Address of Corresponding Author

Respiration 2005;72:395-401 (DOI: 10.1159/000086254)

 Outline

• Key Words
• Abstract
• Introduction
• Patients and Methods
• Patients
• Sleep Study
• Circulating Parameters
• Serum Leptin Measurements
• Serum Ghrelin Measurements
• Statistical Analysis
• Results
• Discussion
• References

• Author Contacts
• Article Information
• Publication Details
• Drug Dosage / Copyright

  Key Words

• Ghrelin
• Hypopnea
• Leptin
• Obesity
• Sleep apnea

  Abstract

Background: Leptin is a hormone with well-investigated functions concerning body composition, energy homeostasis and feeding behavior in humans. The obstructive sleep apnea syndrome (OSAS) is strongly associated with obesity, which is known to be closely associated with hyperleptinemia. More recently, ghrelin, a hormone that also influences appetite and energy homeostasis, has been discovered. Objectives: The aim of this study was to investigate serum leptin and ghrelin levels in obese patients with OSAS in comparison with equally obese controls without OSAS. Methods: Thirty untreated obese patients with moderate-severe OSAS (apnea-hypopnea index: AHI ≥15) and 22 obese controls (AHI <5) were studied. To confirm the diagnosis, all patients underwent standard polysomnography in our sleep disorders center. Serum samples were taken at 08:00 h in the morning after overnight fasting. Results: Significantly higher serum leptin levels were found in OSAS patients compared to controls (p = 0.012), but there was no significant difference in serum ghrelin levels between OSAS patients and controls. Serum leptin levels were significantly correlated with body mass index in both OSAS patients (r = 0.55, p = 0.002) and controls (r = 0.46, p = 0.028), but only in OSAS patients was the leptin level significantly correlated with AHI (r = 0.38, p = 0.036). Conclusion: These data support findings suggesting that leptin is a hormonal factor affected by OSAS and not determined by obesity alone. Further studies are needed to investigate the relationship between serum ghrelin and OSAS.

Copyright © 2005 S. Karger AG, Basel

 Introduction

The etiology of obesity is heterogeneous, with several factors having the potential to cause energy imbalance over long periods. These factors include a high-fat diet, a low level of habitual physical activity, a low resting metabolic rate for a given body mass and body composition and a high respiratory quotient in the fasting state. Interest in the pathophysiology of obesity has recently intensified with the discovery of leptin, the anti-obesity hormone. Leptin, a protein of 167 amino acids, has a structure similar to that of cytokines [1]. The hormone is produced by white adipose and placental tissue. It inhibits neuropeptide Y synthesis in the hypothalamus and downregulates food intake. Leptin is also associated with increased energy expenditure [2].

More recently, ghrelin, a hormone that also influences appetite and energy homeostasis, has been discovered. Since its initial description in 1999, there is a growing body of evidence that this 28-amino-acid gut-strain peptide has a strong effect on appetite, food utilization, body weight and body composition in both animals and humans. It stimulates hunger and food intake when administered intravenously in healthy humans [3].

The obstructive sleep apnea syndrome (OSAS), which is characterized by repeated episodes of upper airway obstruction during sleep leading to significant hypoxemia, is very prevalent particularly amongst middle-aged men, although it is increasingly recognized in women. According to epidemiological studies, 2–5% of the population meet the minimal diagnostic criteria, and two community-based studies have found that about 2% of women and 4% of men are affected by OSAS [4]. Obesity is a major risk factor for OSAS, occurring in up to 50% of obese men [1]. The high prevalence of obstructive sleep apnea in obese humans and the established role of leptin as a respiratory stimulant and appetite suppressant in the mouse raised the possibility that sleep apnea could be a leptin-deficient state. The aim of the present study was to test whether increases of circulating leptin and ghrelin levels were independent of obesity in patients with OSAS. We examined the serum leptin and ghrelin levels, as well as lipid profiles which reflect obesity, in obese control subjects and obese patients with OSAS.

 Patients and Methods

 Patients

Newly diagnosed obese (body mass index, BMI >27 kg/m²) men with moderate-severe OSAS and age- and BMI-matched male obese control subjects were enrolled in this study. Subjects were recruited from patients referred for suspected sleep apnea to the Sleep Disorders Center (Faculty of Medicine, Gazi University, Ankara, Turkey). The study was approved by the institutional ethics committee and performed in accordance with the guidelines of the Declaration of Helsinki. The subjects were examined with polysomnography (PSG) and classified as controls according to data of the apnea-hypopnea index (AHI). All patients with OSAS were also diagnosed with PSG. Before enrollment, all subjects gave written informed consent.

BMI was calculated as weight in kilograms divided by the square of the height. Neck circumference was measured at the level of the superior border of the cricothyroid membrane using a tape measure.

 Sleep Study

Overnight PSG was performed in all patients by a computerized system (Somnostar alpha; Sensormedics, Los Angeles, Calif., USA) and included the following variables: electrooculogram (2 channels), electroencephalogram (4 channels), electromyelogram of submental muscles (2 channels), electromyelogram of the anterior tibialis muscle of both legs (2 channels); electrocardiogram, and airflow (with an oronasal thermistor). Chest and abdominal efforts (2 channels) were recorded using inductive plethysmography, arterial oxyhemoglobin saturation (SaO₂: 1 channel) by pulse oximetry with a finger probe. The recordings were conducted at a paper speed of 10 mm/s, and sleep stages were scored according to the standard criteria of Rechtschaffen and Kales [5]. Arousals were scored according to accepted definitions [6]. Apneas were defined as complete cessation of airflow ≥10 s. Hypopneas were defined as reduction of >50% in one of three respiratory signals, airflow or either respiratory or abdominal signals of respiratory inductance plethysmography, with an associated decrease of ≥3% in oxygen saturation or arousal. AHI was defined as the number of apneas and hypopneas per hour of sleep. Patients with AHI <5 were included in the control group. Patients with AHI ≥5 were considered as OSAS patients. An AHI of ≥5 to <15 indicated mild OSAS, ≥15 to <30 moderate OSAS, and >30 severe OSAS. Patients with sleep disorders, except OSAS, such as upper airway resistance syndrome, periodic leg movements or narcolepsy were excluded.

Pulmonary disorders (except for OSAS) and metabolic diseases such as diabetes mellitus were also excluded.

 Circulating Parameters

All subjects had fasting blood samples taken between 7:00 a.m. and 8:00 a.m. Blood samples were immediately sent to the hospital laboratory for lipid evaluation, while a specimen of clotted blood was centrifuged at 3,000 g for 10 min for serum assessment, which was stored at –70°C until analysis. Cholesterol and triglyceride concentrations in serum and lipoprotein fractions were determined enzymatically using standard laboratory procedures.

 Serum Leptin Measurements

Serum leptin levels were measured with a solid-phase sandwich enzyme-linked immunosorbent assay using a human leptin (h-Leptin) kit (Biosource International, Dallas, Tex., USA). The minimum detectable dose of leptin is 3.5 pg/ml using this assay protocol [7]. Normal leptin levels are 2–11.1 ng/ml.

 Serum Ghrelin Measurements

Serum ghrelin levels were measured using an enzyme immunoassay kit to detect a specific peptide and its related peptides based on the principle of the ‘competitive’ enzyme immunoassay (Phoenix Pharmaceuticals, New Jersey, USA). The minimum detectable concentration of ghrelin is 0.1 ng/ml using this assay technique [8]. The mean normal level of ghrelin is 87.79 (±10.27) pg/ml.

 Statistical Analysis

Means and SEM were determined for continuous variables and percentages for categorical variables. Significant differences between two groups were analyzed by Mann-Whitney U test. Correlations were identified using Spearman’s correlation coefficient.

To assess the relative strength of association of OSAS as well as possible confounding factors with leptin, multiple regression analysis was employed in the patients with OSAS as a single group. In this analysis, we used serum levels of leptin as dependent variable and evaluated the order of inclusion in the model of the following independent variables: AHI, BMI, total cholesterol, high-density lipoprotein, low-density lipoprotein and very-low-density lipoprotein.

All statistical analyses were carried out using statistical software (SPSS, version 11.0 for Windows; SPSS, Chicago, Ill., USA). Differences were considered significant at p < 0.05.

 Results

Thirty of the 65 obese subjects (BMI >27 kg/m²) who underwent PSG were considered to have moderate-severe OSAS. Twenty-two subjects with obesity and snoring were considered not to have OSAS and were enrolled as control subjects.

Serum leptin levels were significantly higher in patients with moderate-severe OSAS compared to control subjects (p = 0.012). There was no difference in serum ghrelin levels or other parameters between both groups (table 1; fig. 1). The patients were statistically divided into four quartiles according to their BMI. The first quartile included patients with a BMI of 27.11–28.68 kg/m², the second of 28.68–31.06 kg/m², the third of 31.06–33.33 kg/m² and the last comprised patients with a BMI of 33.33–43.59 kg/m². In each quartile, serum leptin levels of OSAS patients exceeded those of the controls (fig. 2).

Fig. 1. Serum leptin and ghrelin levels in control subjects and patients with OSAS.

Fig. 2. Serum leptin levels of OSAS patients and controls versus BMI. (The first quartile contained the BMI between 27.11 and 28.68 kg/m2, the second between 28.68 and 31.06 kg/m2, the third between 31.06 and 33.33 kg/m2 and the last between 33.33 and 43.59 kg/m2.)

Table 1. Demographic and clinical parameters in the OSAS patients and controls

Serum leptin levels were significantly and positively correlated with AHI in patients with OSAS (r = 0.38, p = 0.036) but not in control subjects (r = –0.08, p = 0.72), as well as with BMI in patients with OSAS (r = 0.55, p = 0.002) and control subjects (r = 0.46, p = 0.028; fig. 3, 4). Serum ghrelin levels were not correlated with BMI, neither in patients with OSAS nor in control subjects (r = –0.07, p = 0.69, and r = 0.28, p = 0.2, respectively). Serum ghrelin levels were also not correlated with AHI in patients with OSAS and control subjects (r = –0.05, p = 0.8, and r = –0.02, p = 0.91, respectively). However, in all 65 obese subjects, serum ghrelin levels were positively and significantly correlated with BMI (r = 0.35, p = 0.004; fig. 5). There was no correlation between serum lipid levels and AHI or BMI (data not shown).

Fig. 3. Correlation between serum leptin levels and AHI or BMI in patients with OSAS (n = 30).

Fig. 4. Correlation between serum leptin levels and BMI in control subjects (n = 22).

Fig. 5. Correlation between serum ghrelin levels and BMI in all cases (n = 65).

Evaluation of the relative strength of associations using multiple regression analysis between leptin and the severity of OSAS as well as other possible confounding variables showed that AHI and BMI were the two variables that were included in the model (table 2).

Table 2. Linear multiple regression analysis with serum leptin as dependent variable

 Discussion

Leptin regulates fat mass by decreasing food intake and increasing resting energy expenditure. Serum leptin concentrations are highly correlated with body fat content, and its production by adipocytes rapidly declines during starvation [9]. Circulating leptin levels are typically higher than normal in human obesity, indicating that it is a leptin-resistant state [10]. In agreement, our study showed that serum leptin levels were correlated with BMI independently of cholesterol, lipoprotein levels and neck circumference, both in patients with OSAS and in control subjects.

Hyperlipidemia is common in obese subjects. Lipid profiles in OSAS patients have not been widely studied, although clinical observations suggest that dislipidemia is common in patients with OSAS [11]. In our study, there was no correlation between lipid profiles and BMI, which reflects the severity of obesity, and no difference was found between patients with OSAS and control subjects regarding lipid levels. These results may be explained by the fact that both study groups comprised obese individuals with similar BMIs. A limitation of our study was the lack of data on lipid-lowering medications in our patients. Therefore, the lack of a difference between groups may be explained by successful medical treatment of high lipid levels in some patients.

OSAS is a common disease in obese subjects, and it seems to be a consequence of increased visceral fat accumulation. It is believed that increased deposition of fat or soft tissue in the neck and upper airway region predisposes the subject to upper airway collapses during sleep; a larger neck circumference, probably reflecting greater fat or soft tissue deposition, is more significantly associated with sleep apnea. Since obesity has been associated with hyperleptinemia, a possible impact of those factors on the development of OSAS cannot be ruled out. Our study revealed increased serum leptin levels in patients with OSAS compared with the control group. We and others have shown that patients with OSAS have even higher serum leptin levels than subjects without OSAS matched for age and BMI [12, 13]. The positive correlation between serum leptin and AHI, which reflects the severity of OSAS, noted in our study is also in accordance with previous studies. Only Schafer et al. [14] found that serum leptin levels were not related to the degree of OSAS. Earlier studies on continuous positive airway pressure (CPAP) in the treatment of patients with OSAS suggested that OSAS has a significant effect on serum leptin levels, with significantly decreased levels following CPAP treatment [13, 14, 15, 16]. On the other hand, Shimizu et al. [17] reported a correlation between serum leptin levels and circadian rhythm as well as absence of CPAP treatment. According to this finding, independent of the known relationship between obesity and increased serum leptin levels, OSAS represents a leptin-resistant state.

Apart from its anti-obesity effects, leptin exerts important physiological effects on the control of respiration. O’Donnell et al. [18], based on their findings in mice, predicted that obese subjects may be at risk for developing hypoventilation when serum leptin levels are high but cerebral serum fluid leptin levels are proportionately low. Phipps et al. [19] reported that serum leptin is a better predictor than percent body fat for the presence of hypercapnia in patients with obesity-hypoventilation syndrome (OHS) and concluded that in patients with obesity, a higher leptin level predisposes to the development of OHS. According to their findings, it seems that leptin acts to decrease alveolar ventilation in obesity.

OHS occurs in a minority of obese subjects secondary to severe upper-airway obstruction and altered central control of breathing. Therefore, it is difficult to explain the increased leptin levels in OSAS patients independent of obesity with respect to the relationship between leptin and the central respiratory center. Therefore, further studies comparing serum leptin levels between OSAS patients with or without OHS and evaluating the relationship between leptin and the control of the muscles in the upper airways are needed.

Leptin is a protein of 167 amino acids and has a structure similar to that of cytokines. In addition, pro-inflammatory cytokines such as interleukin-6 and tumor necrosis factor-α are increased in the serum of patients with OSAS compared to normal individuals [12, 20, 21, 22]. It can be speculated that leptin release in OSAS is increased because of upper-respiratory-tract inflammation induced by pro-inflammatory cytokines.

Another result of our study was the lack of a difference in serum ghrelin levels between OSAS patients and normal subjects. The discovery of ghrelin as an endogenous ligand for the growth hormone secretor receptor and subsequent studies have widened our understanding of the control of growth hormone secretion and energy homeostasis. Ghrelin increases feeding and weight gain and may have a role to play in the treatment and prevention of obesity [23]. Possible interactions between ghrelin and respiratory mechanisms still have not been investigated. Only Harsch et al. [15] investigated ghrelin levels in a subgroup of patients and found, in contrast to our results, that ghrelin levels were significantly higher in OSAS patients than in BMI-matched controls. This contradiction between our results and those of the study by Harsch et al. [15] can be explained by the different methods used (our enzyme immunoassay kit vs. their radioimmunoassay kit). However, our study showed a positive correlation between serum ghrelin levels and BMI, which confirmed the relationship between ghrelin and obesity.

In conclusion, elevated serum leptin levels in OSAS patients cannot only be explained by increased adipose tissue and may be considered as a marker of OSAS. Although our study needs confirmation by a larger study cohort, the present study revealed that serum ghrelin is not associated with OSAS but has a positive correlation with BMI, which reflects the severity of obesity. Further studies on the relationship between the pathogenesis of OSAS and markers of obesity are required.

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  Author Contacts

Dr. Tansu Ulukavak Ciftci
Yesilyurt sok. 23/5, A. Ayranci
TR–006540 Ankara (Turkey)
Tel. +90 312 2026137, Fax +90 312 2129019
E-Mail tansu.ciftci@gazi.edu.tr

  Article Information

Received: June 22, 2004
Accepted after revision: December 2, 2004
Number of Print Pages : 7
Number of Figures : 5, Number of Tables : 2, Number of References : 23

  Publication Details

Respiration (International Journal of Thoracic Medicine)

Vol. 72, No. 4, Year 2005 (Cover Date: July-August 2005)

Journal Editor: C.T. Bolliger, Cape Town
ISSN: 0025–7931 (print), 1423–0356 (Online)

For additional information: http://www.karger.com/res

  Drug Dosage / Copyright

Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.