“The Effect of Obstructive Sleep Apnea on Peripheral Blood Amino Acid and Biogenic Amine Metabolome at Multiple Time Points Overnight“ Ott Kiens ( [email protected] ) Department of Pulmonary Medicine, University of Tartu, Estonia Egon Taalberg University of Tartu Viktoria Ivanova Tartu University Hospital Ketlin Veeväli Tartu University Hospital Triin Laurits Tartu University Hospital Ragne Tamm Tartu University Hospital Aigar Ottas University of Tartu Kalle Kilk University of Tartu Ursel Soomets University of Tartu Alan Altraja University of Tartu Research Article Keywords: blood serum metabolome, apnea-hypopnea index (AHI), OSA patients, Phenylalanine and proline levels Posted Date: December 9th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-119577/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Title: „The effect of obstructive sleep apnea on peripheral blood amino acid and biogenic amine metabolome at multiple time points overnight“ Authors: Ott Kiens 1,2, Egon Taalberg 3,4, Viktoria Ivanova 2, Ketlin Veeväli 5, Triin Laurits 5, Ragne Tamm 5, Aigar Ottas3,4, Kalle Kilk 3,4, Ursel Soomets 3,4, Alan Altraja 1,2 Affiliations: 1- Department of Pulmonary Medicine, University of Tartu, Estonia 2- Lung Clinic, Tartu University Hospital, Estonia 3- Institute of Biomedicine and Translational Medicine, University of Tartu, Estonia 4- Centre of Excellence for Genomics and Translational Medicine, University of Tartu, Estonia 5- Psychiatry Clinic, Tartu University Hospital, Estonia Corresponding Author: Ott Kiens, Tartu University Lung Clinic, Riia 167, 50411, Tartu, Estonia, [email protected] 1 Abstract There are no clinical studies that have investigated the differences in blood serum metabolome between obstructive sleep apnea (OSA) patients and controls. In a single-center prospective observational study, we compared metabolomic profiles in the peripheral blood of OSA patients with apnea-hypopnea index (AHI) > 15/h and control individuals. Blood was obtained at 3 different time points overnight: 21:00 p.m.; 5:00 a.m. and 7:00 a.m. We used a targeted approach for detecting amino acids and biogenic amines and analyzed the data with ranked general linear model for repeated measures. We recruited 31 patients with moderate- to-severe OSA and 32 controls. Significant elevations in median concentrations of alanine, proline and kynurenine in OSA patients compared to controls were detected. Significant changes in the overnight dynamics of peripheral blood concentrations occurred in OSA: glutamine, serine, threonine, tryptophan, kynurenine and glycine levels increased, whereas a fall occurred in the same biomarker levels in controls. Phenylalanine and proline levels decreased slightly, compared to a steeper fall in controls. The study indicates that serum profiles of amino acid and biogenic amines are significantly altered in patients with OSA referring to vast pathophysiologic shifts reflected in the systemic metabolism. 2 Introduction Obstructive sleep apnea (OSA) is characterized by a history of habitual snoring and the occurrence of obstructed breathing events, hypopneas, and apneas, during sleep. OSA is a highly prevalent sleep disorder, with an estimated prevalence of 10-49.7% among adult men and 3-23.4% among adult women, depending on the study design 1,2. Untreated OSA is associated with an increased risk of motor vehicle accidents, cardiovascular disease 3, atrial fibrillation 4, and exacerbations of chronic airway diseases 5,6. Changes in the metabolome of OSA patients have thus far been studied in search for a biomarker or a set of biomarkers 7. In most studies designed for finding a biomarker in OSA, the samples have been obtained in the morning after sleep, whereas studies, addressing the differences between the evening and morning samples or the samples taken during sleep, are scarce 8,9 Untargeted analysis of the metabolome in morning urine has been used on patients with OSA, which only partly reflects night-time metabolomic profile 7. It is known that the results for untargeted metabolomic analyses are not always interchangeable between laboratories 10 , whereas targeted analysis, on the contrary, has shown better interlaboratory reproducibility 11. The metabolic pathways involved in OSA have not been thoroughly investigated yet. There have been no metabolomic studies that have investigated the metabolomic changes among patients with OSA during night-time or, more importantly, the dynamic changes taking place from the time of going to bed until waking up. We hypothesized that assessing the metabolomic profile at multiple time points in the peripheral blood of OSA patients using a targeted approach could lead to better characterization of obstructive sleep apnea and its systemic effects in particular. This knowledge could improve the treatment and monitoring of OSA patients and lead to a more precise division into subgroups in the future. Together with precise clinical characterization and longitudinal follow-up this approach could lead to better risk assessment among OSA patients. The aim of our study was to assess the amino acid and biogenic amine profile in peripheral blood of OSA patients during multiple time 3 points (evening, night-time, morning) in order to characterize the metabolomic differences between OSA patients and controls. Results A total of 63 individuals were recruited, 31 with moderate-to-severe OSA and 32 controls (Table 1). The patients with OSA had significantly higher plasma concentrations of alanine aminotransferase (ALAT) (p= 0.006) and triglycerides (TG) (p = 0.02) (Table 1). The total sleep time and the time spent in different sleep phases in OSA patients did not differ statistically from that in the control individuals. There was a significant difference in leg movements per hour (p = 0.042) (Table 1). Table 1. OSA patients (n=31) Controls (n=32) *p-value Male gender, n (%) 19 (61.3) 11 (34.4) 0.06 Age, years 57 (48-60) 48 (40.8-56.3) 0.03 BMI (kg/m2) 31.9 (27.7-35.5) 27.7 (23.9-29.4) 0.002 Neck circumference, cm 42.5 (39.8-44.3) 39.0 (36.0-41.0) <0.001 Active smokers, n (%) 10 (32.3) 8 (25) 0.71 STOP BANG score 5.0 (5.0-7.0) 3.5 (2.0-5.0) <0.001 ESS score 10.0 (5.0-12.0) 8.5 (5.75-11.25) 0.73 FBG (mmol/L) 6.0 (5.5-6.6) 5.5 (5.2-6.1) 0.003 ALAT (U/L) 23.0 (21.0-33.0) 20.0 (14.0-26.25) 0.006 ASAT (U/L) 22.0 (20.0-28.5) 22.0 (17.0-24.0) 0.12 Creatinine (μmol/L) 79.0 (70.5-87.5) 74.0 (62.8-79.0) 0.052 Urea (mmol/L) 5.0 (4.0-5.8) 4.9 (4.0-5.4) 0.62 Triglycerides (mmol/L) 1.6 (1.1-2.4) 1.2 (0.8-1.7) 0.02 AHI (/h) 31.1 (20.5-41.4) 6.2 (2.0-11.2) <0.001 ODI < 90% (/h) 8.7 (2.3-18.3) 0.6 (0-2.8) <0.001 ODI ≥ 5% (/h) 3.4 (1.7-12.5) 0.3 (0.1-1.0) <0.001 Sleep-time leg movements (/h) 16.5 (6.9-35.8) 8.25 (5.5-14.4) 0.042 Arousal index (EEG 16.9 (7.8-26.7) 10.9 (6.4-18.3) 0.055 arousals per hour of sleep) 4 The TG values correlated significantly with that of body mass index (BMI) (ρ= 0.288,p = 0.022) and ALAT (ρ = 0.288,p = 0.006). There were no significant differences in high-sensitivity C-reactive protein (hsCRP), haptoglobin or ceruloplasmin concentrations between OSA patients and controls. Significant elevations in the peripheral blood median concentrations of alanine (Ala) (p = 0.016, Fig. 1), proline (Pro) (p = 0.005, Fig. 2) and kynurenine (Kyn) (p = 0.017, Fig. 3) in OSA patients compared to controls were detected (Table 2). Figure 1. 5 Figure 2. Figure 3. 6 Table 2. 21:00 p.m. 5:00 a.m. 7:00 a.m. *p-value Alanine: Controls 331.0 (305.8-415.2) 304.0 (238.8-336.8) 322.0 (247.5-364.8) 0.016 OSA 370.0 (330.0-430.0) 389.0 (290.5-455.0) 380.0 (318.5-449.5) Glutamine: Controls 738.0 (657.8-801.5) 707.0 (670.0-766.8) 699.0 (639.2-765.8) 0.70 OSA 658.0 (613.5-716.0) 685.0 (630.0-800.5) 696.0 (648.5-773.5) Glycine: Controls 232.0 (204.8-263.2) 229.5 (204.0-272.0) 232.0 (207.0-273.0) 0.80 OSA 200.0 (170.5-224.5) 221.0 (194.0-248.5) 233.0 (201.0-269.5) Phenylalanine: Controls 71.3 (62.9-78.7) 59.9 (54.1-65.5) 59.4 (52.6-64.5) 0.74 OSA 69.2 (57.4-76.2) 69.5 (61.3-76.7) 66.2 (60.4-70.8) Proline: Controls 206.0 (184.2-253.2) 170.5 (146.8-201.5) 166.5 (142.5-186.8) 0.005 OSA 235.0 (208.0-268.0) 209.0 (188.0-248.5) 200.0 (168.0-238.5) Serine: Controls 121.0 (107.8-130.0) 115.0 (100.7-127.2) 109.5 (94.6-136.0) 0.99 OSA 102.0 (85.0-123.5) 111.0 (96.1-125.0) 111.0 (101.5-123.5) Threonine: Controls 127.0 (116.5-143.8) 109.5 (96.0-120.3) 110.5 (99.5-133.0) 0.67 OSA 113.0 (97.5-124.0) 120.0 (92.8-135.0) 118.0 (93.0-132.5) Tryptophan: Controls 65.8 (53.9-74.2) 50.3 (44.8-62.2) 55.5 (47.6-59.2) 0.87 OSA 59.6 (51.2-65.3) 62.1 (50.3-68.6) 59.7 (51.0-65.8) Kynurenine: Controls 1.97 (1.72-2.38) 1.78 (1.56-2.35) 1.90 (1.56-2.40) 0.017 OSA 2.47 (1.92-2.81) 2.40 (2.15-2.98) 2.57 (2.19-3.13) Significant changes in the overnight dynamics of peripheral blood concentrations occurred in OSA: glutamine (p = 0.025), serine (p = 0.005), threonine (p = 0.001), tryptophan (Trp) (p <0.001), Kyn (p = 0.001) and glycine (p = 0.007) levels increased, whereas a fall occurred in the same biomarker levels in controls (Figure 4, a-f).