubois et al., 1998), unlike what was seen in our sleeping subjects. A severe reduction in the oxygen concentration of nasal air can produce a significant decrease in nasal NO output (Giraud et al., 1998; Haight et al., 2000). Alveolar hypoxia can lead to decreases in NO production in the lung (Grimminger et al., 1995; Nelin et al., 1996; Carlin et al., 1997). Neither blood gases nor continuous oximetry were measured in our protocol. None of our sleeping subjects were noted to have snoring or to have witnessed apneas during the overnight nasal gas collection. While the majority of the 24-h gas collection subjects were males, none were obese nor had excessive daytime somnolence. While it is unlikely that significant obstructive sleep apnea was an issue amongst our subjects, we cannot rule out the fact that the subtle decreases in oxygenation typical of sleep (Birchfield et al., 1958, 1959; Robin et al., 1958; Muller et al., 1980; Douglas et al., 1982) may have contributed to a decrease in exhaled NO output. While there would not be expected to be a difference in nocturnal oxygen changes between normal men and women (Douglas et al., 1982), the potential exists for a difference in
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NO metabolism between males and females (Liu et al., 2004). While one study suggested no sex-related difference in exhaled NO concentrations (Palm et al., 2000), further study is needed on this possible confounder. While we have demonstrated that exhaled nasal NO output is decreased during the sleep period, we are not yet able to provide a mechanism for this decrease nor can we say whether or not it is pertinent to the physiology of sleep. It is well possible that this is an epiphenomenon and that the decrease in exhaled NO output is simply reflective of the decrease in metabolism similar to the decrease in O2 consumption and CO2 production that we observed. The decrease in exhaled NO output may simply reflect a decrease in nasal mucosal blood flow. While research suggests that NO is important in sleep in the animal brain (Dzoljic and De Vries, 1994; Burlet and Cespuglio, 1997; Faradji et al., 2000), it is not known how nasal NO concentration or output relates to brain NO levels. We therefore cannot identify how exhaled nasal NO output is related to sleep or sleepiness and can only report that a decrease in exhaled NO output occurs in sleep. In a previous study (Sippel et al., 1999) we established that a topical spray of l-NAME (0.5 M, 4 mL) administered to the nasal passages of humans was associated with daytime sleepiness for several hours. The vasoconstrictor oxymetazoline also decreased exhaled nasal NO but did not cause sleepiness suggesting that sleepiness was not due to the mucosal vasoconstriction effects of l-NAME. The mechanism of sleepiness following l-NAME administration is unknown, although uptake of lNAME into the bloodstream and across the blood-brain barrier remains a possibility. Our findings prompted the experiments reported herein, which further suggest an association between sleep or sleepiness and decreased exhaled NO output. At present we can only speculate as to the reasons for such a relationship. One possibility is that decreased exhaled nasal NO at night reflects a circadian variation in nasal NO production, or is linked to other processes with circadian patterns such as body temperature or hormonal variation. Time-dependent variation in urinary NO metabolites has been demonstrated in rats (Borgonio et al., 1999). ten Hacken et al. (1998) measured orally exhaled NO in awake normal and asthmatic subjects and found no significant temporal pattern over a 24 h period. Bartley et al. (1999) performed twice daily nasal NO measurements in awake sitting subjects and concluded there was no circadian variation, but Palm et al. (2003) showed that the concentration of exhaled NO increased from early morning to midday to late afternoon, suggesting a possible circadian relationship. We could identify no consistent pattern in the exhaled NO output in the hourly measurements in our subjects as the day progressed from morning to afternoon. A second possible explanation for a relationship between nasal NO and sleep could be the need for humans to maintain a patent nasal airway during sleep. Humans prefer to breathe nasally during sleep (Fitzpatrick et al., 2003), and decreased nasal patency is associated with sleep-disordered breathing (Zwillich et al., 1981; Lavie et al., 1983). Imada et al. (1996, 2002) have shown an inverse relationship between nasal airway NO and airflow resistance through the nasal passages. On the other hand, despite decreasing nasal volume and increasing air-
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flow resistance, assuming the supine position was not associated with changes in nasal NO output in awake humans (Chatkin et al., 1999) which we now confirm. We speculate that reduced exhaled NO output at night may relate to the need for changes in temperature and humidity conditioning of nasal air during nighttime sleep compared with the awake state. The onset of sleep is associated with a decrease in body temperature. Body temperature is regulated at a lower level during non-REM sleep compared with wakefulness (Glotzbach and Heller, 2000). During REM sleep, the temperature is even more variable with thermoregulatory responses being further inhibited. There is evidence that the nasal passages may participate in thermoregulation in response to changes in temperature locally (Burgess and Whitelaw, 1984), or reflexly with cooling (Speisman, 1936) or heating (White and Cabanec, 1995) of the skin. In a prior study (Holden et al., 1999), we demonstrated that reduced exhaled nasal NO (following topical application of the vasoconstrictor oxymetazoline or l-NAME) was associated with reduced nasal air temperature, and increased exhaled NO (following topical papaverine, a vasodilator) was associated with increased nasal air temperature. These findings support a role of NO in temperature conditioning of nasal air, and by extension, in whole body thermoregulation. In response to a decrease in core body temperature, a reduction in mucosal NO might be expected to reduce the addition of heat to the inhaled air and increase the recovery of heat during exhalation. The balance of these processes could lessen the total loss of heat from the respiratory tract during sleep. 5. Conclusion We report that exhaled nasal NO output is reduced at night during sleep as well as with short nocturnal periods of wakefulness in humans. The reduction during sleep was associated with both decreased minute ventilation and NO concentration in the exhaled air compared with daytime values. Our findings suggest a role of nasal NO in the sleep state or sleepiness, or a role in the physiologic processes that accompany sleep. Further research is needed to develop better mechanistic insight into the association between exhaled NO and sleep. Given the potential bearing of NO to such processes as vasoregulation, gas exchange, and the temperature and humidity conditioning of inspired air, we feel that the finding of decreased exhaled NO output is potentially relevant to understanding the physiology of sleep. References ATS/ERS, 2005. Recommendations for standardized procedures for the online and offline measurement of exhaled lower respiratory nitric oxide and nasal nitric oxide. Am. J. Respir. Crit. Care Med. 171, 912–930. Bartley, J., Fergusson, W., Moody, A., Wells, A.U., Kolbe, J., 1999. Normal adult values, diurnal variation, and repeatability of nasal nitric oxide measurement. Am. J. Rhinol. 13, 401–405. Birchfield, R.I., Sieker, H.O., Heyman, A., 1958. Alterations in blood gases during natural sleep and narcolepsy. Neurology 8, 107–112. Birchfield, R.I., Sieker, H.O., Heyman, A., 1959. Alterations in respiratory function during natural sleep. J. Lab. Clin. Med. 54, 216–222.
Borgonio, A., Witte, K., Stahrenberg, R., Lemmer, B., 1999. Influence of circadian time, ageing, and hypertension on the urinary excretion of nitric oxide metabolites in rats. Mech. Ageing Dev. 111, 23–37. Burgess, K., Whitelaw, W., 1984. Reducing ventilatory response to carbon dioxide by breathing cold air. Am. Rev. Respir. Dis. 129, 687–690. Burlet, S., Cespuglio, R., 1997. Voltammetric detection of nitric oxide (NO) in the rat brain: its variations throughout the sleep–wake cycle. Neurosci. Lett. 226, 131–135. Carlin, R., Ferrario, L., Boyd, J.T., Camporesi, E.M., McGraw, D.J., Hakim, T.S., 1997. Determinants of nitric oxide in exhaled gas in isolated rabbit lung. Am. J. Respir. Crit. Care Med. 155, 922–927. Chambers, D.C., Carpenter, D.A., Ayres, J.G., 2001. Exchange dynamics of nitric oxide in the human nose. J. Appl. Physiol. 91, 1924–1930. Chatkin, J.M., Djupesland, P.G., Qian, W., McClean, P., Furlott, H., Gutierrez, C., Zamel, N., Haight, J.S., 1999. Nasal nitric oxide is independent of nasal cavity volume. Am. J. Rhinol. 13, 179–184. Djupesland, P.G., Chatkin, J.M., Qian, W., Cole, P., Zamel, N., McClean, P., Furlott, H., Haight, J.S., 1999. Aerodynamic influences on nasal nitric oxide output measurements. Acta Otolaryngol. 119, 479–485. Douglas, N.J., White, D.P., Pickett, C.K., Weil, J.V., Zwillich, C.W., 1982. Respiration during sleep in normal man. Thorax 37, 840–844. Dubois, A.B., Douglas, J.S., Stitt, J.T., Mohsenin, V., 1998. Production and absorption of nitric oxide gas in the nose. J. Appl. Physiol. 84, 1217–1224. DuBois, A.B., Kelley, P.M., Douglas, J.S., Mohsenin, V., 1999. Nitric oxide production and absorption in trachea, bronchi, bronchioles, and respiratory bronchioles of humans. J. Appl. Physiol. 86, 159–167. Dzoljic, M.R., De Vries, R., 1994. Nitric oxide synthase inhibition reduces wakefulness. Neuropharmacology 33, 1505–1509. Faradji, H., Rousset, C., Debilly, G., Vergnes, M., Cespuglio, R., 2000. Sleep and epilepsy: a key role for nitric oxide? Epilepsia 41, 794–801. Fitzpatrick, M.F., Driver, H.S., Chatha, N., Voduc, N., Girard, A.M., 2003. Partitioning of inhaled ventilation between the nasal and oral routes during sleep in normal subjects. J. Appl. Physiol. 94, 883–890. Giraud, G.D., Nejadnik, B., Kimberly, B., Holden, W.E., 1998. Physical characteristics and gas composition of nasal air affect nasal nitric oxide release. Respir. Physiol. 114, 285–296. Glotzbach, S., Heller, H., 2000. Temperature regulation. In: Kryger, M., Roth, T., Dement, W. (Eds.), Principles and Practice of Sleep Medicine. W.B. Saunders Co., Phildelphia, PA, pp. 289–304. Grimminger, F., Spriestersbach, R., Weissman, N., Walmrath, D., Seeger, W., 1995. Nitric oxide generation and hypoxic vasoconstriction in bufferperfused rabbit lungs. J. Appl. Physiol. 78, 1509–1515. Haight, J.S.J., Qian, W., Daya, H., Chalmers, P., Zamel, N., 2000. Hypoxia depresses nitric oxide output in the human nasal airways. Laryngoscope 110, 429–433. Holden, W.E., Wilkins, J.P., Harris, M., Milczuk, H.A., Giraud, G.D., 1999. Temperature conditioning of nasal air: effects of vasoactive agents and involvement of nitric oxide. J. Appl. Physiol. 87, 1260–1265. Imada, M., Iwamoto, J., Nonaka, S., Kobayashi, Y., Unno, T., 1996. Measurement of nitric oxide in human nasal airway. Eur. Respir. J. 9, 556–559. Imada, M., Nonaka, S., Kobayashi, Y., Iwamoto, J., 2002. Functional roles of nasal nitric oxide in nasal patency and mucociliary function. Acta Otolaryngol. 122, 513–519. Kimberly, B., Nejadnik, B., Giraud, G.D., Holden, W.E., 1996. Nasal contribution to exhaled nitric oxide at rest and during breathholding in humans. Am. J. Respir. Crit. Care Med. 153, 829–836. Lavie, P., Fischel, N., Zomer, J., Eliaschar, I., 1983. The effects of partial and complete mechanical occlusion of the nasal passages on sleep structure and breathing in sleep. Acta Otolaryngol. 95, 161–166. Liu, L., Yan, Y., Zeng, M., Zhang, J., Hanes, M.A., Ahearn, G., McMahon, T.J., Dickfield, T., Marshall, H.E., Que, L.G., Stamler, J.S., 2004. Essential roles of S-nitrosothiols in vascular homeostasis and endotoxic shock. Cell 116, 617–628. Lundberg, J.O., Weitzberg, E., Nordvall, S.L., Kuylenstierna, R., Lundberg, J.M., Alving, K., 1994. Primarily nasal origin of exhaled nitric oxide and absence in Kartagener’s syndrome. Eur. Respir. J. 7, 1501–1504. Muller, N.L., Francis, P.W., Gurwitz, D., Levison, H., Bryan, A.C., 1980. Mechanism of hemoglobin desaturation during rapid-eye-movement sleep
D.J. O’Hearn et al. / Respiratory Physiology & Neurobiology 156 (2007) 94–101 in normal subjects and in patients with cystic fibrosis. Am. Rev. Respir. Dis. 121, 463–469. Nelin, L.D., Thomas, C.J., Dawson, C.D., 1996. Effect of hypoxia on nitric oxide production in neonatal pig lung. Am. J. Physiol. 271, H8–H14. Neter, J., Wasserman, W., Kutner, M.H., 1990. Applied Linear Statistical Models: Regression, Analysis of Variance, and Experimental Designs. Irwin, Homewood, IL. Palm, J.P., Alving, K., Lundberg, J.O., 2003. Characterization of airway nitric oxide in allergic rhinitis: the effect of intranasal administration of l-NAME. Allergy 58, 885–892. Palm, J.P., Graf, P., Lundberg, J.O., Alving, K., 2000. Characterization of exhaled nitric oxide: introducing a new reproducible method for nasal nitric oxide measurements. Eur. Respir. J. 16, 236–241. Phillipson, E.A., Bowes, G., 1986. Control of breathing during sleep. In: Cherniack, N.S., Widdicombe, J.G. (Eds.), Handbook of Physiology—Section 3: The Respiratory System, vol. II. American Physiological Society, Bethesda, MD, pp. 649–689. Rechtschaffen, A., Kales, A., 1968. A manual of standardized terminology, techniques, and scoring system for sleep stages of human subjects. In: Brain Information Service/Brain Research Institute. UCLA, Los Angeles, CA.
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Robin, E.D., Whaley, R.D., Crump, C.H., Travis, D.H., 1958. Alveolar gas tensions, pulmonary ventilation and blood pH during physiologic sleep in normal subjects. J. Clin. Invest. 37, 981–989. Sippel, J.M., Giraud, G.D., Holden, W.E., 1999. Nasal administration of the nitric oxide synthase inhibitor l-NAME induces daytime somnolence. Sleep 22, 786–788. Speisman, I.G., 1936. Vasomotor responses of the mucosa of the upper respiratory tract to thermal stimuli. Am. J. Physiol. 115, 181–187. ten Hacken, N.H., van der Vaart, H., van der Mark, T.W., Koeter, G.H., Postma, D.S., 1998. Exhaled nitric oxide is higher both at day and night in subjects with nocturnal asthma. Am. J. Respir. Crit. Care Med. 158, 902– 907. Tornberg, D.C., Marteus, H., Schedin, U., Alving, K., Lundberg, J.O., Weitzberg, E., 2002. Nasal and oral contribution to inhaled and exhaled nitric oxide: a study in tracheotomized patients. Eur. Respir. J. 19, 859–864. White, M.D., Cabanec, M., 1995. Nasal mucosal vasodilatation in response to passive hyperthermia in humans. Eur. J. Appl. Physiol. 70, 207–212. Zwillich, C.W., Pickett, C., Hanson, F.N., Weil, J.V., 1981. Disturbed sleep and prolonged apnea during nasal obstruction in normal men. Am. Rev. Respir. Dis. 124, 158–160.
Exhaled nasal nitric oxide output is reduced in humans at night during the sleep period Daniel J. O’Hearn ∗ , George D. Giraud, Jeffrey M. Sippel, Chad Edwards, Benjamin Chan, William E. Holden Medical Service, Portland Veterans Administration Medical Center and Oregon Health and Science University, Portland, OR, USA Accepted 9 August 2006
Abstract The physiologic function of nasal nitric oxide (NO) release is unknown. In prior experiments, topical NG -nitro-l-arginine methyl ester (l-NAME) on nasal mucosa reduced exhaled nasal NO output and caused daytime sleepiness. We hypothesized that nasal NO output is reduced at night during the sleep period. We measured exhaled nasal NO concentration and minute ventilation and calculated nasal NO output in humans over 24 h. Daytime awake NO output was greater than NO output at night during sleep or transient wakefulness. Exhaled NO concentration decreased during sleep along with minute ventilation. A daytime voluntary reduction in minute ventilation also decreased nasal NO output but exhaled NO concentration increased. Nasal NO output was not changed by body position. We conclude that exhaled nasal NO output is decreased at night due to decreased mass flow of NO into nasal air in addition to decreased minute ventilation. Our findings suggest a role of nasal NO in sleep or in the physiologic processes accompanying sleep. © 2006 Elsevier B.V. All rights reserved. Keywords: Sleep; Somnolence; Nitric oxide; Nose
1. Introduction Nitric oxide (NO) is present in the exhaled air of humans. The majority of exhaled NO is released into the nasal passages and paranasal sinuses (Lundberg et al., 1994; Kimberly et al., 1996). The function of NO in the upper airway and factors that modulate its release into nasal air are poorly understood. In prior experiments we have shown that an inhibitor of nitric oxide synthase, NG -nitro-l-arginine methyl ester (l-NAME), delivered by aerosol into the nasal cavity, decreased exhaled NO and altered temperature conditioning and humidity conditioning of inhaled and exhaled air in humans (Holden et al., 1999). Unexpectedly, the subjects complained of sleepiness during and following these experiments. This observation prompted us to study the effects of l-NAME on daytime sleepiness (Sippel et al., 1999). We found that l-NAME reduced exhaled NO and shortened sleep onset latency, indicating increased daytime sleepiness. These
∗ Correspondence to: Pulmonary and Critical Care Medicine, Portland VA Medical Center, P3-PULM, 3710 SW U.S. Veterans Hospital Road, Portland, OR 97201, USA. Tel.: +1 503 220 8262x56625; fax: +1 503 721 7852. E-mail address: [email protected] (D.J. O’Hearn).
1569-9048/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2006.08.002
findings suggested a potential relationship between decreased nasal exhaled NO output and increased sleepiness or sleep in humans, but the mechanism by which the topical l-NAME induced sleepiness remains unknown. Other researchers have shown that inhibition of NO synthase reduces wakefulness in rats (Dzoljic and De Vries, 1994). In addition, rat brain cortical NO may be reduced during sleep (Burlet and Cespuglio, 1997; Faradji et al., 2000). This observation led us to speculate that nasal NO output might be naturally deceased at night when humans are somnolent or asleep. We therefore hypothesized that exhaled nasal NO output is reduced at night. To test this hypothesis, we measured exhaled nasal nitric oxide output in adult humans during daytime wakefulness and nighttime sleep and wakefulness. Nighttime sleep was documented using polysomnography. Since minute ventilation is reduced in sleep compared to the awake state, and since NO output could be a function of minute ventilation alone, we also measured exhaled nasal NO output during daytime voluntary hypoventilation in a range of minute ventilation values similar to those measured during the night. Finally, in order to evaluate the potential variation in NO output due to the supine posture typical of sleep, we compared nasal NO output between the upright and supine positions.
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2. Methods 2.1. Subjects The Investigational Review Board of the Portland VA Medical Center approved the research protocol and subjects gave their written consent before participation. Eight healthy subjects (seven males and one female, mean age 38 years, range 21–61) participated in the daytime awake, nighttime sleep portion of the study. Eleven healthy subjects (five males and six females, mean age 42 years, range 22–62) participated in the voluntary reduction in minute ventilation experiments. Nine healthy subjects (seven males, two females, mean age 48 years, range 29–63) participated in the upright versus supine position experiments. 2.2. Measurement of nasal NO output, O2 consumption and CO2 output Subjects, with mouth closed, breathed into a nasal continuous positive airway pressure mask (Respironics, Inc., Murrysville, PA, USA) connected to a two-way valve (Hans–Rudolph, Inc., Kansas City, KS, USA) that allowed for inhalation of room air through one port and collection of exhaled air through the other port. The exhalation port of the valve was attached to a Mylar bag known to be impermeable to and non-reactive with NO. We measured NO concentration (ppb) using a chemiluminescence analyzer (Sievers Instruments, Inc., model 270B, Boulder, CO, USA) calibrated using precise dilutions of a certified gas mixture (45 ppm NO in air, Sievers Instruments, Inc.) in NO-free air using a calibrated two-liter syringe. With each exhaled gas measurement, concurrent ambient levels of NO were also measured. In general, ambient NO levels are negligible in Portland, OR, at night, and were consistently found to be ≤5 ppb during the daytime experiments reported herein. To determine minute ventilation, the volume of each timed collection of exhaled air was measured in a Tissot gasometer. Nasal NO output (nL/min) was calculated as the product of NO concentration in the collection bag and minute ventilation: NO output (nL/ min) = [NO] (ppb or nL/L) × minute ventilation (L/ min) Because we have previously noted a direct relationship between body mass and exhaled nasal NO output (unpublished observation), the results are expressed per m2 of body surface area. Oxygen consumption and carbon dioxide output were measured to document during the awake and asleep states during the day and at night. The percentage of O2 and of CO2 in exhaled air was measured using a calibrated (certified gas mixtures of O2 and CO2 in nitrogen) mass spectrometer (MGA 100, Perkin-Elmer, Inc., Pomona, CA, USA) or metabolic cart (Sensormedics, Inc., Yorba Linda, CA, USA). 2.3. Nasal gas collections over a 24-h period Hourly daytime measurements were made with the subjects seated and resting quietly for 2 min before exhaled nasal gas col-
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lections were collected over 2 min. Similar nighttime exhaled gas collections were made every 20 min while subjects were supine in bed during the sleep period (approximately from 11 p.m. to 6 a.m.). Sleep stages were monitored at night using surface electroencephalographic (EEG) activity of the central and occipital regions and submental electromyography. The sleep state (wake versus sleep) was determined from the EEG record using standard criteria (Rechtschaffen and Kales, 1968). While the subjects were supine in bed, a chinstrap was used during the nocturnal recordings to ensure mouth closure and nasal breathing. Subjects were monitored for oral breathing during the night by observation via video camera and oral air breathing was not observed during the nocturnal recordings. 2.4. Measurements of exhaled nasal NO output during voluntary changes in minute ventilation Minute ventilation is reduced during sleep compared with wakefulness, primarily due to decreased tidal volume and mean inhaled airflow (Phillipson and Bowes, 1986). In a previous study we observed that decreases in airflow in a closed nasal circuit decreased nasal NO output slightly over the range of airflow that occurs with breathing (Giraud et al., 1998). To determine if changes in minute ventilation could account for the differences in nasal NO output between daytime wakefulness and nighttime sleep in the 24-h measurements, we measured exhaled nasal NO output during the day in 11 awake, seated subjects during resting normal ventilation and voluntary hypoventilation. The subjects voluntarily controlled their minute ventilation by altering respiratory rate and/or tidal volume in whatever way they found most comfortable over a range of 5–10 L/min (the range of minute ventilation observed in our 24-h sleep wake study). No attempt was made to selectively decrease tidal volume, respiratory rate or airflow rate during voluntary hypoventilation over the 2-min gas collection. 2.5. Measurements of exhaled nasal NO output with change in position The supine posture of sleep typically differs from the upright status characteristic of daytime wakefulness. To determine the potential contribution of body position on the difference in nasal NO output between wakefulness and sleep, we measured nasal NO output during the day in nine awake subjects after a 30-min period of rest in both the seated and supine positions. 2.6. Statistical analysis All numeric data in the text is expressed as mean with standard deviation and in the illustrations as mean with standard error of the mean. The unit of analysis was each exhaled gas collection. Linear mixed models were used to fit the data where sleep state was the fixed effect and study subject was the random effect (Neter et al., 1990). Tukey-Kramer adjustments were used for multiple comparisons with an alpha level of 0.05. Paired Student’s t-tests were used in the analyses of ventilation state and body position data with statistical significance set at p < 0.05.
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Fig. 1. Exhaled nasal NO concentration (upper panel), minute ventilation (middle panel) and exhaled nasal NO output (lower panel) during the daytime with subjects awake and sitting (open bars) and during the night with subjects supine and in bed (solid bars) in eight subjects. * p < 0.05 comparing daytime awake to night values. During the night, exhaled NO concentration and minute ventilation were reduced, leading to a decrease in calculated nasal NO output.
3. Results 3.1. Nasal NO output over a 24-h period The major finding of this study was that exhaled nasal NO output was reduced when subjects were supine in bed at night compared with the daytime awake state (Fig. 1, lower panel). Reduced nasal NO output was due to reductions in both exhaled nasal NO concentration and minute ventilation (Fig. 1, upper and middle panels) during the night. Exhaled nasal nitric oxide concentration decreased from 28.0 ppb (S.D. 10.5) in the daytime awake state to 22.2 ppb (S.D. 10.5) at night (p < 0.001). Minute ventilation decreased from 8.8 L/min (S.D. 0.8) in the daytime awake state to 6.1 L/min (S.D. 0.8) at night (p < 0.001). These reductions in NO concentration and minute ventilation led to a 46% reduction of exhaled nasal
Fig. 2. Exhaled nasal NO output in two subjects over 24 h. Measurements were made hourly during the day with the subjects awake and sitting (open circles) and every 20 min during the night when the subjects were supine in bed. During the night, the awake (shaded circles) vs. asleep (solid circles) state was confirmed by EEG analysis. The shaded portion of the figure indicates the supine position in bed in the dark. Values of NO output were less at night when the subjects were supine and in bed, in both the awake and asleep state. Note that NO output decreased promptly when the subjects assumed the supine position (and before falling asleep) and increased promptly in the morning when the subjects rose from bed.
NO output from 136.3 nL/min/m2 (S.D. 48.8) in the daytime awake state to 77.4 nL/min/m2 (S.D. 48.8) during the night (p < 0.001). The second major finding of this study was that exhaled nasal NO output was reduced at night during periods of brief wakefulness as well as during EEG-documented sleep (Figs. 2 and 3). Fig. 2 illustrates the relationship between nasal nitric oxide output in the sitting daytime awake, supine nighttime awake and nighttime asleep states in two representative subjects. Interestingly, exhaled nasal NO output decreased shortly after the subjects assumed the supine position at bedtime while the subjects were still awake, and then continued to be decreased (compared with daytime awake values) until the final awakening in the morning. When the subjects awoke in the middle of the night, exhaled nasal NO output remained reduced compared to the daytime awake level. Fig. 3 shows group values of exhaled NO concentration (upper panel), minute ventilation (middle panel) and NO output (lower panel) for eight subjects. Similar to the findings of Fig. 1, exhaled NO concentration, minute ventilation
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3.2. Nasal NO output and voluntary hypoventilation Since calculated exhaled nasal NO output is the product of minute ventilation and the NO concentration of exhaled air, the finding of reduced exhaled nasal NO output at night could potentially be due to decreased NO concentration, decreased minute ventilation, or reductions in both. We observed that both minute ventilation and NO concentration were reduced at night compared with daytime values. In an additional experiment we examined the effect of daytime awake voluntary hypoventilation on exhaled nasal NO output (Fig. 4). Voluntary hypoventilation during the day reduced minute ventilation to a level (Fig. 4
Fig. 3. Exhaled nasal NO concentration (upper panel), minute ventilation (middle panel) and NO output (lower panel) during the sitting daytime awake (open bars), EEG-confirmed nighttime awake (diagonal bars) and nighttime asleep (solid bars) condition in eight subjects. * p < 0.05 comparing nighttime awake or asleep to daytime awake, # p < 0.05 comparing nighttime asleep to nighttime awake. Similar to the results of Fig. 1, during EEG-verified sleep, exhaled NO, minute ventilation and exhaled NO output decreased in comparison with the daytime awake condition. During the nighttime awake condition, NO output and minute ventilation were also reduced compared with the daytime awake state, but NO concentration was not changed.
and exhaled nasal NO output during EEG-documented sleep were reduced compared with the daytime awake state. However, the findings during nighttime awake periods suggested an intermediary state between daytime wakefulness and nighttime sleep, with decreased minute ventilation and NO output but unchanged exhaled NO concentration compared with the daytime awake state. As expected, oxygen uptake and carbon dioxide output during daytime hours (0.35 L/min, S.D. 0.07, and 0.30 L/min, S.D. 0.05, respectively) were significantly greater relative to the nighttime awake values (0.24 L/min, S.D. 0.08, and 0.21 L/min, S.D. 0.06, each p < 0.0001) and nighttime asleep values (0.21 L/min, S.D. 0.07, and 0.17 L/min, S.D. 0.05, each p < 0.0001).
Fig. 4. Exhaled nasal NO concentration (upper panel), minute ventilation (middle panel) and NO output (lower panel) in the daytime control sitting and resting condition (open bars) and during daytime voluntary hypoventilation (solid bars) in 11 subjects. * p < 0.05 comparing control normal resting ventilation to hypoventilation. The value of minute ventilation during voluntary hypoventilation was comparable to values of minute ventilation during the night in the studies of Figs. 1 and 3. During hypoventilation minute ventilation was reduced (p < 0.0001) but exhaled NO concentration increased (p < 0.01). Calculated nasal NO output also decreased (p < 0.006), but the reduction was less marked than that which occurred during sleep or the nighttime awake state.
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upper panel, 5.3 L/min, S.D. 1.3) that was comparable to nighttime minute ventilation in subjects either asleep (6.0 L/min, S.D. 1.1) or awake (6.8 L/min, S.D. 1.4) (values from Fig. 3, middle panel). Exhaled nasal NO output was reduced by approximately 14% (p < 0.05) during voluntary hypoventilation from 127.2 nL/min/m2 (S.D. 47.8) to 109.9 nL/min/m2 (S.D. 34.6) (Fig. 4, lower panel). Hence, at least some of the observed decrease in exhaled nasal NO output at night can be explained by a reduction in minute ventilation that occurs during nocturnal wakefulness and sleep. However, it is important to note that exhaled NO concentration increased significantly during daytime voluntary hypoventilation (Fig. 4, middle panel), whereas either no significant change or a decrease in exhaled NO concentration was observed at night in the awake or sleeping state respectively (Fig. 3, upper panel). This indicates that the mass output of NO into nasal air is reduced at night, causing a greater reduction in NO output than can be explained by reduced minute ventilation alone.
Fig. 5. Exhaled nasal NO concentration (upper panel), minute ventilation (middle panel) and NO output (lower panel) in the seated position (open bars) and in the supine position (solid bars) in nine subjects. No significant differences were noted between the seated and supine positioning.
3.3. The effect of posture on nasal NO output In order to assess the potential contribution of the typical difference in posture between sleep and wakefulness, we measured exhaled nasal NO output in both the seated and supine positions during the day after each of the subjects had rested in that position for 30 min while awake (Fig. 5). Exhaled NO output, NO concentration or minute ventilation did not differ in any appreciable manner in our subjects between the seated and supine positions. 4. Discussion The significant finding of this study was that exhaled nasal NO was reduced at night when humans are generally sleepy or asleep. Exhaled nasal NO output was reduced both during sleep and during periods of brief awakening compared to daytime awake levels. The decrease in exhaled nasal NO output during sleep was due to decreases in both of the factors which determine exhaled nasal NO output: minute ventilation and NO concentration in the exhaled nasal air. Daytime voluntary hypoventilation also decreased exhaled nasal NO output, but the decrease was less than the decrease seen during either sleep or wakefulness at night despite comparable changes in minute ventilation. In addition, voluntary hypoventilation was associated with an increase in exhaled nasal NO concentration, unlike during the nighttime in which exhaled NO concentration decreased. The observation that exhaled nasal NO concentration decreased at night along with decreased exhaled minute ventilation indicates that the mass flow of NO from the nasal mucosa into nasal air decreased at night. This finding also suggests that either sleep or sleepiness (or both) is associated with changes in NO output into nasal air, and raises the possibility that NO is involved in some way with the physiologic mechanisms of sleep or sleepiness, or with the physiologic changes that occur in the nose during nighttime sleep. An interesting observation is that exhaled nasal NO output often decreased following lights out for the study, and before the subjects entered sleep as measured by polysomnography. NO output increased in the morning promptly with the subject’s awakening, and usually prior to rising from bed. There are some methodological issues requiring consideration in our study. First, measurement of exhaled nasal NO output does not represent total NO output into nasal air. Nasal air NO is in equilibrium with NO in the nasal mucosa and NO is both excreted and quickly absorbed by the nasal mucosa (Dubois et al., 1998) as well as the lower airways (DuBois et al., 1999). Our group (Kimberly et al., 1996) and others (Tornberg et al., 2002) have shown that NO is continuously released into the nasal passages during respiration. During nose breathing with the mouth closed, nasal NO is inhaled into the lower respiratory tract and largely taken up by the lungs. Exhaled air from the lungs contains decreased concentrations of NO relative to nasal exhaled air and entrains additional nasal NO in the exhalant. Also, there is evidence that nasal NO output during inspiration is greater than during expiration in awake subjects (Tornberg et al., 2002). Hence, total nasal NO output is likely underestimated by our measurements of exhaled nasal NO output in this study,
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although the precise proportion of inhaled and exhaled nasal NO output under conditions of sleep is unknown. Measurement of total nasal NO output during sleep would require the relatively invasive placement of catheters in the posterior nasopharynx or oropharynx with potential impact on the quantity and quality of sleep. The use of a fixed flow rate and fixed expiratory resistance as suggested by the 2005 ATS/ERS Nitric Oxide Joint Consensus Statement (ATS/ERS, 2005) was not possible in our tidal breathing sleeping subjects. Therefore, we chose to measure NO output only during exhalation rather than throughout the respiratory cycle. Measurement of exhaled NO output during single breath exhalations is a reproducible and convenient method for clinical measurements in humans (Palm et al., 2000). A second methodological issue in our study is that accurate collection of exhaled nasal NO output requires nasal breathing with the mouth closed. Humans prefer nasal breathing during sleep (Fitzpatrick et al., 2003), and we assisted mouth closure during sleep with use of a chinstrap. Subjects were also monitored closely by video camera, especially during exhaled nasal air collections, to assess whether mouth breathing occurred. Nevertheless, if transient mouth breathing went undetected, it would be expected to lower the measured minute ventilation. A third methodological issue is that we did not independently measure respiratory airflow or tidal volumes in our study. It is thus unclear if the pattern of breathing leading to decreased minute ventilation at night was equivalent to that used by subjects who voluntarily decreased their minute ventilation during the day over a 2-min period. If differing patterns of breathing occurred – i.e., different inhalation or exhalation flows with the potential for variance in the mucosal shear stress between the two conditions – NO output measurements might be affected. Other groups (Dubois et al., 1998; Djupesland et al., 1999; Chambers et al., 2001) and we (Giraud et al., 1998) have shown a direct relationship between airflow through the nasal passages and nasal NO output. This effect is typically small over the range of airflow during tidal breathing (Giraud et al., 1998). In addition, when nasal airflow is decreased in awake subjects, exhaled NO concentration typically increases (Dubois et al., 1998), unlike what was seen in our sleeping subjects. A severe reduction in the oxygen concentration of nasal air can produce a significant decrease in nasal NO output (Giraud et al., 1998; Haight et al., 2000). Alveolar hypoxia can lead to decreases in NO production in the lung (Grimminger et al., 1995; Nelin et al., 1996; Carlin et al., 1997). Neither blood gases nor continuous oximetry were measured in our protocol. None of our sleeping subjects were noted to have snoring or to have witnessed apneas during the overnight nasal gas collection. While the majority of the 24-h gas collection subjects were males, none were obese nor had excessive daytime somnolence. While it is unlikely that significant obstructive sleep apnea was an issue amongst our subjects, we cannot rule out the fact that the subtle decreases in oxygenation typical of sleep (Birchfield et al., 1958, 1959; Robin et al., 1958; Muller et al., 1980; Douglas et al., 1982) may have contributed to a decrease in exhaled NO output. While there would not be expected to be a difference in nocturnal oxygen changes between normal men and women (Douglas et al., 1982), the potential exists for a difference in
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NO metabolism between males and females (Liu et al., 2004). While one study suggested no sex-related difference in exhaled NO concentrations (Palm et al., 2000), further study is needed on this possible confounder. While we have demonstrated that exhaled nasal NO output is decreased during the sleep period, we are not yet able to provide a mechanism for this decrease nor can we say whether or not it is pertinent to the physiology of sleep. It is well possible that this is an epiphenomenon and that the decrease in exhaled NO output is simply reflective of the decrease in metabolism similar to the decrease in O2 consumption and CO2 production that we observed. The decrease in exhaled NO output may simply reflect a decrease in nasal mucosal blood flow. While research suggests that NO is important in sleep in the animal brain (Dzoljic and De Vries, 1994; Burlet and Cespuglio, 1997; Faradji et al., 2000), it is not known how nasal NO concentration or output relates to brain NO levels. We therefore cannot identify how exhaled nasal NO output is related to sleep or sleepiness and can only report that a decrease in exhaled NO output occurs in sleep. In a previous study (Sippel et al., 1999) we established that a topical spray of l-NAME (0.5 M, 4 mL) administered to the nasal passages of humans was associated with daytime sleepiness for several hours. The vasoconstrictor oxymetazoline also decreased exhaled nasal NO but did not cause sleepiness suggesting that sleepiness was not due to the mucosal vasoconstriction effects of l-NAME. The mechanism of sleepiness following l-NAME administration is unknown, although uptake of lNAME into the bloodstream and across the blood-brain barrier remains a possibility. Our findings prompted the experiments reported herein, which further suggest an association between sleep or sleepiness and decreased exhaled NO output. At present we can only speculate as to the reasons for such a relationship. One possibility is that decreased exhaled nasal NO at night reflects a circadian variation in nasal NO production, or is linked to other processes with circadian patterns such as body temperature or hormonal variation. Time-dependent variation in urinary NO metabolites has been demonstrated in rats (Borgonio et al., 1999). ten Hacken et al. (1998) measured orally exhaled NO in awake normal and asthmatic subjects and found no significant temporal pattern over a 24 h period. Bartley et al. (1999) performed twice daily nasal NO measurements in awake sitting subjects and concluded there was no circadian variation, but Palm et al. (2003) showed that the concentration of exhaled NO increased from early morning to midday to late afternoon, suggesting a possible circadian relationship. We could identify no consistent pattern in the exhaled NO output in the hourly measurements in our subjects as the day progressed from morning to afternoon. A second possible explanation for a relationship between nasal NO and sleep could be the need for humans to maintain a patent nasal airway during sleep. Humans prefer to breathe nasally during sleep (Fitzpatrick et al., 2003), and decreased nasal patency is associated with sleep-disordered breathing (Zwillich et al., 1981; Lavie et al., 1983). Imada et al. (1996, 2002) have shown an inverse relationship between nasal airway NO and airflow resistance through the nasal passages. On the other hand, despite decreasing nasal volume and increasing air-
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flow resistance, assuming the supine position was not associated with changes in nasal NO output in awake humans (Chatkin et al., 1999) which we now confirm. We speculate that reduced exhaled NO output at night may relate to the need for changes in temperature and humidity conditioning of nasal air during nighttime sleep compared with the awake state. The onset of sleep is associated with a decrease in body temperature. Body temperature is regulated at a lower level during non-REM sleep compared with wakefulness (Glotzbach and Heller, 2000). During REM sleep, the temperature is even more variable with thermoregulatory responses being further inhibited. There is evidence that the nasal passages may participate in thermoregulation in response to changes in temperature locally (Burgess and Whitelaw, 1984), or reflexly with cooling (Speisman, 1936) or heating (White and Cabanec, 1995) of the skin. In a prior study (Holden et al., 1999), we demonstrated that reduced exhaled nasal NO (following topical application of the vasoconstrictor oxymetazoline or l-NAME) was associated with reduced nasal air temperature, and increased exhaled NO (following topical papaverine, a vasodilator) was associated with increased nasal air temperature. These findings support a role of NO in temperature conditioning of nasal air, and by extension, in whole body thermoregulation. In response to a decrease in core body temperature, a reduction in mucosal NO might be expected to reduce the addition of heat to the inhaled air and increase the recovery of heat during exhalation. The balance of these processes could lessen the total loss of heat from the respiratory tract during sleep. 5. Conclusion We report that exhaled nasal NO output is reduced at night during sleep as well as with short nocturnal periods of wakefulness in humans. The reduction during sleep was associated with both decreased minute ventilation and NO concentration in the exhaled air compared with daytime values. Our findings suggest a role of nasal NO in the sleep state or sleepiness, or a role in the physiologic processes that accompany sleep. Further research is needed to develop better mechanistic insight into the association between exhaled NO and sleep. Given the potential bearing of NO to such processes as vasoregulation, gas exchange, and the temperature and humidity conditioning of inspired air, we feel that the finding of decreased exhaled NO output is potentially relevant to understanding the physiology of sleep. References ATS/ERS, 2005. Recommendations for standardized procedures for the online and offline measurement of exhaled lower respiratory nitric oxide and nasal nitric oxide. Am. J. Respir. Crit. Care Med. 171, 912–930. Bartley, J., Fergusson, W., Moody, A., Wells, A.U., Kolbe, J., 1999. Normal adult values, diurnal variation, and repeatability of nasal nitric oxide measurement. Am. J. Rhinol. 13, 401–405. Birchfield, R.I., Sieker, H.O., Heyman, A., 1958. Alterations in blood gases during natural sleep and narcolepsy. Neurology 8, 107–112. Birchfield, R.I., Sieker, H.O., Heyman, A., 1959. Alterations in respiratory function during natural sleep. J. Lab. Clin. Med. 54, 216–222.
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