Substance P, Thyrotropin-releasing Hormone, and Monoamine Metabolites in Cerebrospinal Fluid in Sleep Apnea Patients':'
THORARINN GISLASON, JAN HEDNER, LARS TERENIUS, GARTH BISETTE, and CHARLES B. NEMEROFF
The etiology and pathogenesis of the sleep apnea syndrome (SAS) are unclear. However, central nervous mechanisms involved in respiratory regulation and/or upper airway muscle function may be important in the development of SAS (1). The concentrations of endogenous neurotransmitters or their metabolites in the cerebrospinal fluid (CSF) have frequently been used as an estimate of neuronal activity in the central nervous system (2, 3). Elevated CSF levels of the serotonin metabolite 5-hydroxyindoleacetic acid (5HIAA) (4, 5) and the dopamine metabolite homovanillic acid (HVA) (6) have also been reported in patients with SAS. In one patient, the CSF concentrations of both 5-HIAA and HVA were found to decrease after operative treatment (6). There are no previous reports on CSF levels of neuropeptides such as thyrotropin-r-eleasing hormone (TRH) and substance P (SP), which both potentially may interfere with central respiratory regulation (7, 8). We have previously (9) reported on CSF endogenous opioids in 15 consecutive patients with SAS. The present study was undertaken to measure CSF concentrations of TRH and SP and also the monoamine metabolites HVA, 5-HIAA, and 3-methoxy-4-hydroxyphenyl glycol (MHPG) because they may be potentially involved in the genesis of SAS. All patients were investigated in accordance with a protocol previouslyapproved by the Ethics Committee of the Medical Faculty of Uppsala University. The study comprised 14 men and one woman 39 to 63 yr of age, with a mean age of 53 yr (table 1). Five patients (Patients 1, 3, 6, 12, and 14)were habitual smokers, and six weretreated for systemic hypertension. None of the patients exhibitedclinical or laboratory signs of hypothyroidism. Most patients were overweight (table 1). During 4 to 5 days in hospital all patients were investigated extensively. In 10 of the patients with SAS the measurements were repeated 6 months after uvulopalatopharyngoplasty (UPPP) (10). Standard spirometry and plethysmography wereperformed, and arterial blood gas determinations were obtained. The ventilatoryresponseto hyperoxichypercapnea was determined by a modified Read rebreathing test (11) during rest in the awake state. Five patients had lower airway obstruction, as evidenced by an FEV! ~ 75010 (table 1). Two of these patients (Patients 1 and 3) also had increased residual volume, decreased ventilatory response to CO 2 , polycythemia, and clinical symptoms of chronic bronchitis. An all-nightpolygraphicrecording was made using standard techniques (1,9). Oxygen saturation was monitored continuously by a BIOX III oximeter(Ohmeda, Louisville, KY).Sleep stages were scored, and respiratory tracings were
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SUMMARY The cerebrospinal fluid (CSF) concentrations of thyrotropin-releasing hormone (TRH), substance P (SP), 5-hydroxylndoleacetic acid (5-HIAA), homovanilllc acid (HVA), and 3-methoxy-4hydroxyphenyl glycol (MHPG) were measured in 15 consecutive patients with the sleep apnea syndrome (SAS) and in healthy control subjects. Second measurements were performed 6 months after surgical treatment in 10of the patients. The mean (± SO)concentration of TRH-Ilke Immunoreactive material (TRH-L1M)(pg/ml) did not differ significantly between patients with SAS (8.1 ± 2.8) and control subjects (7.5 ± 2.2). However, postoperatively, this concentration was increased In the six clinically cured patients with SAS, from 6.9 ± 2.7 to 9.4 ± 1.6(p < 0.03). Substance P-llke immunoreactive material (SP-L1M) was higher in untreated patients with SAS than In control subjects: 19.2 ± 6.7 versus 14.4 ± 4.2 fmollml (p < 0.02), and the level remained high after operation in the group treated surgically. The HVA, 5·HIAA, and MHPG concentrations were similar In patients with SAS and control subjects, and no consistent changes were found postoperatively. The CSF deviations in TRH-L1Mand SP-LIM concentrations in the patients may reflect a primary central nervous system defect or they may be secondary to intermittent nocturnal hypoxia, progressive hypercapnia, and/or sleep fragmentation. In this sense, both these systems may be markers of SAS-SP as a ''trait'' marker AM REV RESPIR DIS 1992; 146:784-786 and TRH as an indicator of the current state.
evaluatedfor the presenceof apneas and hypopneas (1). An apneas and hypopneas (A+H) index was calculated as the total number of events per hour of sleep (1). Lumbar puncture was performed in the decubitus position in the early morning, during the 4 to 5 days the patients werehospitalized. The CSF was collected by free flow into plastic tubes and immediately frozen and stored in aliquots at -70 0 C until analyzed. An initial CSF sample was obtained from all SAS patients, and the 12 patients undergoing operation were asked for a repeated sample 6 months after the UPPP. This was agreed to by 10 of the 12 patients. Two different control groups were included in the investigation, but they did not undergo sleep studies and were not obese. For the comparison of the SP concentrations, a group of 18 healthy volunteers, 10 women and eight men with a mean age of 30 yr (range, 22 to 45 yr) (12) was used. To match the patient samples analyzed for HVA, 5-HIAA, MHPG, and TRH-LIM, another group of 12male patients with a mean age of 39yr (range, 23 to 71 yr) was selected from among patients undergoing diagnostic lumbar puncture because of a suspected neurologic disease.These control samples were included only if the CSF was clear and did not displayany signsof barrier damage in terms of the glucose, cell, or protein content. The control analyses were performed during the same time as those of the patient samples. SP-LIMand TRH weredetermined according to methods described previously (12, 13). The CSF concentrationsof HVAand 5-HIAA weremeasured by means of a reverse-phasehigh-performance liquid chromatography procedureas describedby Kilts and coworkers (14). MHPG was determined separately with the same technique. Allvaluesare given ± 1SD.Conventionalstatistical methods were used. The two-tailed t test was employed for comparison of group means.Student's t test on paired values was used for comparison of preoperative and postoperative levels. The
strength of the relationships betweendifferent measurementswasassessedby least-squares linearcorrelation analysis. The mean total duration of sleep in the 15 patients with SAS during the control night was 332 ± 49 min, and the mean A + H index was 47 ± 34 (table 1). All 12 surgically treated patients were followed for 6 months postoperatively (10). The A + H index decreased from an initial value of 42 ± 28 to 17 ± 25 (p < 0.01, n = 12) postoperatively. Ten of the 12 operated patients showed a 50% or greater reduction in this index postoperatively (table 1) and were considered as responders. Only one patient (Patient 1) showed some weight reduction (12 kg). Only minor individual changes in pulmonary function and in the ventilatory response to CO 2 were observed postoperatively (after 6 months) except in Patients 1 and 3. These patients increased the ventilatory responsiveness to CO 2
(Receivedin originalform September 26, 1991 and in revised form March 25, 1992) ! From the Departments of Lung Medicine and Pharmacology, Uppsala University, Uppsala, the Department of Clinical Pharmacology, Sahlgren's University Hospital, Goteborg, Sweden, and the Departments of Psychiatry and Pharmacology and the Center for Aging and Human Development, Duke University Medical Center, Durham, North Carolina. 2 Correspondence and requests for reprints should be addressed to Thorarinn Gislason, M.D., Department of Lung Medicine, Vifilsstaoir, 210 Garoabaer, Iceland.
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TABLE 1 CHARACTERISTICS OF THE STUDY GROUP OF 14 MEN AND ONE WOMAN (PATIENT 7) Age (yr)
BMI (kglm 2 )
FEV 1 (% pred)
RV (% pred)
Paco 2 (kPa)
AV/APC0 2 (LiminlkPa)
A + H Index* (nih)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
61 57 51 54 58 52 60 39 51 50 64 39 60 61 45
32.8 32.5 39.0 36.3 30.4 24.5 29.0 28.4 27.0 33.6 29.0 36.0 28.7 34.3 38.4
71 109 67 72 97 95 134 106 81 96 85 91 86 70 64
141 89 156 113 81 83 111 71 98 105 110 86 119 123 102
7.5 4.5 5.7 4.8 4.9 5.3 4.7 4.9 5.1 5.2 4.5 3.3 4.3 5.4 5.4
1.8 32.7 9.8 21.8 21.6 29.8 36.6 26.1 25.1 24.7 30.2 16.2 20.2 17.8 20.3
29 (100) 23 (92) 16 (100) 49 79 (15) 25 (100) 55 (65) 10 (91) 37 (82) 84 16 (74) 112 7 74 (51) 96 (32)
Mean SD
53 8
32.0 4.3
88 19
106 22
5.0 0.9
22.3 8.9
47 34
Patient No.
Medical History COPD COPD HT HT HT
HT, DM DD COPD HT HT
Definition of abbreviations: BMI = body massindex;RV = residual volume;A + H Index = apneaplus hypopneaindex;COPO = chronic obstructivepulmonary disease; HT = systemic hypertension; DO = depressive disorder; OM = diabetesmellitus.
• Numbers in parentheses indicate the percent postoperative reduction of the A + H index.
from 1.8 to 7.3 L/min/kPa and 9.8 to 13.4 L/min/kPa, respectively. No significant differences in the initial CSF concentrations (ug/ml) of HVA (21.7 ± 9.4 versus 15.5 ± 8.8), 5-HIAA (17.5 ± 5.3 versus 22.8 ± 13.2), or MHPG (5.4 ± 3.4 versus 7.3 ± 5.3) were found between the SAS group (n = 14) and the 12 control subjects. In the untreated SAS group both HVA and 5-HIAA werepositively correlated to resting Pco., and negatively to the ventilatory response to COl as well as resting POl (table 2). CSF concentrations of the studied monoamine metabolites werenot statistically related to the A + H index (table 2), and no consistent changes were found between the preoperative and postoperative concentrations of these metabolites in
patients undergoing UPPP, regardless of postoperative outcome. The CSF concentration of SP-LIM was higher in 14 of the patients with SAS compared with the healthy control subjects, 19.2 ± 6.7 and 14.4 ± 4.2 fmol/ml, respectively (p < 0.02). The patients with SAS with lower airway obstruction had significantly lower SPLIM (table 2) (p < 0.01), and among the five patients with FEV 1 of ~ 75% predicted only one patient (Patient 1) had an SP-LIM value of > 15 fmol/ml. There was a tendency (p = 0.05) towards lower SP-LIM values with increasing obesity, but no statistical correlation between SP-LIM and resting Pco, or POland A + H index (table 2). No consistent changes compared with the preoperative values were
TABLE 2 CORRELATION COEFFICIENTS BETWEEN CLINICAL DATA AND CSF NEUROTRANSMITIERS SP-L1M Age BMI FEV 1 RV Paco 2 AV/APC0 2 Pa02 A + H index TRH HVA 5-HIAA MHPG
(+) 0.21 (-) 0.53* (+) 0.71t (-) 0.23 (+) 0.18 (+) 0.35 (+) 0.23 (-)0.46 (+) 0.28 (-) 0.19 (+) 0.30 (+) 0.15
TRH
(+) (-) (-) (+) (+) (-) (-) (-)
0.26 0.12 0.08 0.05 0.30 0.10 0.24 0.10
HVA (-) 0.27 (-) 0.20 (-)0.42 (+) 0.60:t: (+) 0.84t (-) 0.82t (-) 0.59:t: (-) 0.06 (+) 0.55§
5-HIAA (-) (-) (-) (-) (+) (-) (-) (-) (+) (+)
0.26 0.13 0.35 0.54 0.86t O.77t 0.55* 0.01 0.5911 0.8St
MHPG (-) 0.56 (-) 0.03 (-) 0.22 (+) 0.67:t: (-) 0.22 (-) 0.05 (-) 0.55 (-)0.49 (+) 0.81t (-) 0.54§ (-)0.47
Definition of abbreviations: TRH = thyrotropin releasing hormone; SP = substance P; 5-HIAA = 5-hydroxyindoleacetic acid; HVA = homovanillicacid;MHPG = 3-methoxy-4-hydroxyphenyl glycoland clinical findings. For other definitions, see table 1. • P = 0.05. t p < 0.01. :j: P < 0.05. § p = 0.08. II p = 0.06.
found in CSF samples obtained postoperatively from 10 patients. Among seven postoperative responders, the SP level remained unchanged in one patient, increased in three, and decreased in three. The mean level of TRH-LIM in CSF was similar in 14 patients with SAS (8.1 ± 3.0 pg/ml) and in 10control subjects (7.5 ± 2.2 pg/ml). The TRH-LIM levels were not significantly correlated to age, weight, pulmonary function, or the severity of SAS (table 2). Both preoperative and postoperative TRHLIM were measured in six patients, all responders. In these patients TRH-LIM increased by an average of 36070 postoperatively (p < 0.03). The monoamine metabolites HVA and 5-HIAA weresignificantly positivelycorrelated, whereas there was a negative correlation between HVAand MHPG (table 2). TRH was positivelycorrelated to MHPG, and there was a tendency towards a statistically positive correlation between TRH and 5-HIAA (p = 0.08) (table 2). SP-LIM concentrations were unrelated to the other studied transmitters and metabolites. Similarly, CSF endorphin levels previously reported from this patient material (9) were unrelated.
* * * The SASis usually associated with some kind of upper airway narrowing, but for unknown reasons these predisposing anatomic factors seem to cause sleep-related apneic episodes in some patients, but not in others. Thus, in SAS there appears to be a delicate interaction between predisposing anatomic factors and apnea-related pressure changes in the upper airway. Clearly, the way by which the central respiratory and upper airway muscle tone controlling mechanisms adapt to this delicate balance in each individual may be important in the expression of apnea occurrence. The primary determinants for the central nervous system(CNS) adaption may be increased work of breathing, upper airway receptor sensitivity hypoxia, and/or sleep fragmentation. One way to investigate the possible involvement of a central neurochemical disorder is to measure the CSF concentration of neurotransmitters or their metabolites (15). However, some other events might be taking place within the CNS that cannot be identified by measuring CSF samples. The present study group was heterogeneous with respect to the severity of the disease, and it was therefore extensivelyinvestigated. With the benefit of hindsight a well-matched control group undergoing both a full polysomnographic study and a lumbar puncture may have been more desirable, but such subjects are difficult to obtain for both practical and ethical reasons. No previous reports on the CSF monoamine metabolites HVA and 5-HIAA have clearly indicated the nature or source of the control subjects (4-6). Still, in these studies, the 5-HIAA levels were claimed to be increased in all patients with SAS, whereas high
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HVAlevelswerefound in only one case. These observations werenot confirmed in the present study. If anything, a tendency towards lower mean value was noted in the SAS group, and there were no general changes in the postoperative valuesof the UPPP responders. In addition CSF levels of the major noradrenergic metabolite MHPG were similar in patients with SAS and control subjects. One reason for the discrepancy between these previous reports and the present findings may relate to concomitant daytime hypoventilation. In previous studies daytime blood gases or spirometry data werenot addressed. However, among our patients with SAS both HVA and 5-HIAA were correlated to both resting Pc02 and the ventilatory response to CO 2 (table 2). In addition, we have previously reported 2.4 times higher HVAlevelsthan expected for age in two children with chronic central alveolar hypoventilation (16). High HVAand 5-HIAA levels may therefore reflect all the actual pulmonary and ventilatory situation rather than an SAS-related CNS mechanism. The SP concentration was significantly higher in patients with SAS than in control subjects. In different animal models SP appears to act as a powerful respiratory stimulant by exerting its effects mainly in the ventrolateral region ofthe medulla oblongata (3, 17).Moreover, a specificantagonist of SP was found to readily induce apnea in experimental animals (18). Infants at risk for sudden infant death syndrome were found to have significantly lower plasma SP-LIM (19), and in this study higher SP levels weresignificantly correlated to a shorter mean apnea duration. Finally, patients with SAS and concomitant hypercapnia have been found to have significantly lower SP-immunoreactivity density in certain brainstem regions when compared with eucapnic patients with SAS (20). These data indicate that SP may act to sustain the central ventilatory drive in the medulla oblongata. In the present context, high SP-LIM concentrations in patients with SAS would imply a compensatory increase to overcome the impairment of normal ventilation. Another possible explanation for the high SP concentration in patients with SAS may be chronic hypoxia and/or hypercapnia. SPLIM was found to increase during hypoxia in the dorsal medulla oblongata of artificially ventilated cats (21). However, in our study there was no significant correlation between SP-LIM and blood gas measurements or apnea index. Moreover, SP-LIM remained unchanged postoperatively in our patients who had undergone successful UPPP, indicating that hypoxia or sleep disturbances or any other factor secondary to the repetitive apneas was not the cause of the high SP concentrations. The third possibility may be an involve-
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ment of SP in the complex mechanism regulating the satiety system. In 14obese children plasma SP levels were twice those of a healthy control group (22). The degree of obesity (body mass index)in our patients with SAS was, however, negatively related to the CSF SP-LIM in the present study (table 2). Clearly, CSF and plasma levels may differ, and a comparison between data cannot easily be performed. Still, our findings do not indicate that obesity per se could explain the increased CSF SP-LIM in patients with SAS. The TRH-LIM levelsin the CSF were not different between patients and control subjects, but they increased significantly postoperatively. The elevated TRH-LIM concentration might therefore reflect the previously repeated hypoxia, progressivehypercapnia, or chronic sleep disturbances. To our knowledge, the effect of chronic blood gas changes on the CSF level of TRH-LIM is not known. However, TRH may well be intimately involved in sleep regulation, as this tripeptide has been shown to affect physiologic sleep (23) and pharmacologically induced narcosis (24). Moreover, TRH acts as a powerful respiratory stimulant in the rat through an effect in the hypothalamic region (7). Development of SAS has also been described in association with some hypothalamic lesions (25), which may imply a connection between this syndrome and modified TRH activity in the CNS. Acknowledgment The writers thank the Swedish National Association against Heart and Chest Diseases, Stockholm, and the King Oscar II Jubilee Fund, Stockholm.
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