Metabolomics-based search for lung cancer markers among patients with different smoking status.

Tobacco smoking is the main etiological factor of lung cancer (LC), which can also cause metabolome disruption. This study aimed to investigate whether the observed metabolic shift in LC patients was also associated with their smoking status. Untargeted metabolomics profiling was applied for the initial screening of changes in serum metabolic profile between LC and chronic obstructive pulmonary disease (COPD) patients, selected as a non-cancer group. Differences in metabolite profiles between current and former smokers were also tested. Then, targeted metabolomics methods were applied to verify and validate the proposed LC biomarkers. For untargeted metabolomics, a single extraction-dual separation workflow was applied. The samples were analyzed using a liquid chromatograph-high resolution quadrupole time-of-flight mass spectrometer. Next, the selected metabolites were quantified using liquid chromatography-triple-quadrupole mass spectrometry. The acquired data confirmed that patients' stratification based on smoking status impacted the discriminating ability of the identified LC marker candidates. Analyzing a validation set of samples enabled us to determine if the putative LC markers were truly robust. It demonstrated significant differences in the case of four metabolites: allantoin, glutamic acid, succinic acid, and sphingosine-1-phosphate. Our research showed that studying the influence of strong environmental factors, such as tobacco smoking, should be considered in cancer marker research since it reduces the risk of false positives and improves understanding of the metabolite shifts in cancer patients.

Serum S1P level in interstitial lung disease (ILD) is a potential biomarker reflecting the severity of pulmonary function.

BACKGROUND: sphingosine-1-phosphate (S1P), a naturally occurring sphingolipid, has been involved in pulmonary interstitial remodeling signaling. However, no study has examined its clinical merits for interstitial lung disease (ILD). This study aimed to investigate the serum level of S1P in ILD patients and its clinical correlation with the severity of disease in the two main types of ILDs: the IPF and the CTD-ILD patients. METHODS: This retrospective observational pilot study included 67 ILD patients and 26 healthy controls. These patients were stratified into the IPF group (35) and the CTD-ILD group (32). The severity of ILD was evaluated through pulmonary function indicators and the length of hospital stay. RESULTS: Serum S1P level was statistically higher in ILD patients than in health control (p = 0.002), while the Serum S1P levels in CTD-ILD and IPF patients were comparable. Serum S1P level further showed statistically negative correlation with pulmonary function indexes (TLC% pred, FVC% pred and FEV1% pred) and positive correlation with length of hospital stay (r = -0.38, p = 0.04; r = -0.41, p = 0.02, r = -0.37, p = 0.04; r = 0.42, p = 0.02, respectively) in CTD-ILD patients, although serum S1P level was not significantly correlated with inflammatory indexes. The IPF patients failed to exhibit a significant correlation of serum S1P level with pulmonary function and length of hospital stay. CONCLUSIONS: Serum S1P level might be a clinically useful biomarker in evaluating the severity of CTD-ILD patients rather than IPF patients.

Sphingolipid changes in mouse brain and plasma after mild traumatic brain injury at the acute phases.

BACKGROUND: Traumatic brain injury (TBI) causes neuroinflammation and can lead to long-term neurological dysfunction, even in cases of mild TBI (mTBI). Despite the substantial burden of this disease, the management of TBI is precluded by an incomplete understanding of its cellular mechanisms. Sphingolipids (SPL) and their metabolites have emerged as key orchestrators of biological processes related to tissue injury, neuroinflammation, and inflammation resolution. No study so far has investigated comprehensive sphingolipid profile changes immediately following TBI in animal models or human cases. In this study, sphingolipid metabolite composition was examined during the acute phases in brain tissue and plasma of mice following mTBI. METHODS: Wildtype mice were exposed to air-blast-mediated mTBI, with blast exposure set at 50-psi on the left cranium and 0-psi designated as Sham. Sphingolipid profile was analyzed in brain tissue and plasma during the acute phases of 1, 3, and 7 days post-TBI via liquid-chromatography-mass spectrometry. Simultaneously, gene expression of sphingolipid metabolic markers within brain tissue was analyzed using quantitative reverse transcription-polymerase chain reaction. Significance (P-values) was determined by non-parametric t-test (Mann-Whitney test) and by Tukey's correction for multiple comparisons. RESULTS: In post-TBI brain tissue, there was a significant elevation of 1) acid sphingomyelinase (aSMase) at 1- and 3-days, 2) neutral sphingomyelinase (nSMase) at 7-days, 3) ceramide-1-phosphate levels at 1 day, and 4) monohexosylceramide (MHC) and sphingosine at 7-days. Among individual species, the study found an increase in C18:0 and a decrease in C24:1 ceramides (Cer) at 1 day; an increase in C20:0 MHC at 3 days; decrease in MHC C18:0 and increase in MHC C24:1, sphingomyelins (SM) C18:0, and C24:0 at 7 days. Moreover, many sphingolipid metabolic genes were elevated at 1 day, followed by a reduction at 3 days and an absence at 7-days post-TBI. In post-TBI plasma, there was 1) a significant reduction in Cer and MHC C22:0, and an increase in MHC C16:0 at 1 day; 2) a very significant increase in long-chain Cer C24:1 accompanied by significant decreases in Cer C24:0 and C22:0 in MHC and SM at 3 days; and 3) a significant increase of C22:0 in all classes of SPL (Cer, MHC and SM) as well as a decrease in Cer C24:1, MHC C24:1 and MHC C24:0 at 7 days. CONCLUSIONS: Alterations in sphingolipid metabolite composition, particularly sphingomyelinases and short-chain ceramides, may contribute to the induction and regulation of neuroinflammatory events in the early stages of TBI, suggesting potential targets for novel diagnostic, prognostic, and therapeutic strategies in the future.

Comparative analysis of lysophospholipid metabolism profiles and clinical characteristics in patients with high vs. low C-reactive protein levels in acute exacerbations of chronic obstructive pulmonary disease.

BACKGROUND: The precise role of lysophospholipids (LysoPLs) in the pathogenesis of acute exacerbations of Chronic Obstructive Pulmonary Disease (AECOPD) remains unclear. In this study, we sought to elucidate the differences in serum LysoPL metabolite profiles and their correlation with clinical features between patients with low versus high CRP levels. METHODS: A total of 58 patients with AECOPD were enrolled in the study. Patients were classified into two groups: low CRP group (CRP < 20 mg/L, n = 34) and high CRP group (CRP ≥ 20 mg/L, n = 24). Clinical data were collected, and the LysoPL metabolite profiles were analyzed using Liquid Chromatography-Mass Spectrometry (LC-MS) and identified by matching with the LipidBlast library. RESULTS: Nineteen differential LysoPLs were initially identified through Student's t-test (p < 0.05 and VIP > 1). Subsequently, four LysoPLs, LPC(16:0), LPE(18:2), LPC(22:0), and LPC(24:0), were identified by FDR adjustment (adjusted p < 0.05). These four lysoPLs had a significant negative correlation with CRP. Integrative analysis revealed that LPC (16:0) and LPC (22:0) correlated with less hypercapnic respiratory failure and ICU admission. CONCLUSION: AECOPD patients with high CRP levels demonstrated a distinctive LysoPL metabolism profile, with LPC (16:0), LPE(18:2), LPC(22:0), and LPC(24:0) being the most significantly altered lipid molecules. These alterations were associated with poorer clinical outcomes.

Platelet Function is Independent of Sphingolipid Manipulation.

INTRODUCTION: Previous literature suggests that sphingolipids may impact systemic coagulation and platelet aggregation, thus modulating the risks of thrombotic events. The goal of this investigation was to evaluate the role of serum sphingolipids on intrinsic platelet function to assess whether pharmacologic manipulation of sphingolipid metabolites would impact platelet aggregability. METHODS: C57BL/6J mice were injected with either normal saline, 1 mg/kg FTY720 (synthetic sphingosine-1-phosphate [S1P] receptor analog), or 5 mg/kg SLM6031434 (sphingosine kinase two inhibitor). Mice were sacrificed at 6 h and whole blood (WB) was collected for impedance aggregometry assessing platelet responsiveness to arachidonic acid or adenosine diphosphate. Ex vivo studies utilized WB or platelet-rich plasma that was pretreated with S1P, FTY720, amitriptyline, or d-sphingosine then analyzed by aggregability and flow cytometry for platelet and platelet-derived microvesicle characteristics. RESULTS: FTY720 and SLM6031434 pretreated induced similar arachidonic acid and adenosine diphosphate-mediated platelet aggregation as controls. Ex vivo WB and platelet-rich plasma treatment with S1P, FTY720, amitriptyline and d-sphingosine did not impact platelet aggregation. The percentages of CD41+, CD62P+ and CD41+/ceramide+, CD62P+/ceramide + platelets, and platelet-derived microvesicle were not significantly different between amitriptyline-treated and normal saline-treated cohorts. CONCLUSIONS: Sphingolipid modulating agents, such as FTY720, SLM6031434, S1P, amitriptyline, ceramide, and d-sphingosine do not appear to independently impact platelet aggregation in murine models.

Plasma Metabolite Profiling Identifies Nondiabetic Chronic Pancreatitis Patients With Metabolic Alterations Progressing to Prediabetes Before HbA1c.

INTRODUCTION: Diabetes (T3cDM) secondary to chronic pancreatitis (CP) arises due to endocrine dysfunction and metabolic dysregulations. Currently, diagnostic tests are not available to identify patients who may progress from normoglycemia to hyperglycemia in CP. We conducted plasma metabolomic profiling to diagnose glycemic alterations early in the course of disease. METHODS: Liquid chromatography-tandem mass spectrometry was used to generate untargeted, targeted plasma metabolomic profiles in patients with CP, controls (n = 445) following TRIPOD guidelines. Patients were stratified based on glucose tolerance tests following ADA guidelines. Multivariate analysis was performed using partial least squares discriminant analysis to assess discriminatory ability of metabolites among stratified groups. COMBIROC and logistic regression were used to derive biomarker signatures. AI-ML tool (Rapidminer) was used to verify these preliminary results. RESULTS: Ceramide, lysophosphatidylethanolamine, phosphatidylcholine, lysophosphatidic acid (LPA), phosphatidylethanolamine, carnitine, and lysophosphatidylcholine discriminated T3cDM CP patients from healthy controls with AUC 93% (95% CI 0.81-0.98, P < 0.0001), and integration with pancreatic morphology improved AUC to 100% (95% CI 0.93-1.00, P < 0.0001). LPA, phosphatidylinositol, and ceramide discriminated nondiabetic CP with glycemic alterations (pre-diabetic CP); AUC 66% (95% CI 0.55-0.76, P = 0.1), and integration enhanced AUC to 74% (95% CI 0.55-0.88, P = 0.86). T3cDM was distinguished from prediabetic by LPA, phosphatidylinositol, and sphinganine (AUC 70%; 95% CI 0.54-0.83, P = 0.08), and integration improved AUC to 83% (95% CI 0.68-0.93, P = 0.05). CombiROC cutoff identified 75% and 78% prediabetes in validation 1 and 2 cohorts. Random forest algorithm assessed performance of integrated panel demonstrating AUC of 72% in predicting glycemic alterations. DISCUSSION: We report for the first time that a panel of metabolites integrated with pancreatic morphology detects glycemia progression before HbA1c in patients with CP.

Preservation of recipient plasma sphingosine-1-phosphate levels reduces transfusion-related acute lung injury.

Cold-stored (CS) platelets are once again being reintroduced for clinical use. Transfused CS platelets offer benefits over room temperature-stored (RTS) platelets such as increased hemostatic effects and prolongation of shelf-life. Despite these advantages little is known about their association with transfusion-related acute lung injury (TRALI). TRALI is associated with prolonged storage of RTS platelets and has a mortality of >15%. Determining the safety of CS platelets is important considering their proposed use in TRALI-vulnerable populations with inflammation such as surgical patients or patients with trauma. Donor platelet-derived ceramide causes TRALI, whereas donor platelet sphingosine-1-phosphate (S1P) is barrier protective. Females have higher plasma levels of S1P than males. Cold temperatures increase S1P levels in cells. Therefore, we hypothesized that female (donors or recipients) and/or CS platelets would decrease TRALI. To test this, we compared how male and female donor and recipient allogeneic platelet transfusions of CS (4°C) versus RTS (23°C) platelets stored for 5 days influence murine TRALI. Transfusion of CS platelets significantly reduced recipient lung tissue wet-to-dry ratios, bronchoalveolar lavage total protein, lung tissue myeloperoxidase enzyme activity, histological lung injury scores, and increased plasma sphingosine-1-phosphate (S1P) levels compared with RTS platelet transfusions. Female as opposed to male recipients had less TRALI and higher plasma S1P levels. Female donor mouse platelets had higher S1P levels than males. Mouse and human CS platelets had increased S1P levels compared with RTS platelets. Higher recipient plasma S1P levels appear protective considering females, and males receiving platelets from females or male CS platelets had less TRALI.NEW & NOTEWORTHY Transfusion-related acute lung injury (TRALI) though relatively rare represents a severe lung injury. The sphingolipid sphingosine-1-phosphate (S1P) regulates the severity of platelet-mediated TRALI. Female platelet transfusion recipient plasmas or stored platelets from female donors have higher S1P levels than males, which reduces TRALI. Cold storage of murine platelets preserves platelet-S1P, which reduces TRALI in platelet-transfused recipients.

Identification of lysophosphatidic acid in serum as a factor that promotes epithelial apical junctional complex organization.

The apical junctional complex (AJC) consists of adherens junctions (AJs) and tight junctions and regulates epithelial integrity and remodeling. However, it is unclear how AJC organization is regulated based on environmental cues. We found here using cultured EpH4 mouse mammary epithelial cells that fetal bovine serum (FBS) in a culture medium showed an activity to promote AJC organization and that FBS showed an activity to promote tight junction formation even in the absence of AJ proteins, such as E-cadherin, αE-catenin, and afadin. Furthermore, we purified the individual factor responsible for these functions from FBS and identified this molecule as lysophosphatidic acid (LPA). In validation experiments, purified LPA elicited the same activity as FBS. In addition, we found that the AJC organization-promoting activity of LPA was mediated through the LPA receptor 1/5 via diacylglycerol-novel PKC and Rho-ROCK pathway activation in a mutually independent, but complementary, manner. We demonstrated that the Rho-ROCK pathway activation-mediated AJC organization was independent of myosin II-induced actomyosin contraction, although this signaling pathway was previously shown to induce myosin II activation. These findings are in contrast to the literature, as previous results suggested an AJC organization-disrupting activity of LPA. The present results indicate that LPA in serum has an AJC organization-promoting activity in a manner dependent on or independent of AJ proteins.

Sphingosine-1-phosphate and CRP as potential combination biomarkers in discrimination of COPD with community-acquired pneumonia and acute exacerbation of COPD.

BACKGROUND:  Chronic obstructive pulmonary disease (COPD) is a significant public health concern. The patients with acute exacerbations of COPD (AECOPD) and pneumonia have similar clinical presentations. The use of conventional diagnostic markers, such as complete blood count with differential and C-reactive protein (CRP), is the current mainstream method for differentiating clinically relevant pneumonia from other mimics. However, those conventional methods have suboptimal sensitivity and specificity for patients with a clinical suspicion of infection. The limitations often cause the ambiguity of the initiation of antibiotic treatment. Recently, our pilot study suggested that the patients with pneumonia have significantly higher plasma Sphingosine-1-phosphate (S1P) levels than controls. The initial findings suggest that plasma S1P is a potential biomarker for predicting prognosis in pneumonia. The aim of this study was to evaluate the value of S1P and CRP for discriminating COPD with pneumonia and AECOPD in an Emergency Department (ED) setting. METHODS:  Patients diagnosed with AECOPD or COPD with pneumonia were recruited from the Emergency Department of Wan Fang Hospital. The clinical data, demographics, and blood samples were collected upon ED admission. The concentration of plasma S1P was measured by ELISA. RESULTS:  Thirty-nine patients with AECOPD and 78 with COPD plus pneumonia were enrolled in this observational study. The levels of blood S1P and CRP were significantly higher in patients with COPD plus CAP compared to those in AE COPD patients. The area under the receiver operator characteristic (ROC) curve for the S1P and CRP for distinguishing between patients with COPD plus CAP and AECOPD is 0.939 (95% CI: 0.894-0.984) and 0.886 (95% CI: 0.826-0.945), whereas the combination of S1P and CRP yielded a value of 0.994 (95% CI: 0.897-1.000). By comparing with CRP or S1P, combining CRP and S1P had significantly higher AUC value for differentiating between the COPD with pneumonia group and the AECOPD group. CONCLUSIONS: Our findings suggest that S1P is a potential diagnostic biomarker in distinguishing COPD with CAP from AECOPD. Additionally, the diagnostic ability of S1P can be improved when used in combination with CRP.

Serum lysophospholipidome of dietary origin as a suitable susceptibility/risk biomarker of human hypercholesterolemia: A cross-sectional study.

BACKGROUND & AIMS: Whether bioactive lysophospholipids (lyso-PLs) and trimethylamine-N-oxide (TMAO) serve as non-invasive biomarkers in early human hypercholesterolemia (HC) is unknown. This study aimed to assess whether serum lyso-PLs and plasma TMAO may be suitable susceptibility/risk biomarkers of HC in humans. Secondarily, we aimed to evaluate the relationships between targeted metabolites, diet composition and circulating liver transaminases, and verify these results in hamsters. METHODS: A targeted metabolomics and lipidomics approach determined plasma TMAO and serum lysophosphatidylcholines (lyso-PCs) and lysophosphatidylethanolamines (lyso-PEs) in low (L-LDL-c) and moderate to high (MH-LDL-c) LDL-cholesterol subjects. Additionally, the relationships between targeted metabolites, liver transaminases and diet, particularly fatty acid intake, were tested. In parallel, plasma and liver lyso-PL profiles were studied in 16 hamsters fed a moderate high-fat (HFD) or low-fat (LFD) diet for 30 days. RESULTS: Predictive models identified lyso-PC15:0 and lyso-PE18:2 as the most discriminant lyso-PLs among groups. In MH-LDL-c (n = 48), LDL-cholesterol and saturated FAs were positively associated with lyso-PC15:0, whereas in L-LDL-c (n = 70), LDL-cholesterol and polyunsaturated fatty acids (PUFAs) were negatively and positively related to lyso-PE18:2, respectively. Interestingly, in MH-LDL-c, the lower lyso-PE 18:2 concentrations were indicative of higher LDL-cholesterol levels. Intrahepatic accumulation of lyso-PLs-containing essential n-6 PUFAs, including lyso-PE18:2, were higher in HFD-fed hamsters than LFD-fed hamsters. CONCLUSIONS: Overall, results revealed a possible hepatic adaptive mechanism to counteract diet-induced steatosis in animal and hypercholesterolemia progression in humans. In particular, low serum lyso-PE18:2 suggests a suitable susceptibility/risk biomarker of HC in humans.

Therapeutic CFTR Correction Normalizes Systemic and Lung-Specific S1P Level Alterations Associated with Heart Failure.

Heart failure (HF) is among the main causes of death worldwide. Alterations of sphingosine-1-phosphate (S1P) signaling have been linked to HF as well as to target organ damage that is often associated with HF. S1P's availability is controlled by the cystic fibrosis transmembrane regulator (CFTR), which acts as a critical bottleneck for intracellular S1P degradation. HF induces CFTR downregulation in cells, tissues and organs, including the lung. Whether CFTR alterations during HF also affect systemic and tissue-specific S1P concentrations has not been investigated. Here, we set out to study the relationship between S1P and CFTR expression in the HF lung. Mice with HF, induced by myocardial infarction, were treated with the CFTR corrector compound C18 starting ten weeks post-myocardial infarction for two consecutive weeks. CFTR expression, S1P concentrations, and immune cell frequencies were determined in vehicle- and C18-treated HF mice and sham controls using Western blotting, flow cytometry, mass spectrometry, and qPCR. HF led to decreased pulmonary CFTR expression, which was accompanied by elevated S1P concentrations and a pro-inflammatory state in the lungs. Systemically, HF associated with higher S1P plasma levels compared to sham-operated controls and presented with higher S1P receptor 1-positive immune cells in the spleen. CFTR correction with C18 attenuated the HF-associated alterations in pulmonary CFTR expression and, hence, led to lower pulmonary S1P levels, which was accompanied by reduced lung inflammation. Collectively, these data suggest an important role for the CFTR-S1P axis in HF-mediated systemic and pulmonary inflammation.

Endothelial-Specific Loss of Sphingosine-1-Phosphate Receptor 1 Increases Vascular Permeability and Exacerbates Bleomycin-induced Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive disease which leads to significant morbidity and mortality from respiratory failure. The two drugs currently approved for clinical use slow the rate of decline in lung function but have not been shown to halt disease progression or reverse established fibrosis. Thus, new therapeutic targets are needed. Endothelial injury and the resultant vascular permeability are critical components in the response to tissue injury and are present in patients with IPF. However, it remains unclear how vascular permeability affects lung repair and fibrosis following injury. Lipid mediators such as sphingosine-1-phosphate (S1P) are known to regulate multiple homeostatic processes in the lung including vascular permeability. We demonstrate that endothelial cell-(EC) specific deletion of the S1P receptor 1 (S1PR1) in mice (EC-S1pr1-/-) results in increased lung vascular permeability at baseline. Following a low-dose intratracheal bleomycin challenge, EC-S1pr1-/- mice had increased and persistent vascular permeability compared with wild-type mice, which was strongly correlated with the amount and localization of resulting pulmonary fibrosis. EC-S1pr1-/- mice also had increased immune cell infiltration and activation of the coagulation cascade within the lung. However, increased circulating S1P ligand in ApoM-overexpressing mice was insufficient to protect against bleomycin-induced pulmonary fibrosis. Overall, these data demonstrate that endothelial cell S1PR1 controls vascular permeability in the lung, is associated with changes in immune cell infiltration and extravascular coagulation, and modulates the fibrotic response to lung injury.

Hypogelsolinemia and Decrease in Blood Plasma Sphingosine-1-Phosphate in Patients Diagnosed with Severe Acute Pancreatitis.

BACKGROUND: Acute pancreatitis (AP) is a frequent hospitalization cause of patients suffering from gastrointestinal disorders. Gelsolin has an ability to bind bioactive lipids including different sphingolipids engaged in inflammatory response. Importantly, hypogelsolinemia was observed in patients with different states of acute and chronic inflammation. AIMS: The aim of the present study was to assess the interplay of blood plasma gelsolin and blood plasma sphingosine-1-phosphate (S1P) concentration in patients diagnosed with acute pancreatitis. MATERIALS AND METHODS: To assess the concentration of gelsolin and S1P, immunoblotting and HPLC technique were employed, respectively. Additionally, the concentrations of amylase, lipase, C-reactive protein (CRP), procalcitonin (PCT) and the number of white blood cells (WBC) and platelet (PLT) were recorded. RESULTS: We found that both pGSN and S1P concentrations in the plasma of the AP patients were significantly lower (pGSN ~ 15-165 mg/L; S1P ~ 100-360 pmol/mL) when compared to the levels of pGSN and S1P in a control group (pGSN ~ 130-240 mg/L; S1P ~ 260-400 pmol/mL). Additionally, higher concentrations of CRP, WBC, amylase and lipase were associated with low level of gelsolin in the blood of AP patients. No correlations between the level of PCT and PLT with gelsolin concentration were noticed. CONCLUSION: Plasma gelsolin and S1P levels decrease during severe acute pancreatitis. Simultaneous assessment of pGSN and S1P can be useful in development of more accurate diagnostic strategies for patients with severe acute pancreatitis.

An improved functional assay in blood spot to diagnose Barth syndrome using the monolysocardiolipin/cardiolipin ratio.

Barth syndrome is an X-linked disorder characterized by cardiomyopathy, skeletal myopathy, and neutropenia, caused by deleterious variants in TAFAZZIN. This gene encodes a phospholipid-lysophospholipid transacylase that is required for the remodeling of the mitochondrial phospholipid cardiolipin (CL). Biochemically, individuals with Barth syndrome have a deficiency of mature CL and accumulation of the remodeling intermediate monolysocardiolipin (MLCL). Diagnosis typically relies on mass spectrometric measurement of CL and MLCL in cells or tissues, and we previously described a method in blood spot that uses a specific MLCL/CL ratio as diagnostic biomarker. Here, we describe the evolution of our blood spot assay that is based on the implementation of reversed phase-UHPLC separation followed by full scan high resolution mass spectrometry. In addition to the MLCL/CL ratio, our improved method also generates a complete CL spectrum allowing the interrogation of the CL fatty acid composition, which considerably enhances the diagnostic reliability. This addition negates the need for a confirmatory test in lymphocytes thereby providing a shorter turn-around-time while achieving a more certain test result. As one of the few laboratories that offer this assay, we also evaluated the diagnostic yield and performance from 2006 to 2021 encompassing the use of both the original and improved assay. In this period, we performed 796 diagnostic analyses of which 117 (15%) were characteristic of Barth syndrome. In total, we diagnosed 93 unique individuals with Barth syndrome, including three females, which together amounts to about 40% of all reported individuals with Barth syndrome in the world.

Erythrocytes increase endogenous sphingosine 1-phosphate levels as an adaptive response to SARS-CoV-2 infection.

Low plasma levels of the signaling lipid metabolite sphingosine 1-phosphate (S1P) are associated with disrupted endothelial cell (EC) barriers, lymphopenia and reduced responsivity to hypoxia. Total S1P levels were also reduced in 23 critically ill patients with coronavirus disease 2019 (COVID-19), and the two main S1P carriers, serum albumin (SA) and high-density lipoprotein (HDL) were dramatically low. Surprisingly, we observed a carrier-changing shift from SA to HDL, which probably prevented an even further drop in S1P levels. Furthermore, intracellular S1P levels in red blood cells (RBCs) were significantly increased in COVID-19 patients compared with healthy controls due to up-regulation of S1P producing sphingosine kinase 1 and down-regulation of S1P degrading lyase expression. Cell culture experiments supported increased sphingosine kinase activity and unchanged S1P release from RBC stores of COVID-19 patients. These observations suggest adaptive mechanisms for maintenance of the vasculature and immunity as well as prevention of tissue hypoxia in COVID-19 patients.

Plasma Sphingosine-1-Phosphate Levels Are Associated with Progression of Estrogen Receptor-Positive Breast Cancer.

Although numerous experiments revealed an essential role of a lipid mediator, sphingosine-1-phosphate (S1P), in breast cancer (BC) progression, the clinical significance of S1P remains unclear due to the difficulty of measuring lipids in patients. The aim of this study was to determine the plasma concentration of S1P in estrogen receptor (ER)-positive BC patients, as well as to investigate its clinical significance. We further explored the possibility of a treatment strategy targeting S1P in ER-positive BC patients by examining the effect of FTY720, a functional antagonist of S1P receptors, on hormone therapy-resistant cells. Plasma S1P levels were significantly higher in patients negative for progesterone receptor (PgR) expression than in those positive for expression (p = 0.003). Plasma S1P levels were also significantly higher in patients with larger tumor size (p = 0.012), lymph node metastasis (p = 0.014), and advanced cancer stage (p = 0.003), suggesting that higher levels of plasma S1P are associated with cancer progression. FTY720 suppressed the viability of not only wildtype MCF-7 cells, but also hormone therapy-resistant MCF-7 cells. Targeting S1P signaling in ER-positive BC appears to be a possible new treatment strategy, even for hormone therapy-resistant patients.

Dynamics of Vascular Protective and Immune Supportive Sphingosine-1-Phosphate During Cardiac Surgery.

Introduction: Sphingosine-1-phosphate (S1P) is a signaling lipid and crucial in vascular protection and immune response. S1P mediated processes involve regulation of the endothelial barrier, blood pressure and S1P is the only known inducer of lymphocyte migration. Low levels of circulatory S1P correlate with severe systemic inflammatory syndromes such as sepsis and shock states, which are associated with endothelial barrier breakdown and immunosuppression. We investigated whether S1P levels are affected by sterile inflammation induced by cardiac surgery. Materials and Methods: In this prospective observational study we included 46 cardiac surgery patients, with cardiopulmonary bypass (CPB, n=31) and without CPB (off-pump, n=15). Serum-S1P, S1P-sources and carriers, von-Willebrand factor (vWF), C-reactive protein (CRP), procalcitonin (PCT) and interleukin-6 (IL-6) were measured at baseline, post-surgery and at day 1 (POD 1) and day 4 (POD 4) after surgical stimulus. Results: Median S1P levels at baseline were 0.77 nmol/mL (IQR 0.61-0.99) and dropped significantly post-surgery. S1P was lowest post-surgery with median levels of 0.37 nmol/mL (IQR 0.31-0.47) after CPB and 0.46 nmol/mL (IQR 0.36-0.51) after off-pump procedures (P<0.001). The decrease of S1P was independent of surgical technique and observed in all individuals. In patients, in which S1P levels did not recover to preoperative baseline ICU stay was longer and postoperative inflammation was more severe. S1P levels are associated with its sources and carriers and vWF, as a more specific endothelial injury marker, in different phases of the postoperative course. Determination of S1P levels during surgery suggested that also the anticoagulative effect of heparin might influence systemic S1P. Discussion: In summary, serum-S1P levels are disrupted by major cardiac surgery. Low S1P levels post-surgery may play a role as a new marker for severity of cardiac surgery induced inflammation. Due to well-known protective effects of S1P, low S1P levels may further contribute to the observed prolonged ICU stay and worse clinical status. Moreover, we cannot exclude a potential inhibitory effect on circulating S1P levels by heparin anticoagulation during surgery, which would be a new pro-inflammatory pleiotropic effect of high dose heparin in patients undergoing cardiac surgery.

Lysophosphatidic acid species are associated with exacerbation in chronic obstructive pulmonary disease.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) exacerbations are heterogenous and profoundly impact the disease trajectory. Bioactive lipid lysophosphatidic acid (LPA) has been implicated in airway inflammation but the significance of LPA in COPD exacerbation is not known. The aim of the study was to investigate the utility of serum LPA species (LPA16:0, 18:0, 18:1, 18:2, 20:4) as biomarkers of COPD exacerbation. PATIENTS AND METHODS: LPA species were measured in the baseline placebo sera of a COPD randomized controlled trial. Tertile levels of each LPA were used to assign patients into biomarker high, medium, and low subgroups. Exacerbation rate and risk were compared among the LPA subgroups. RESULTS: The levels of LPA species were intercorrelated (rho 0.29-0.91). Patients with low and medium levels of LPA (LPA16:0, 20:4) had significantly higher exacerbation rate compared to the respective LPA-high patients [estimated rate per patient per year (95% CI)]: LPA16:0-low = 1.2 (0.8-1.9) (p = 0.019), LPA16:0-medium = 1.3 (0.8-2.0) (p = 0.013), LPA16:0-high = 0.5 (0.2-0.9); LPA20:4-low = 1.4 (0.9-2.1) (p = 0.0033), LPA20:4-medium = 1.2 (0.8-1.8) (p = 0.0089), LPA20:4-high = 0.4 (0.2-0.8). These patients also had earlier time to first exacerbation (hazard ratio (95% CI): LPA16:0-low = 2.6 (1.1-6.0) (p = 0.028), LPA16:0-medium = 2.7 (1.2-6.3) (p = 0.020); LPA20.4-low = 2.8 (1.2-6.6) (p = 0.017), LPA20:4-medium = 2.7 (1.2-6.4) (p = 0.021). Accordingly, these patients had a significant increased exacerbation risk compared to the respective LPA-high subgroups [odd ratio (95% CI)]: LPA16:0-low = 3.1 (1.1-8.8) (p = 0.030), LPA16:0-medium = 3.0 (1.1-8.3) (p = 0.031); LPA20:4-low = 3.8 (1.3-10.9) (p = 0.012), LPA20:4-medium = 3.3 (1.2-9.5) (p = 0.025). For the other LPA species (LPA18:0, 18:1, 18:2), the results were mixed; patients with low and medium levels of LPA18:0 and 18:2 had increased exacerbation rate, but only LPA18:0-low patients had significant increase in exacerbation risk and earlier time to first exacerbation compared to the LPA18:0-high subgroup. CONCLUSIONS: The study provided evidence of association between systemic LPA levels and exacerbation in COPD. Patients with low and medium levels of specific LPA species (LPA16:0, 20:4) had increased exacerbation rate, risk, and earlier time to first exacerbation. These non-invasive biomarkers may aid in identifying high risk patients with dysregulated LPA pathway to inform risk management and drug development.

Serum Sphingosine-1-Phosphate Levels Are Associated With Severity and Outcome in Patients With Cerebral Ischemia.

BACKGROUND AND PURPOSE: The aim of this study was to examine whether sphingosine-1-phosphate (S1P) levels in patients with acute stroke are associated with stroke severity and outcome. METHODS: In a prospective stroke cohort (MARK-STROKE), 374 patients with acute ischemic stroke or transient ischemic attack were enrolled (mean age: 67.9±13.0 years, sex: 64.7% male), and serum-S1P at admission was analyzed with tandem mass spectrometry. In addition to cross-sectional analyses, 79 adverse events (death, stroke, myocardial infarction, rehospitalization) were recorded in 270 patients during follow-up. Regression analyses were adjusted for age, sex, low-density lipoprotein cholesterol, and vascular risk factors. Results were validated in an independent stroke cohort with 219 patients with acute ischemic stroke (CIRCULAS). RESULTS: Low serum-S1P was associated with higher National Institutes of Health Stroke Scale score at admission and with anterior circulation nonlacunar infarcts determined by multivariate regression analyses. During a follow-up of 294±170 days, patients with S1P in the lowest tertile (<1.33 µmol/L) had more adverse events (Kaplan-Meier analysis, P=0.048 for trend). In adjusted Cox regression analysis, the lowest S1P tertile was associated with a worse outcome after stroke (hazard ratio, HR 0.51 [95% confidence interval 0.28-0.92]). Results were confirmed in an independent cohort, ie, low S1P levels were associated with higher National Institutes of Health Stroke Scale, larger infarct volumes and worse outcome after 90 days (ß-coefficient: -0.03, P=0.026; ß-coefficient: -0.099, P=0.009 and odds ratio 0.52 [0.28-0.96], respectively). CONCLUSIONS: Our findings imply a detrimental role of low S1P levels in acute stroke and therefore underpin the therapeutic potential of S1P-mimics.

The lysophospholipid-binding molecule CD1D is not required for the alloimmunization response to fresh or stored RBCs in mice despite RBC storage driving alterations in lysophospholipids.

BACKGROUND: Despite the significant adverse clinical consequences of RBC alloimmunization, our understanding of the signals that induce immune responses to transfused RBCs remains incomplete. Though RBC storage has been shown to enhance alloimmunization in the hen egg lysozyme, ovalbumin, and human Duffy (HOD) RBC alloantigen mouse model, the molecular signals leading to immune activation in this system remain unclear. Given that the nonclassical major histocompatibility complex (MHC) Class I molecule CD1D can bind to multiple different lysophospholipids and direct immune activation, we hypothesized that storage of RBCs increases lysophospholipids known to bind CD1D, and further that recipient CD1D recognition of these altered lipids mediates storage-induced alloimmunization responses. STUDY DESIGN AND METHODS: We used a mass spectrometry-based approach to analyze the changes in lysophospholipids that are induced during storage of mouse RBCs. CD1D knockout (CD1D-KO) and wild-type (WT) control mice were transfused with stored HOD RBCs to measure the impact of CD1D deficiency on RBC alloimmunization. RESULTS: RBC storage results in alterations in multiple lysophospholipid species known to bind to CD1D and activate the immune system. Prior to transfusion, CD1D-deficient mice had lower baseline levels of polyclonal immunoglobulin (IgG) relative to WT mice. In response to stored RBC transfusion, CD1D-deficient mice generated similar levels of anti-HOD IgM and anti-HOD IgG. CONCLUSION: Although storage of RBCs leads to alteration of several lysophospholipids known to be capable of binding CD1D, storage-induced RBC alloimmunization responses are not impacted by recipient CD1D deficiency.