Association of salivary biochemical parameters with etiopathogenetic variants of laryngopharyngeal reflux in young adult men: a single-centre cross-sectional observational study
DOI:
https://doi.org/10.14739/2409-2932.2025.3.341786Keywords:
laryngopharyngeal reflux, salivary pH, nitrites, nitric oxide synthase, thiol groups, protein carbonyls, cardiac rate, biochemical parameters, oxidative stress, nitrosative stress, lipid peroxidationAbstract
Aim. To verify salivary biochemical parameters (pH, NO2–/NOS, –SH groups, protein carbonyls – aldehyde and ketone phenylhydrazones (APH, KPH)) and heart rate variability (HRV) indices (SDNN, SI, LF/HF, TP, PARS) according to etiopathogenetic LPR phenotypes in young adult men.
Materials and methods. A single-center, cross-sectional, comparative observational study was conducted focusing on salivary biochemical parameters (pH, NOx/NOS, –SH groups, protein carbonyls) and HRV indices as tools for phenotype-oriented stratification. Two groups of young men were formed: the main LPR group (MG; n = 91) and a conditionally healthy control group without LPR signs (CG; n = 64). Examinations were performed between July 2024 and June 2025.
Results. In the MG, sympathetic predominance (28.6 %) and vagotonia / parasympathetic predominance (24.2 %) prevailed, whereas 13.2 % exhibited a breakdown of autonomic regulation – an indicator of depleted reserves – unlike the CG. No statistically significant differences between autonomic subtypes (by HRV) were found within the CG, whereas the MG showed significant inter-subgroup differences in salivary biochemical parameters; subsequent analyses were performed within subgroups (SG1–SG5).
Compared with controls, LPR patients demonstrated significantly higher salivary pH, –SH groups, NOS activity, NO2–, total protein, and protein carbonyls. Salivary pH varied by autonomic regulation type: SG1 remained close to control; SG2 showed acidification; SG3–SG4 exhibited an alkaline shift, maximal in SG4. The greatest increases in NOS activity and NO2– were observed in SG3–SG4, indicating hyperactivation of NO-dependent processes. These subgroups also showed maximal –SH groups and protein carbonyls, reflecting enhancement of thiol-dependent antioxidant defense and oxidative protein modification. SG2 displayed signs of adaptive strain – low pH with moderate increases in NOS and APH/KPH. SG5 tended toward reduced NOx and –SH, interpreted as depletion of antioxidant potential and transition of nitrosative stress to a destructive phase. The observed changes support saliva’s role as an indicator of oxidative – antioxidant balance and the barrier function of the upper airways.
Conclusions. Patients with LPR exhibit elevated salivary pH, NOS activity, NO2–, –SH, APH/KPH, and total protein, indicating nitrosative stress with strained antioxidant mechanisms. The magnitude of changes depends substantially on the autonomic regulation type: the most pronounced shifts occur in parasympathetic and humoral-metabolic phenotypes, which show maximal increases in NOS activity, NO2–, protein carbonyls, and salivary pH, whereas regulation breakdown features decreases in NO-related and antioxidant markers, reflecting enzymatic exhaustion and a transition from compensatory to destructive nitrosative stress. LPR patients thus display distinct biochemical phenotypes determined by autonomic profile, which should be considered for proper interpretation of results and for forecasting the adaptive reserves of the antioxidant system.
References
Lechien JR, Vaezi MF, Chan WW, Allen JE, Karkos PD, Saussez S, et al. The Dubai Definition and Diagnostic Criteria of Laryngopharyngeal Reflux: The IFOS Consensus. Laryngoscope. 2024;134(4):1614-24. doi: https://doi.org/10.1002/lary.31134
Brar S, Watters C, Watson N, Birchall M, Karagama Y. Ear, nose and throat (ENT) manifestations and complications of reflux. Frontline Gastroenterol. 2022;13(e1):e57-e64. doi: https://doi.org/10.1136/flgastro-2022-102184
Tan JJ, Dai YF, Wang F, Lv ZH, Huang LJ, Peng LY, et al. Pepsin-mediated inflammation in laryngopharyngeal reflux via the ROS/NLRP3/IL-1β signaling pathway. Cytokine. 2024;178:156568. doi: https://doi.org/10.1016/j.cyto.2024.156568
Li Y, Xu G, Zhou B, Tang Y, Liu X, Wu Y, et al. Effects of acids, pepsin, bile acids, and trypsin on laryngopharyngeal reflux diseases: physiopathology and therapeutic targets. Eur Arch Otorhinolaryngol. 2022;279(6):2743-52. doi: https://doi.org/10.1007/s00405-021-07201-w
Lippincott M, Velanovich V. The upper esophageal sphincter in gastroesophageal reflux disease. Ann Esophagus. 2022;5:26. doi: https://doi.org/10.21037/aoe-21-3
Yadlapati R, Weissbrod P, Walsh E, Carroll TL, Chan WW, Gartner-Schmidt J, et al. The San Diego Consensus for Laryngopharyngeal Symptoms and Laryngopharyngeal Reflux Disease. Am J Gastroenterol. 2025 Apr 8:10.14309/ajg.0000000000003482. doi: https://doi.org/10.14309/ajg.0000000000003482
Lechien JR, Chiesa-Estomba CM, Hans S, Nacci A, Schindler A, Bohlender JE, et al. European clinical practice guideline: managing and treating laryngopharyngeal reflux disease. Eur Arch Otorhinolaryngol. 2024 Dec 24. doi: https://doi.org/10.1007/s00405-024-09181-z. Epub ahead of print. Erratum in: Eur Arch Otorhinolaryngol. 2025 Mar 27. doi: https://doi.org/10.1007/s00405-025-09307-x
Kryshtal VM, Melnikova OV, Khorolets OV, Hancheva OV. [Laryngopharyngeal reflux: current perspectives and controversies]. Modern Medical Technology. 2025;17(3):205-16. Ukrainian. doi: https://doi.org/10.14739/mmt.2025.3.335612
Lechien JR, Bobin F, Rodriguez A, Dequanter D, Muls V, Huet K, et al. Development and validation of the short version of the Reflux Symptom Score: Reflux Symptom Score-12. Otolaryngol Head Neck Surg. 2021;164(1):166-74. doi: https://doi.org/10.1177/0194599820941003
Lechien JR, Rodriguez Ruiz A, Dequanter D, Bobin F, Mouawad F, Muls V, et al. Validity and Reliability of the Reflux Sign Assessment. Ann Otol Rhinol Laryngol. 2020;129(4):313-25. doi: https://doi.org/10.1177/0003489419888947
Quigley KS, Gianaros PJ, Norman GJ, Jennings JR, Berntson GG, de Geus EJ. Publication guidelines for human heart rate and heart rate variability studies in psychophysiology-Part 1: Physiological underpinnings and foundations of measurement. Psychophysiology. 2024;61(9):e14604. doi: https://doi.org/10.1111/psyp.14604
Huizinga JD, Chen JH, Hussain A, Zheng D, Liu L, Lui H, et al. Determining autonomic sympathetic tone and reactivity using Baevsky's stress index. Am J Physiol Regul Integr Comp Physiol. 2025;328(5):R562-77. doi: https://doi.org/10.1152/ajpregu.00243.2024
Pang B, Qi X, Zhang H. Salivary-Gland-Mediated Nitrate Recirculation as a Modulator for Cardiovascular Diseases. Biomolecules. 2025;15(3):439. doi: https://doi.org/10.3390/biom15030439
Maciejczyk M, Bielas M, Zalewska A, Gerreth K. Salivary Biomarkers of Oxidative Stress and Inflammation in Stroke Patients: From Basic Research to Clinical Practice. Oxid Med Cell Longev. 2021;2021:5545330. doi: https://doi.org/10.1155/2021/5545330
Bechir F, Pacurar M, Tohati A, Bataga SM. Comparative Study of Salivary pH, Buffer Capacity, and Flow in Patients with and without Gastroesophageal Reflux Disease. Int J Environ Res Public Health. 2021;19(1):201. doi: https://doi.org/10.3390/ijerph19010201
Hatipoğlu Ö, Yıldırım Ö, Hatipoğlu FP. Impact of Gastroesophageal Reflux Disease on Salivary Flow Rate, pH and Buffer Capacity: A Systematic Review and Meta-Analysis. J Oral Rehabil. 2025;52(10):1682-98. doi: https://doi.org/10.1111/joor.14025
Cui N, Dai T, Liu Y, Wang YY, Lin JY, Zheng QF, et al. Laryngopharyngeal reflux disease: Updated examination of mechanisms, pathophysiology, treatment, and association with gastroesophageal reflux disease. World J Gastroenterol. 2024;30(16):2209-19. doi: https://doi.org/10.3748/wjg.v30.i16.2209
Lechien JR, De Marrez LG, Hans S, Muls V, Spinato L, Briganti G, et al. Digestive Biomarkers of Laryngopharyngeal Reflux: A Preliminary Prospective Controlled Study. Otolaryngol Head Neck Surg. 2024;170(5):1364-71. doi: https://doi.org/10.1002/ohn.674
Jeon SY, Rim HS, Lee Y, Park JM, Lee MK, Il Kim S, et al. The Association Between Salivary Function and Laryngopharyngeal Reflux Measured Objectively. J Voice. 2025:S0892-1997(25)00368-6. doi: https://doi.org/10.1016/j.jvoice.2025.09.003
Schwerdt G, Schulz MC, Kopf M, Mildenberger S, Reime S, Gekle M. Physiological regulation of oral saliva ion composition and flow rate are not coupled in healthy humans-Partial revision of our current knowledge required. Pflugers Arch. 2025;477(1):55-65. doi: https://doi.org/10.1007/s00424-024-03025-9
Mortazavi H, Yousefi-Koma AA, Yousefi-Koma H. Extensive comparison of salivary collection, transportation, preparation, and storage methods: a systematic review. BMC Oral Health. 2024;24(1):168. doi: https://doi.org/10.1186/s12903-024-03902-w
Basilicata M, Pieri M, Marrone G, Nicolai E, Di Lauro M, Paolino V, et al. Saliva as Biomarker for Oral and Chronic Degenerative Non-Communicable Diseases. Metabolites. 2023;13(8):889. doi: https://doi.org/10.3390/metabo13080889
Lechien JR, El Ayoubi M, Muls V, Hans S, Saussez S, De Vos N. Accuracy and Clinical Findings of Saliva Digestive Biomarkers in Laryngopharyngeal Reflux Disease. Laryngoscope. 2025 Aug 20. doi: https://doi.org/10.1002/lary.70062
Ghasemi A. Quantitative aspects of nitric oxide production from nitrate and nitrite. EXCLI J. 2022;21:470-86. doi: https://doi.org/10.17179/excli2022-4727
Akimov OY, Kostenko VO. Functioning of nitric oxide cycle in gastric mucosa of rats under excessive combined intake of sodium nitrate and fluoride. Ukr Biochem J. 2016;88(6):70-5. doi: https://doi.org/10.15407/ubj88.06.070
Sasaki CT, Doukas SG, Doukas PG, Vageli DP. Weakly Acidic Bile Is a Risk Factor for Hypopharyngeal Carcinogenesis Evidenced by DNA Damage, Antiapoptotic Function, and Premalignant Dysplastic Lesions In Vivo. Cancers (Basel). 2021;13(4):852. doi: https://doi.org/10.3390/cancers13040852
Lechien JR, De Vos N, Saussez S. Predictive Value of Digestive Enzymes in Patients With Reflux-Induced Chronic Cough. Otolaryngol Head Neck Surg. 2025;173(2):453-60. doi: https://doi.org/10.1002/ohn.1283
Vašková J, Kočan L, Vaško L, Perjési P. Glutathione-Related Enzymes and Proteins: A Review. Molecules. 2023;28(3):1447. doi: https://doi.org/10.3390/molecules28031447
Del Valle-Mondragón L, Becerra-Luna B, Cartas-Rosado R, Infante O, Pérez-Grovas H, Lima-Zapata LI, et al. Correlation between Angiotensin Serum Levels and Very-Low-Frequency Spectral Power of Heart Rate Variability during Hemodialysis. Life (Basel). 2022;12(7):1020. doi: https://doi.org/10.3390/life12071020
Lee CH, Shin HW, Shin DG. Impact of Oxidative Stress on Long-Term Heart Rate Variability: Linear Versus Non-Linear Heart Rate Dynamics. Heart Lung Circ. 2020;29(8):1164-73. doi: https://doi.org/10.1016/j.hlc.2019.06.726
Alen NV. The cholinergic anti-inflammatory pathway in humans: State-of-the-art review and future directions. Neurosci Biobehav Rev. 2022;136:104622. doi: https://doi.org/10.1016/j.neubiorev.2022.104622
Koopman FA, Chavan SS, Miljko S, Grazio S, Sokolovic S, Schuurman PR, et al. Vagus nerve stimulation inhibits cytokine production and attenuates disease severity in rheumatoid arthritis. Proc Natl Acad Sci U S A. 2016;113(29):8284-9. doi: https://doi.org/10.1073/pnas.1605635113
Subramanian M, Edwards L, Melton A, Branen L, Herron A, Sivasubramanian MK, et al. Non-invasive vagus nerve stimulation attenuates proinflammatory cytokines and augments antioxidant levels in the brainstem and forebrain regions of Dahl salt sensitive rats. Sci Rep. 2020;10(1):17576. doi: https://doi.org/10.1038/s41598-020-74257-9
Jurado-Castro JM, Casanova-Rodriguez D, Campos-Perez J, Llorente-Cantarero FJ, De La Florida-Villagran CA, Diaz-Bernier VM, et al. Beetroot Juice Produces Changes in Heart Rate Variability and Reduces Internal Load during Resistance Training in Men: A Randomized Double-Blind Crossover. Nutrients. 2022;14(23):5119. doi: https://doi.org/10.3390/nu14235119
Prolo C, Piacenza L, Radi R. Peroxynitrite: a multifaceted oxidizing and nitrating metabolite. Curr Opin Chem Biol. 2024;80:102459. doi: https://doi.org/10.1016/j.cbpa.2024.102459
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