2.1

Study Population

Thirty-one patients (age range 25–41 years (mean ± SD age, 33.7 ± 7.1)) who presented with ENP at the Otolaryngology Clinic of Erzurum Education and Research Hospital between February 2008 and January 2010 were included in this study. Twenty-seven age and gender matched control subjects (age range 24–39 years (mean ± SD age, 32.9 ± 6.8)) were picked from among healthy patients attending the same hospital during the same study period.

At study entry, routine blood and urine analyses, cardiopulmonary graphs, and chest X-rays were performed in all subjects. The patients and controls both underwent endoscopic nasal examination, and only the patients received paranasal sinus computed tomography for the diagnosis of nasal polyposis. The Epworth Sleepiness Scale which is a simple and validated questionnaire for assessing the sleep disorders was used to appreciate the obstructive sleep apnea (OSA) in all patients and controls. In both groups none of the subjects did not have OSA.

Study patients and control subjects with any clinical or laboratory evidence of inflammation, OSA or infection, those who had received hormone therapy and/or steroid therapy in the one month prior to the study, or those who were taking any drugs that might affect melatonin and cortisol levels (including antidepressants, antipsychotic medications, benzodiazepines, calcium channel blockers, beta-blockers, anticoagulants, interleukin-2, non-steroidal anti-inflammatory drugs) were also excluded from the study.

All subjects had given written informed consent to participate and the aim of the study and possible risks were fully explained. This study was acknowledged by the Ethical Committee of Erzurum Education and Research Hospital.

2.2

Saliva collection

The sampling process was started at 12:00 in all cases and samples were taken at 4 hourly intervals. Saliva samples were taken under indoor light conditions. The intensity of light was limited to 300 lx in full light, but at 00:00 and 04:00 only flashlight (about 50 lx) was used to light up the mouth. Approximately 10–20 ml of saliva was taken from each subject. Saliva was collected prior to meals using a sugarless gum to stimulate saliva flow if necessary. The samples were inserted into 50 ml tubes and placed in the refrigerator at ± 4 °C for 24 h. The samples were then centrifuged for 10 min at 2000×g to remove mucins from the saliva and all samples were kept at − 40 °C until appreciation.

2.3

Saliva Assay

Melatonin in the saliva was evaluated by a radioimmunoassay (RIA) using kits obtained from BÜHLMANN Laboratories AG (Baselstr. 55 CH-4124, Schönenbuch, Switzerland) (RK-DSM2). The standard range of melatonin in this kit was 0.5–50 pg/ml. Day time (baseline) level, night time (peak) level, acrophase (clock time at which the melatonin reaches peak level), amplitude (difference between peak and baseline level) and peak duration (time interval during the periodic curve deviates from the baseline level) were used as parameters to assess the melatonin rhythm.

Salivary cortisol was evaluated by ELISA. The kit is manufactured by Eagle Biosciences, Inc. (82 Broad Street, Suite 383, Boston, USA) (COR32-K01). The standard range of the cortisol kit was 0.1–30 ng/ml. The 24-h mean level, amplitude (distance from mean to peak levels) and acrophase (clock time at which the cortisol levels reaches highest level) were used as parameters to assess the cortisol rhythm.

2.4

Statistics

Statistical analyses were performed by using the SPSS® software package, version 17.0 (SPSS Inc., Chicago, IL, USA) for Windows®. Categorical variables are presented as percentages and continuous variables are presented as mean ± SD. Data continuous variables were analyzed statistically using nonparametric tests, using the Friedman two-way ANOVA to establish whether melatonin levels differed in the samples taken at the various times to evaluate both the ENP patient and control groups. After confirmation, the Wilcoxon matched-pairs signed-rank test was used to determine the differences between sample times, and a repeated-measures ANOVA with between subject factors was used to compare the cases with the control group. Finally, we used the Mann–Whitney test to compare peak values between the patient and control groups. A value of p < 0.05 was considered to be statistically significant.

2.1

Study Population

Thirty-one patients (age range 25–41 years (mean ± SD age, 33.7 ± 7.1)) who presented with ENP at the Otolaryngology Clinic of Erzurum Education and Research Hospital between February 2008 and January 2010 were included in this study. Twenty-seven age and gender matched control subjects (age range 24–39 years (mean ± SD age, 32.9 ± 6.8)) were picked from among healthy patients attending the same hospital during the same study period.

At study entry, routine blood and urine analyses, cardiopulmonary graphs, and chest X-rays were performed in all subjects. The patients and controls both underwent endoscopic nasal examination, and only the patients received paranasal sinus computed tomography for the diagnosis of nasal polyposis. The Epworth Sleepiness Scale which is a simple and validated questionnaire for assessing the sleep disorders was used to appreciate the obstructive sleep apnea (OSA) in all patients and controls. In both groups none of the subjects did not have OSA.

Study patients and control subjects with any clinical or laboratory evidence of inflammation, OSA or infection, those who had received hormone therapy and/or steroid therapy in the one month prior to the study, or those who were taking any drugs that might affect melatonin and cortisol levels (including antidepressants, antipsychotic medications, benzodiazepines, calcium channel blockers, beta-blockers, anticoagulants, interleukin-2, non-steroidal anti-inflammatory drugs) were also excluded from the study.

All subjects had given written informed consent to participate and the aim of the study and possible risks were fully explained. This study was acknowledged by the Ethical Committee of Erzurum Education and Research Hospital.

2.2

Saliva collection

The sampling process was started at 12:00 in all cases and samples were taken at 4 hourly intervals. Saliva samples were taken under indoor light conditions. The intensity of light was limited to 300 lx in full light, but at 00:00 and 04:00 only flashlight (about 50 lx) was used to light up the mouth. Approximately 10–20 ml of saliva was taken from each subject. Saliva was collected prior to meals using a sugarless gum to stimulate saliva flow if necessary. The samples were inserted into 50 ml tubes and placed in the refrigerator at ± 4 °C for 24 h. The samples were then centrifuged for 10 min at 2000×g to remove mucins from the saliva and all samples were kept at − 40 °C until appreciation.

2.3

Saliva Assay

Melatonin in the saliva was evaluated by a radioimmunoassay (RIA) using kits obtained from BÜHLMANN Laboratories AG (Baselstr. 55 CH-4124, Schönenbuch, Switzerland) (RK-DSM2). The standard range of melatonin in this kit was 0.5–50 pg/ml. Day time (baseline) level, night time (peak) level, acrophase (clock time at which the melatonin reaches peak level), amplitude (difference between peak and baseline level) and peak duration (time interval during the periodic curve deviates from the baseline level) were used as parameters to assess the melatonin rhythm.

Salivary cortisol was evaluated by ELISA. The kit is manufactured by Eagle Biosciences, Inc. (82 Broad Street, Suite 383, Boston, USA) (COR32-K01). The standard range of the cortisol kit was 0.1–30 ng/ml. The 24-h mean level, amplitude (distance from mean to peak levels) and acrophase (clock time at which the cortisol levels reaches highest level) were used as parameters to assess the cortisol rhythm.

2.4

Statistics

Statistical analyses were performed by using the SPSS® software package, version 17.0 (SPSS Inc., Chicago, IL, USA) for Windows®. Categorical variables are presented as percentages and continuous variables are presented as mean ± SD. Data continuous variables were analyzed statistically using nonparametric tests, using the Friedman two-way ANOVA to establish whether melatonin levels differed in the samples taken at the various times to evaluate both the ENP patient and control groups. After confirmation, the Wilcoxon matched-pairs signed-rank test was used to determine the differences between sample times, and a repeated-measures ANOVA with between subject factors was used to compare the cases with the control group. Finally, we used the Mann–Whitney test to compare peak values between the patient and control groups. A value of p < 0.05 was considered to be statistically significant.