Surface Active Salivary Metabolites Indicate Oxidative Stress and Inflammation in Obstructive Sleep Apnea

Abstract

Purpose: Obstructive sleep apnea (OSA), a highly prevalent and potentially serious sleep disorder, requires effective screening tools. Saliva is a useful biological fluid with various metabolites that might also influence upper airway patency by affecting surface tension in the upper airway. However, little is known about the composition and role of salivary metabolites in OSA. Therefore, we investigated the metabolomics signature in saliva from the OSA patients and evaluated the associations between identified metabolites and salivary surface tension.

Methods: We studied 68 subjects who visited sleep clinic due to the symptoms of OSA. All underwent full-night in-lab polysomnography. Patients with apnea-hypopnea index (AHI) < 10 were classified to the control, and those with AHI ≥ 10 were the OSA groups. Saliva samples were collected before and after sleep. The centrifuged saliva samples were analyzed by liquid chromatography with high-resolution mass spectrometry (ultra-performance liquid chromatography-tandem mass spectrometry; UPLC-MS/MS). Differentially expressed salivary metabolites were identified using open source software (XCMS) and Compound Discoverer 2.1. Metabolite set enrichment analysis (MSEA) was performed using MetaboAnalyst 5.0. The surface tension of the saliva samples was determined by the pendant drop method.

Results: Three human-derived metabolites (1-palmitoyl-2-[5-hydroxyl-8-oxo-6-octenoyl]-sn-glycerol-3-phosphatidylcholine [PHOOA-PC], 1-palmitoyl-2-[5-keto-8-oxo-6-octenoyl]-sn-glycerol-3-phosphatidylcholine [KPOO-PC], and 9-nitrooleate) were significantly upregulated in the after-sleep salivary samples from the OSA patients compared to the control group samples. Among the candidate metabolites, only PHOOA-PC was correlated with the AHI. In OSA samples, salivary surface tension decreased after sleep. The differences in surface tension were negatively correlated with PHOOA-PC and 9-nitrooleate concentrations. Furthermore, MSEA revealed that arachidonic acid-related metabolism pathways were upregulated in the after-sleep samples from the OSA group.

Conclusions: This study revealed that salivary PHOOA-PC was correlated positively with the AHI and negatively with salivary surface tension in the OSA group. Salivary metabolomic analysis may improve our understanding of upper airway dynamics and provide new insights into novel biomarkers and therapeutic targets in OSA.

Keywords: Sleep apnea, obstructive; airway resistance; biomarkers; metabolomics; phosphatidylcholines; saliva; sleep; surface tension.

Fig. 1. Flow chart of subject selection and identification of metabolites. (A) Flow chart of subject selection and exclusion. (B) Workflow of untargeted metabolomic analysis. (C) Study flow of metabolite set enrichment analysis.

AHI, apnea-hypopnea index; PHOOA-PC, 1-palmitoyl-2-(5-hydroxyl-8-oxo-6-octenoyl)-sn-glycerol-3-phosphatidylcholine; KPOO-PC, 1-palmitoyl-2-(5-keto-8-oxo-6-octenoyl)-sn-glycerol-3-phosphatidylcholine; OSA, obstructive sleep apnea; MSEA, metabolite set enrichment analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Fig. 2. Intensity comparison of identified metabolites in the OSA group.

Comparison of (A) PHOOA-PC, (B) KPOO-PC, and (C) 9-nitrooleate intensities between control and OSA in both the before-sleep and after-sleep samples. (D) PHOOA-PC, (E) KPOO-PC, and (F) 9-nitrooleate intensities according to the severity of OSA in both the before-sleep and after-sleep samples. The control group (n = 21) was defined as AHI < 10; the OSA group (n = 47) was 10 ≤ AHI in before-sleep and after-sleep groups (A-C). The OSA group was divided according to OSA severity: control (AHI < 10, n = 21); mild to moderate OSA (10 ≤ AHI < 30, n = 21); and severe OSA (30 ≤ AHI, n = 26) in before-sleep and after-sleep groups (D-F). Data are presented as mean ± standard deviation with P values. OSA, obstructive sleep apnea; PHOOA-PC, 1-palmitoyl-2-(5-hydroxyl-8-oxo-6-octenoyl)-sn-glycerol-3-phosphatidylcholine; KPOO-PC, 1-palmitoyl-2-(5-keto-8-oxo-6-octenoyl)-sn-glycerol-3-phosphatidylcholine; AHI, apnea-hypopnea index. The 2-sided unpaired t-test was used for comparative analyses within the before- and after-sleep sample groups, respectively (statistical significance is denoted with asterisks, ^*P < 0.05; ^**P < 0.01; ^***P < 0.001). The paired t-test was used for comparative analyses between the control and patient groups (statistical significance is denoted with daggers, ^†P < 0.05, ^††P < 0.01; ^†††P < 0.001).

Fig. 3. Analysis of enriched metabolic pathways in OSA group.

Significantly upregulated pathways were analyzed using MetaboAnalyst 5.0. Pathway analysis was performed between the before-sleep samples from the OSA and the control groups using (A) the mummichog algorithm, or (B) the GSEA algorithm. After-sleep samples from OSA were compared to control subjects using (C) the mummichog algorithm (figure shows the top 5 pathways), or (D) the GSEA algorithm. Upregulated metabolites were mapped on the KEGG global network map. Solid circles denote significant metabolites in (E) before-sleep samples, and (F) after-sleep samples from OSA group compared to the control group. Combined P values (combination of mummichog and GSEA P values) were calculated by the Fisher combined probability test. OSA, obstructive sleep apnea; GSEA, gene set enrichment analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes; EPA, eicosapentaenoic acid; NES, normalized enrichment score; Pcomb, combined P value.

Fig. 4. Analysis of enriched metabolic pathways in the after-sleep samples from the control and OSA groups.

Pathway analysis was performed between paired samples. In the control group, altered pathways in the after-sleep samples compared to the before-sleep samples were analyzed using (A) the mummichog, or (B) the GSEA algorithm. In the OSA group, altered pathways in the after-sleep samples compared to the before-sleep samples were analyzed using (C) the mummichog, or (D) the GSEA algorithm. Upregulated metabolites were mapped on the KEGG global network map. Solid circles denote significant metabolites in the after-sleep samples from (E). the control group, or (F) OSA group. Combined P values (combination of mummichog and GSEA P values) were calculated by the Fisher combined probability test. OSA, obstructive sleep apnea; GSEA, gene set enrichment analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes; EPA, eicosapentaenoic acid; NES, normalized enrichment score; Pcomb, combined P value.

Fig. 5. Decreased surface tension of saliva in the after-sleep samples of the OSA group.

(A) Geometry of the pendant drop for the calculation of surface tension. (B) Validation check of the pendant drop model theory with deionized water (up to 18.3 MΩ). (C) Measured surface tension of patients’ saliva before and after sleep. (D) Comparison of the surface tension of saliva samples between the control and OSA groups in both the before-sleep and after-sleep samples. The control group (n = 14) was defined as AHI < 10; the OSA group (n = 36) was 10 ≤ AHI in before-sleep and after-sleep groups. OSA, obstructive sleep apnea; AHI, apnea-hypopnea index; DI, deionized; Ctrl, control group; n.s., not significant. ^†Denotes P < 0.05 between the indicated groups by the paired t-test.

Fig. 6. Altered metabolic patterns of before- and after-sleep saliva samples in the OSA group.

Pathway analysis was performed between before-sleep samples from the OSA and control groups using (A) the mummichog algorithm, or (B) the GSEA algorithm. After-sleep samples from the OSA group was compared to the control subjects using (C) the mummichog, or (D) the GSEA algorithm. Upregulated metabolites were mapped on the KEGG global network map. Solid circles denote significant metabolites in (E) the before-sleep samples, and (F) the after-sleep samples from the OSA group compared to the control group. Combined P values (combination of mummichog and GSEA P values) were calculated by the Fisher combined probability test. Highly correlated (r > 0.7 for positive correlations; r < −0.7 for negative correlations, with P values < 0.05) metabolites were used in MSEA. OSA, obstructive sleep apnea; GSEA, gene set enrichment analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes; NES, normalized enrichment score; Pcomb, combined P value.