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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.

The interest in neonatal screening for lysosomal storage disorders has increased substantially because of newly developed enzyme replacement therapies, the need for early diagnosis, and technical advances. We tested for Gaucher's disease, Pompe's disease, Fabry's disease, and Niemann-Pick disease types A and B in an anonymous prospective nationwide screening study that included genetic mutation analysis to assess the practicality and appropriateness of including these disorders in neonatal screening panels. Specimens from dried blood spots of 34 736 newborn babies were collected consecutively from January, 2010 to July, 2010, as part of the national routine Austrian newborn screening programme. Anonymised samples were analysed for enzyme activities of acid β-glucocerebrosidase, α-galactosidase, α-glucosidase, and acid sphingomyelinase by electrospray ionisation tandem mass spectrometry. Genetic mutation analyses were done in samples with suspected enzyme deficiency. All 34 736 samples were analysed successfully by the multiplex screening assay. Low enzyme activities were detected in 38 babies. Mutation analysis confirmed lysosomal storage disorders in 15 of them. The most frequent mutations were found for Fabry's disease (1 per 3859 births), followed by Pompe's disease (1 per 8684), and Gaucher's disease (1 per 17 368). The positive predictive values were 32% (95% CI 16–52), 80% (28–99), and 50% (7–93), respectively. Mutational analysis detected predominantly missense mutations associated with a late-onset phenotype. The combined overall proportion of infants carrying a mutation for lysosomal storage disorders was higher than expected. Neonatal screening for lysosomal storage disorders is likely to raise challenges for primary health-care providers. Furthermore, the high frequency of late-onset mutations makes lysosomal storage disorders a broad health problem beyond childhood. Austrian Ministry of Health, Family, and Women.

Effective treatment strategies for severe coronavirus disease (COVID-19) remain scarce. Hydrolysis of membrane-embedded, inert sphingomyelin by stress responsive sphingomyelinases is a hallmark of adaptive responses and cellular repair. As demonstrated in experimental and observational clinical studies, the transient and stress-triggered release of a sphingomyelinase, SMPD1, into circulation and subsequent ceramide generation provides a promising target for FDA-approved drugs. Here, we report the activation of sphingomyelinase-ceramide pathway in 23 intensive care patients with severe COVID-19. We observed an increase of circulating activity of sphingomyelinase with subsequent derangement of sphingolipids in serum lipoproteins and from red blood cells (RBC). Consistent with increased ceramide levels derived from the inert membrane constituent sphingomyelin, increased activity of acid sphingomyelinase (ASM) accurately distinguished the patient cohort undergoing intensive care from healthy controls. Positive correlational analyses with biomarkers of severe clinical phenotype support the concept of an essential pathophysiological role of ASM in the course of SARS-CoV-2 infection as well as of a promising role for functional inhibition with anti-inflammatory agents in SARS-CoV-2 infection as also proposed in independent observational studies. We conclude that large-sized multicenter, interventional trials are now needed to evaluate the potential benefit of functional inhibition of this sphingomyelinase in critically ill patients with COVID-19.

The sphingolipidoses Fabry disease, Gaucher disease and Acid sphingomyelinase deficiency (ASMD) are the three most common lysosomal storage diseases for which treatment is currently available. Timely diagnosis with estimation of the disease severity and possibilities of follow-up of patients, whether or not under therapy, is crucial for providing good care and for the prevention of possible lethal complications. With this research we provide an efficient and sensitive detection method; its implementation in clinical practice could optimize the diagnosis and follow-up of patients with Gaucher, Fabry and ASMD. This detection method on dried blood spots (DBS) was validated according to the international Clinical and Laboratory Standards Institute (CLSI) guidelines, looking at reproducibility, linearity, carry-over and lower limit of quantification. Analogously, validation and subsequent comparison of the method validation results using another matrix, the Capitainer blood sampling cards (Capitainers), was fulfilled. The results showed that this detection method is fully applicable clinically when using DBS as well as Capitainers. In addition, even additional improvements of some validation parameters were found when using the Capitainers. Twenty-six patient samples and fifteen healthy samples were analyzed for case finding control. All patient cases were detected without ambiguity. We present a high-resolution mass spectrometry method that provides an accurate analysis for targeted screening, aiming for improved/accelerated diagnosis when added in the diagnostic pathway and monitoring of Fabry, Gaucher and ASMD in DBS as well as in Capitainers, with the main advantages of a small volume of blood samples, guaranteeing stability and easy transportation from the collection site to the laboratory.

Wilson disease is caused by accumulation of Cu 2+ in cells, which results in liver cirrhosis and, occasionally, anemia. Here, we show that Cu 2+ triggers hepatocyte apoptosis through activation of acid sphingomyelinase (Asm) and release of ceramide. Genetic deficiency or pharmacological inhibition of Asm prevented Cu 2+ -induced hepatocyte apoptosis and protected rats, genetically prone to develop Wilson disease, from acute hepatocyte death, liver failure and early death. Cu 2+ induced the secretion of activated Asm from leukocytes, leading to ceramide release in and phosphatidylserine exposure on erythrocytes, events also prevented by inhibition of Asm. Phosphatidylserine exposure resulted in immediate clearance of affected erythrocytes from the blood in mice. Accordingly, individuals with Wilson disease showed elevated plasma levels of Asm, and displayed a constitutive increase of ceramide- and phosphatidylserine-positive erythrocytes. Our data suggest a previously unidentified mechanism for liver cirrhosis and anemia in Wilson disease.

The activity of acid sphingomyelinase (ASMase), a key enzyme in sphingolipid metabolism, has been found to be increased in a variety of human diseases. Studies conducted on animal and cellular models showed that sphingolipids and ASMase play a central role in the pathogenesis of type 1 diabetes (T1D) and T1D-related vascular damage. Currently, no studies have investigated the role of ASMase activity in pediatric patients with T1D. Therefore, we conducted a cross-sectional study to evaluate the activity of the secretory form of ASMase (S-ASMase) in the serum of patients with T1D aged 2-16 years in comparison with a control group (healthy subjects matched for age, gender, and pubertal stage). We recruited children and adolescents affected by T1D (including patients with new-onset and established T1D) aged 2-16 years and healthy normal-weight subjects with normal timing of puberty (matched for age, gender, and pubertal stage), who were consecutively admitted-as outpatients-to our institution for screening purposes. Serum lipid profile, glycated hemoglobin (HbA1c), and urine albumin-creatinine ratio (uACR) were assessed in all T1D patients. S-ASMase activity was measured in all study participants through a colorimetric assay. In total, 68 T1D patients and 51 healthy controls were recruited in this study. None of the T1D patients had T1D-related complications. No difference in S-ASMase activity was observed between subjects with T1D and healthy controls. However, when T1D patients were stratified according to the duration of diabetes, we found a significantly higher activity of S-ASMase in patients with new-onset T1D (recruited within 1 week after the disease diagnosis) as compared to that observed in patients with established T1D. In all patients with T1D, S-ASMase activity correlated positively with HbA1c and triglyceride levels, while it correlated negatively with total cholesterol (TC) and high-density lipoprotein cholesterol (HDL-C) levels. However, there were no significant differences in S-ASMase activity between T1D patients with new-onset disease who presented with diabetic ketoacidosis (DKA; n = 12) and T1D patients with new-onset disease who did not present with DKA (n = 13). Our study evaluated, for the first time, the in vivo activity of S-ASMase in a pediatric cohort of patients with T1D. In pediatric patients with new-onset T1D, we found a significantly higher S-ASMase activity as compared to that observed in patients with established T1D. In all T1D patients, the positive correlation between S-ASMase activity, HbA1c, and triglyceride levels, as well as the negative correlation between S-ASMase activity and HDL-C levels, suggests a potential role played by sphingolipids in T1D pathophysiology. Further mechanistic studies are needed to better elucidate the role of S-ASMase in patients with T1D at different stages of the disease.

Extracellular vesicles (EVs), including exosomes and microvesicles, function in cell-to-cell communication through delivery of proteins, lipids and microRNAs to target cells via endocytosis and membrane fusion. These vesicles are enriched in ceramide, a sphingolipid associated with the promotion of cell senescence and apoptosis. We investigated the ceramide profile of serum exosomes from young (24–40 yrs.) and older (75–90 yrs.) women and young (6–10 yrs.) and older (25–30 yrs.) rhesus macaques to define the role of circulating ceramides in the aging process. EVs were isolated using size-exclusion chromatography. Proteomic analysis was used to validate known exosome markers from Exocarta and nanoparticle tracking analysis used to characterize particle size and concentration. Specific ceramide species were identified with lipidomic analysis. Results show a significant increase in the average amount of C24:1 ceramide in EVs from older women (15.4 pmol/sample) compared to those from younger women (3.8 pmol/sample). Results were similar in non-human primate serum samples with increased amounts of C24:1 ceramide (9.3 pmol/sample) in older monkeys compared to the younger monkeys (1.8 pmol/sample). In vitro studies showed that primary bone-derived mesenchymal stem cells (BMSCs) readily endocytose serum EVs, and serum EVs loaded with C24:1 ceramide can induce BMSC senescence. Elevated ceramide levels have been associated with poor cardiovascular health and memory impairment in older adults. Our data suggest that circulating EVs carrying C24:1 ceramide may contribute directly to cell non-autonomous aging.

Acid sphingomyelinase (ASM) and acid β-glucosidase 1 (GBA1) catalyze ceramide formation through different routes, and both are involved in rheumatoid arthritis (RA) pathogenesis as well as IL-6 production. However, whether ASM and GBA1 regulate IL-6 production in RA remains unknown. Serum ASM, GBA1, and ceramide levels were measured in RA patients and healthy controls by enzyme-linked immunosorbent assay, and their correlations with clinical indicators of patients were evaluated. Pharmacologic inhibitors or small hairpin RNAs of ASM and GBA1 were employed to explore the roles of ASM and GBA1 in IL-6 production, cell behavior, and MAPK signaling in fibroblast-like synoviocytes from RA patients (RAFLS). ASM, GBA1, and ceramide serum levels were significantly elevated in patients with RA. GBA1 and ceramide serum levels were negatively and positively correlated with IL-6 serum level in RA patients, respectively. ASM inhibitor or knockdown of ASM abolished IL-1β-induced IL-6 expression and secretion. Functionally, ASM inhibitor suppressed IL-1β-induced cell proliferation, migration, and invasion in RAFLS. Mechanistically, ASM inhibitor or knockdown of ASM effectively countered IL-1β-induced activation of p38 MAPK signaling. The pharmacologic inhibitor or knockdown of GBA1 exhibited the opposite effects. Importantly, p38 inhibitor blocked IL-1β-induced IL-6 production in RAFLS. ASM plays a pathogenic role in RA, whereas GBA1 plays a protective role in RA possibly by regulating IL-6 production in RAFLS at least partially via p38 signaling, serving as potential therapeutic targets in RA treatment.

Background: Newborn screening for deficiency in the lysosomal enzymes that cause Fabry, Gaucher, Krabbe, Niemann–Pick A/B, and Pompe diseases is warranted because treatment for these syndromes is now available or anticipated in the near feature. We describe a multiplex screening method for all five lysosomal enzymes that uses newborn-screening cards containing dried blood spots as the enzyme source. Methods: We used a cassette of substrates and internal standards to directly quantify the enzymatic activities, and tandem mass spectrometry for enzymatic product detection. Rehydrated dried blood spots were incubated with the enzyme substrates. We used liquid-liquid extraction followed by solid-phase extraction with silica gel to remove buffer components. Acarbose served as inhibitor of an interfering acid α-glucosidase present in neutrophils, which allowed the lysosomal enzyme implicated in Pompe disease to be selectively analyzed. Results: We analyzed dried blood spots from 5 patients with Gaucher, 5 with Niemann–Pick A/B, 11 with Pompe, 5 with Fabry, and 12 with Krabbe disease, and in all cases the enzyme activities were below the minimum activities measured in a collection of heterozygous carriers and healthy noncarrier individuals. The enzyme activities measured in 5–9 heterozygous carriers were approximately one-half those measured with 15–32 healthy individuals, but there was partial overlap of each condition between the data sets for carriers and healthy individuals. Conclusion: For all five diseases, the affected individuals were detected. The assay can be readily automated, and the anticipated reagent and supply costs are well within the budget limits of newborn-screening centers.

Acid sphingomyelinase is involved in lipid signalling pathways and regulation of apoptosis by the generation of ceramide and plays an important role during the host response to infectious stimuli. It thus has the potential to be used as a novel diagnostic marker in the management of critically ill patients. The objective of our study was to evaluate acid sphingomyelinase serum activity (ASM) as a diagnostic and prognostic marker in a mixed intensive care unit population before, during, and after systemic inflammation. 40 patients admitted to the intensive care unit at risk for developing systemic inflammation (defined as systemic inflammatory response syndrome plus a significant procalcitonin [PCT] increase) were included. ASM was analysed on ICU admission, before (PCT before), during (PCT peak) and after (PCT low) onset of SIRS. Patients undergoing elective surgery served as control (N = 8). Receiver-operating characteristics curves were computed. ASM significantly increased after surgery in the eight control patients. Patients from the intensive care unit had significantly higher ASM on admission than control patients after surgery. 19 out of 40 patients admitted to the intensive care unit developed systemic inflammation and 21 did not, with no differences in ASM between these two groups on admission. In patients with SIRS and PCT peak, ASM between admission and PCT before was not different, but further increased at PCT peak in non-survivors and was significantly higher at PCT low compared to survivors. Survivors exhibited decreased ASM at PCT peak and PCT low. Receiver operating curve analysis on discrimination of ICU mortality showed an area under the curve of 0.79 for ASM at PCT low. In summary, ASM was generally higher in patients admitted to the intensive care unit compared to patients undergoing uncomplicated surgery. ASM did not indicate onset of systemic inflammation. In contrast to PCT however, it remained high in non-surviving ICU patients after systemic inflammation.