Obstructive sleep apnea (OSA) is a common sleep disorder characterized by repetitive partial or complete collapse of the upper airway during sleep, resulting in apnea or hypopnea (Kohler and Stradling, 2010). This leads to many consequences, the most important of which are intermittent hypoxia and arousals from sleep causing sleep fragmentation (Lv et al., 2023; Osman et al., 2018). OSA is commonly associated with a wide range of cardiovascular diseases (CVD), including coronary artery disease (CAD), hypertension, heart failure, arrhythmia, stroke, and pulmonary hypertension (Li and Ren, 2022). The main pathomechanisms that contribute to the elevated cardiovascular risk in sleep apnea syndrome include chronic activation of the sympathetic nervous system, oxidative stress, chronic inflammation and endothelial dysfunction (Unnikrishnan et al., 2015; Javaheri et al., 2017; Harańczyk et al., 2022; Peracaula et al., 2022). It has been demonstrated that repetitive hypoxia/reoxygenation cycles and sleep fragmentation impair endothelial function. In particular, in OSA, endothelial nitric oxide production and repair capacity are restricted, whereas oxidative stress and inflammation are intensified (Atkeson et al., 2009).

Sphingolipids are both structural components in the plasma membranes of eukaryotic cells and signaling molecules regulating a variety of biological functions (Sasset and Di Lorenzo, 2022). Proper sphingolipid metabolism is crucial for maintaining endothelial cell homeostasis (Lai et al., 2022). Of all sphingolipids, ceramide and sphingosine-1-phosphate (S1P) in particular are able to differentially regulate cellular functions by modulating opposing signaling pathways. In this context, the mutual, dynamic balance of these two interconnected lipid mediators has been termed the ceramide/S1P rheostat (Piccoli et al., 2023).

Ceramides are a family of bioactive sphingolipids acting as second messengers in cell signaling pathways. They can activate various kinases and transcription factors, leading to the regulation of cell growth, proliferation, differentiation, and apoptosis (Shen et al., 2025). Endothelium (Cantalupo et al., 2020), hepatocytes (Merrill et al., 1995) and adipose tissue (Akawi et al., 2021) are the sources of circulating ceramides. Elevated plasma concentrations of ceramides have been associated with multiple risk factors for coronary artery disease (Poss et al., 2020), obesity (Haus et al., 2009), diabetes mellitus (Fretts et al., 2020), hypertension (Spijkers et al., 2011), as well as cardiac remodeling and dysfunction (Ji et al., 2017). Moreover, circulating ceramides positively correlate with systemic insulin resistance and inflammation (Haus et al., 2009; de Mello VD et al., 2009). Interestingly, ceramide levels are notably higher within atherosclerotic plaques (Schissel et al., 1996), which may contribute to atherosclerosis by promoting the infiltration of low-density lipoproteins (LDLs) into the endothelium and their aggregation within the intima of artery walls (Meeusen et al., 2020).

S1P is a potent lipid mediator that regulates various physiological as well as pathological processes in the vasculature and immune system (Winkler et al., 2015). This important molecule is produced intracellularly from ceramide. Firstly, ceramide is converted to sphingosine by ceramidase, and then sphingosine is phosphorylated into S1P by sphingosine kinase (SphK) 1 or 2 (Kurano and Yatomi, 2018). After extracellular release, S1P exerts pleiotropic effects through binding to specific G protein-coupled receptors S1PR1-5 (Liu et al., 2012). These five receptors are expressed on various cell types from the immune, respiratory, cardiovascular, hepatic, and neurological system (Thuy et al., 2014). Through differential binding to its receptors, S1P regulates many physiological and pathological processes including, blood pressure (Intapad, 2019), vascular endothelial function (Weigel et al. 2023), atherosclerosis (Soltau et al., 2016), coagulation and inflammation (Obinata and Hla, 2012). S1P is detected at high concentrations in plasma where it is bound mainly to high-density-lipoprotein (HDL) via apolipoprotein M (ApoM), or to albumin (Christoffersen et al., 2011). The main sources of S1P in circulation are erythrocytes, platelets, and the endothelium (Kurano and Yatomi, 2018). With regards to endothelium dysfunction (a known complication in OSA), in vitro studies have shown that sphingosine-1-phosphate exerts a protective effect against endothelial cell damage induced by hypoxemia (Yu et al., 2018).

The roles of bioactive sphingolipids, including the most well studied ones - ceramide and S1P, remain poorly understood in the context of obstructive sleep apnea. Until now, only two reports have been published on this subject. In the first, Lebkuchen et al. (2018) in a comprehensive metabolomic analysis, described a modest increase in the concentration of certain ceramides (d18:1/24:4) in OSA male patients. In the second study, Horváth et al., 2023 reported a significantly increased concentration of anti-ceramide antibodies (ceramide-Ab) as well as S1P in OSA patients compared with healthy controls. Considering the abovementioned reports, we sought to investigate whether serum S1P and ceramide-Ab are altered in our well characterized cohort of OSA patients and whether they are associated with OSA severity, specific clinical parameters, and comorbidities such as coronary heart disease, diabetes mellitus and hypertension. Additionally, we intended to explore whether S1P and ceramide-Ab could serve as reliable biomarkers for the diagnosis of OSA and the assessment of disease severity.

The primary aim of this study was to evaluate the concentrations of two key sphingolipids, sphingosine-1-phosphate and ceramide in serum samples collected from OSA patients and healthy individuals. Instead, however, in order to detect ceramide we decided to measure the level of anti-ceramide antibodies. We believe that their levels simply correspond to the levels of ceramides present in the blood (higher levels of ceramide-Ab likely reflect higher levels of ceramide). There are several publications in which the authors evaluated antibodies against ceramides using the ELISA technique in relation to diseases, such as leprosy (Singh et al., 2010), peripheral neuropathies (Sykam et al., 2017), non-small cell like cancer (Bűdi et al., 2025), and finally in OSA (Horváth et al., 2023). To the best of our knowledge, 2 years ago during the time of planning our study, no commercially available ELISA test for ceramide was available. Therefore instead of using an ELISA test, ceramides were often evaluated by more sophisticated and sensitive techniques such as liquid chromatography in connection with mass spectrometry (LC-MS/MS). Unfortunately, we were unable to apply them. Therefore, we decided to use an indirect method of ceramide assessment (by detecting ceramide-Ab) in the form of an ELISA test.

In our study, ceramide-Ab levels were significantly increased in OSA patients compared to controls. A very similar result was reported by Horváth et al. (2023) although they noted a larger difference in antibody concentration between patients and controls (∼4-fold). In this report, it was 1.3-fold. As mentioned in the introduction, elevated levels of specific ceramides (d18:1/24:4) were also reported in male OSA patients in comprehensive lipidomic analysis (Lebkuchen et al., 2018). This indirectly indicates that the level of ceramide-Ab may actually reflect the amount of ceramide in circulation in the case of OSA. As also mentioned in the introduction, increased ceramide levels have been reported in several conditions frequently accompanying obstructive sleep apnea, such as obesity (Haus et al., 2009), coronary artery disease (Poss et al., 2020), insulin resistance (Boon et al., 2013), diabetes (Fretts et al., 2020), hypertension (Spijkers et al., 2011) or heart failure (Ji et al., 2017). Moreover, some studies showed that specific plasma ceramide ratios, including C24:0/C16:0 and C22:0/C16:0, independently correlate with major adverse cardiovascular events in patients with and without coronary artery diseases (Laaksonen et al., 2016).

In parallel, our study showed that OSA patients had significantly reduced S1P levels compared to the healthy control group. In our opinion, the results obtained in this study may indicate dysregulation of the ceramide/S1P rheostat in the course of OSA and a potential reduction in the conversion of ceramide to S1P. Under normal conditions, ceramides are produced acutely via sphingomyelinases (NSmases) in the endothelium and subsequently commonly converted to S1P with the use of ceramidases and sphingosine kinase. Among the ceramide metabolites, S1P is well known to regulate vascular endothelial function by stimulating nitric oxide (NO) production via endothelial nitric oxide synthase (eNOS) (Kerage et al., 2021; SenthilKumar et al., 2024). NO exerts well-documented vasoprotective effects through acute vasodilation, reducing proliferation, migration, thrombosis and inflammation (Su, 2015). However, under chronic pathological conditions when ceramide is produced in excess or endothelial enzymes that metabolize this sphingolipid are impaired, abnormal accumulation of ceramide may occur. Increased cellular levels of ceramide coupled with insufficient conversion to S1P result in the direct activation of protein phosphatase 2A and protein kinase C, of which the former dephosphorylates and inactivates eNOS, while the latter simultaneously phosphorylates and stimulates NADPH oxidase (NOX) to produce reactive oxygen species (ROS) (Zhang et al., 2012; Fox et al., 2007; Cosentino-Gomes et al., 2012; SenthilKumar et al., 2024). Chronically elevated ROS levels result in oxidative stress leading to endothelial dysfunction manifested by increased endothelial permeability, inflammation, and alterations in thrombotic or fibrinolytic mechanisms (Daiber and Chlopicki, 2020). Of note, patients with OSA demonstrate endothelial dysfunction even in the absence of any manifested vascular disease (Ip et al., 2004; Patt et al., 2010).

It is noteworthy that reciprocal changes in plasma ceramide and S1P levels have been observed in patients affected by cardiovascular disease. While S1P is generally reduced in patients affected by CAD (Sattler et al., 2010; Kurano and Yatomi, 2018), those patients affected by familial CAD have higher levels of some ceramide species in plasma compared to healthy controls (Poss et al., 2020). The shift towards ceramides over S1P observed in CAD does not seem surprising, as OSA is one of the strongest risk factors for CAD (Yacoub et al., 2017). Moreover, in CAD, dysfunction and inflammatory activation of the endothelium are pivotal events in the development of atherosclerosis and are associated with an elevated risk of cardiovascular events (Medina-Leyte et al., 2021). There are more examples of disorders connected with OSA in which a dysregulated sphingolipid rheostat may play a role. (i) Lipidomic analysis revealed increased total levels of ceramides alongside very long–chain ceramides in both the myocardium and serum of patients with advanced heart failure (Ji et al., 2017). (ii) A sustained decrease in plasma S1P concentrations in acute myocardial infarction (AMI) patients has been observed. The authors of the study suggested a cardioprotective effect of S1P in AMI, but attributed the most likely reason for S1P reduction to its poor release or increased degradation (Knapp et al., 2013). (iii) S1P levels were significantly lower in patients with pre-diabetes and diabetes mellitus type 2 (T2DM) compared with those without diabetes (Vaisar et al., 2018; Sui et al., 2019). (iv) Patients with ischemic stroke showed lower serum S1P concentrations compared with hemorrhagic stroke patients or healthy controls (Liu et al., 2020).

Despite many reports of the association of both S1P and ceramide with OSA-associated comorbidities, we did not show such a relationship in our study for S1P and ceramide-Ab (as a surrogate for ceramide). This was demonstrated both by bivariate analysis for more frequent comorbidities, such as hypertension, diabetes, and CHD, as well as by using multiple linear regression models that also included COPD and stroke. Increased ceramide-Ab and decreased S1P levels appear to be characteristic of OSA in our patients and not a result of common comorbidities. However, due to the small number of patients with COPD or a history of stroke, the statistical power for these variables may be limited.

Importantly, the results presented in our study regarding S1P are contradictory to the results recently published by Horvath et al. (2023). They noted significantly elevated concentrations of S1P in their OSA patients. The explanation behind this discrepancy is quite puzzling. It may be due to the smaller number of OSA patients (N = 31) and controls (N = 37) recruited by Horvath et al. or a different study design as the authors used a S1P ELISA test from a different manufacturer. Additionally, the medications taken by patients or differences in treatment regimens may play a role. Either way, further studies on representative and well matched cohorts are needed to precisely determine the S1P levels in OSA. Significantly, our study was conducted on larger cohorts (N = 109 and N = 49) and has sufficient power (∼100%) and sample size to detect statistical differences between the tested groups. In addition, our data supports previous reports of lower levels of S1P concentration in patients suffering from comorbidities related to OSA (discussed above).

Interestingly, we observed a trend toward a significant positive correlation between ceramide-Ab levels and the desaturation index. While the correlation is not statistically significant, it suggests the involvement of anti-ceramide antibodies (or ceramide itself) in the pathogenesis of intermittent hypoxia associated with OSA. Moreno et al. (2014) presented a possible explanation for this hypothesis in their publication. They found that ceramide content and ROS production increased in pulmonary arteries (PA) following pulmonary vascular hypoxia. Furthermore, the nSMase inhibitor GW4869 and the anti-ceramide antibody reduced hypoxic pulmonary vasoconstriction (HPV) in chicken PA. The authors concluded that nSMase-derived ceramide could play a critical role in acute oxygen sensing in specialized vascular tissues (Moreno et al., 2014). It is unclear whether the excess anti-ceramide antibodies detected in the serum of OSA patients affect the concentration of active ceramide involved in HPV. Hypothetically, if the antibodies bind to ceramide, they could inhibit pulmonary vasoconstriction induced by intermittent hypoxia. Nevertheless, the question remains as to whether this would constitute a favorable or unfavorable outcome. HPV is an intrinsic homeostatic mechanism of the pulmonary vasculature. In response to alveolar hypoxia, intrapulmonary arteries constrict, diverting blood to better-oxygenated lung segments. This optimizes ventilation/perfusion matching and systemic oxygen delivery (Dunham-Snary et al., 2017). This mechanism, initially protective in nature, becomes detrimental in the chronic setting of OSA, contributing to increased pulmonary artery pressure and vascular remodeling (Balcan et al., 2024). Thus, “switching off” HPV with ceramide antibodies may initially have negative effects, such as disturbing gas exchange in the lungs. However, in the long term, it may prevent pulmonary hypertension and vascular remodeling.

Conversely, anti-ceramide antibodies may block the conversion of ceramide to “protective” S1P, thereby exacerbating the pathological processes in OSA and contributing to endothelial dysfunction (as discussed above). Therefore, we believe it is crucial to investigate this issue in future research.

The literature on the potential physiological role of anti-ceramide antibodies is limited. However, it is known that manufactured ceramide-Abs can bind ceramide and block its functions. For example, Rotolo et al. (2012) demonstrated that ceramide-Ab (2A2) protects against endothelial apoptosis in the small intestinal lamina propria and promotes recovery of crypt stem cell clonogens. This prevented the death of mice from radiation GI syndrome after high radiation doses. Similarly, in a recent publication, Dorweiler et al. (2024) demonstrated that an anti-ceramide antibody is able to reverse the effects of diabetic retinopathy. In this case, ceramide-Ab protects endothelial retinal cells from apoptosis, which is initiated by ceramide induced by TNF-α and IL-1β. Given that elevated levels of ceramide have been well documented in the course of various diseases including cardiovascular, neurodegenerative (Alzheimer’s disease, motor neuron disease), metabolic conditions (obesity, type II diabetes, insulin resistance, impaired glucose tolerance), it is potentially possible to use anti-ceramide antibodies in their diagnosis and treatment (Shen et al. 2025).

Interestingly, in the present study, body mass index was positively correlated with OSA severity (AHI parameter) and with the desaturation index (DI). On the other hand, we detected a negative correlation between BMI and mean SaO₂ and minimum SaO₂. Very similar correlations between BMI vs. AHI, DI, as well as mean SaO_2, were also recently described by Pau et al. (2023) in Italian OSA patients. These results highlight the very important observation that the higher the BMI, the more severe the episodes of obstruction and desaturation and, consequently, the lower the oxygen saturation during sleep. Positive correlation between BMI and AHI was reported also for patients from China (Liu et al., 2021) and Mauritius (Sant Bakshsingh et al., 2024). Additionally, we found a strong positive correlation between BMI and serum CRP level (r = 0.60). Notably, a similar magnitude of positive correlation between these two parameters was noted by Suša et al., 2021 for Serbian patients (r = 0.633), and a slightly weaker correlation was detected by Guilleminault et al. (2004) (r = 0.459) after adjusting for other coefficients. The correlation between BMI and CRP may be due to the fact that adipose tissue secretes a variety of bioactive mediators including adipocytokines such as adiponectin, leptin, resistin, visfatin or classical cytokines such as tumor necrosis factor α (TNFα), interleukin 1 (IL-1), CC-chemokine ligand 2 (CCL2), and interleukin 6 (IL-6) (Tilg and Moschen, 2006). Among these cytokines, IL-6 is the primary factor driving hepatic CRP production. Importantly, almost one-third of the IL-6 concentration in circulation of obese patients originates from adipose tissue (Mohamed-Ali et al., 1997). Moreover, a significant positive association between IL-6 concentration and fat mass (adipose tissue percentage) was also recently found (Suša et al., 2021).

There are a few limitations of the current study: (1) Testing of anti-ceramide antibodies rather than ceramide itself. Although the measurement of anti-ceramide antibodies is valuable and informative in itself, in the next phase of our research we plan to examine ceramide levels by using the LC-MS/MS technique in our study groups and correlate the obtained results with those obtained from ELISA. Given that two reports (ours and Horváth et al., 2023) are consistent with the elevated levels of anti-ceramide antibodies in OSA patients, we suggest that these antibodies may turn out to be a valuable biomarker for the diagnosis of OSA. This is all the more possible given that increased levels of these antibodies in our patients were not related to presence/absence of comorbidities such as CHD, diabetes, or hypertension. (2) Underrepresentation of women in our control group. Currently, we have a male-to-female ratio of about 6:1, whereas it should be around 3:1 (similar to the patient group). OSA is generally estimated to have a male-to-female ratio of between 3:1 and 5:1 in the general population (Wimms et al., 2016), so we should recruit at least seven more women. However, the gender imbalance in our study did not prevent statistical analysis. (3) Lack of assessment of the concentrations of molecules that bind and transport S1P in the bloodstream, i.e., HDL together with apolipoprotein M or albumin, as their variations may affect the levels of S1P detected in serum.

In conclusion, we demonstrated that anti-ceramide antibody levels were elevated, while S1P concentrations were decreased in patients with obstructive sleep apnea compared to subjects without OSA. Patient BMI was positively correlated with OSA severity, desaturation index, and CRP levels, and negatively correlated with mean SaO₂ and minimum SaO₂. We did not find any association of ceramide-Ab and S1P concentrations with the presence of comorbidities such as hypertension, coronary heart disease, diabetes, COPD, and stroke.