Quantification Of Ceramides Research Articles

Sphingolipid profiling reveals differential functions of sphingolipid biosynthesis isozymes of Caenorhabditis elegans

Multiple isozymes are encoded in the Caenorhabditis elegans genome for the various sphingolipid biosynthesis reactions, but the contributions of individual isozymes are characterized only in part. We developed a simple but effective reversed-phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS) method that enables simultaneous identification and quantification of ceramides (Cer), glucosylceramides (GlcCer), and sphingomyelins (SM) from the same MS run. Validating this sphingolipid profiling method, we show that nearly all 47 quantifiable sphingolipid species found in young adult worms were reduced upon RNA interference (RNAi) of sptl-1 or elo-5, which are both required for synthesis of the id17:1 sphingoid base. We also confirm that HYL-1 and HYL-2, but not LAGR-1, constitute the major ceramide synthase activity with different preference for fatty acid substrates, and that CGT-3, but not CGT-1 and CGT-2, plays a major role in producing GlcCers. Deletion of sms-5 hardly affected SM levels. RNAi of sms-1, sms-2, and sms-3 all lowered the abundance of certain SMs with an odd-numbered N-acyl chains (mostly C21 and C23, with or without hydroxylation). Unexpectedly, sms-2 RNAi and sms-3 RNAi elevated a subset of SM species containing even-numbered N-acyls. This suggests that sphingolipids containing even-numbered N-acyls could be regulated separately, sometimes in opposite directions, from those containing odd-numbered N-acyls, which are presumably monomethyl branched chain fatty acyls. We also find that ceramide levels are kept in balance with those of GlcCers and SMs. These findings underscore the effectiveness of this RPLC-MS/MS method in studies of C. elegans sphingolipid biology.

Hydrogen alleviates acute lung injury induced by limb ischaemia/reperfusion in mice

AimsLimb ischaemia/reperfusion (LIR) occurs in various clinical conditions including critical limb ischaemia, abdominal aortic aneurysm, and traumatic arterial injury. Reperfusion of the acutely ischemic limb can lead to a systemic inflammation response and multiple organ dysfunction syndrome, further resulting in significant morbidity and mortality. Molecular hydrogen exhibits therapeutic activity for the treatment and prevention of many diseases. Our study investigated the possible therapeutic effects of hydrogen and its mechanism of action in a LIR-induced acute lung injury (ALI) model. Materials and methodsLimb ischaemia/-reperfusion model was established in mice. The hydrogen-saturated saline was administered by intraperitoneal injection. Protein level of nuclear factor erythroid 2-related factor 2 (Nrf2), haem oxygenase-1 (HO1) and nicotinamide adenine dinucleotide phosphate quinone oxidoreductase 1 (NQO1) was evaluated by immunohistochemistry staining and western blotting. Autophagy-related molecules were evaluated by western blotting. Malondialdehyde (MDA) and superoxide dismutase (SOD) were determined by assay kits. Quantification of ceramides in lung was performed by high-performance liquid chromatography-tandem mass spectrometry. Key findingsMolecular hydrogen exhibited a protective effect on the LIR-induced ALI model. Hydrogen decreased malondialdehyde and increased superoxide dismutase activity in lung tissues. Additionally, hydrogen activated Nrf2 signalling in lung tissues. Hydrogen could inhibit the upregulation of autophagy in the present rodent model. Furthermore, ceramide was accumulated in lung tissues because of LIR; however, hydrogen altered the accumulation status. SignificanceMolecular hydrogen was found to be therapeutically effective in the LIR-induced ALI model; the mechanisms of action included modulation of antioxidation and autophagy.

Interferon-Beta Increases Plasma Ceramides of Specific Chain Length in Multiple Sclerosis Patients, Unlike Fingolimod or Natalizumab.

Fingolimod is used for the treatment of multiple sclerosis (MS) and targets receptors for the bioactive sphingolipid sphingosine-1-phosphate (S1P). Whether fingolimod or other MS therapies conversely affect plasma concentrations of sphingolipids has, however, not yet been analyzed. Herein, we quantified 15 representative sphingolipid species by mass spectrometry in plasma from relapsing-remitting MS patients currently under fingolimod (n = 24), natalizumab (n = 16), or IFN-β (n = 18) treatment. Healthy controls (n = 21) and untreated MS patients (n = 11) served as control groups. IFN-ß treatment strongly increased plasma level of C16:0, C18:0, C20:0, and C24:1 ceramides compared to healthy controls, untreated patients, or patients receiving fingolimod or natalizumab medication. Natalizumab treatment increased plasma concentrations of both S1P and sphinganine-1-phosphate, whereas fingolimod treatment did not affect any of these lipids. Correlations of sphingolipids with the Expanded Disability Status Scale and other disease specific parameters revealed no systemic change of sphingolipids in MS, independent of the respective treatment regime. These results indicate type I interferon treatment to cause a strong and specific increase in ceramide level. If confirmed in larger cohorts, these data have implications for the efficacy and adverse effects of IFN-β. Moreover, quantification of ceramides soon after therapy initiation may help to identify therapy-responsive patients.

Metabolic profiling and quantification of ceramide by liquid chromatography mass spectrometry

ObjectiveCeramides are putative mediators of insulin resistance and lipotoxicity; an up‐regulation of ceramide biosynthesis has been well described in rodent models of metabolic disease. A precise, robust and specific LC/MS method was developed to quantify ceramide profiles and support determinations of ceramide flux. The LC/MS method was used to determine the effects of diet‐induced obesity (DIO) in C57B1/6J mice ±treatment with myriocin (1mg/kg, over 7 days), an inhibitor of ceramide synthesis.MethodPlasma and tissue samples were collected from mice (lean, high‐fat fed ± myriocin) at various times following administration of stable isotope tracers. The ceramides were extracted with organic solvents and analyzed using electrospray ionization tandem mass spectrometry; concentrations were determined by normalization to an internal standard (d31‐c16:0 ceramide).ResultsIn contrast to lean mice, DIO‐mice demonstrated an increased concentration and synthesis of circulating ceramides. Myriocin treatment inhibited ceramide synthesis and led to a normalization of circulating levels.ConclusionLC/MS analyses can enable characterization of circulating concentrations of ceramide subspecies and enable stable isotope determinations of in vivo kinetics.Support or Funding InformationAll authors are employed by Merck.

Inhibition of human tumour prostate PC-3 cell growth by cannabinoids R(+)-Methanandamide and JWH-015: Involvement of CB2

Background:We have previously shown that cannabinoids induce growth inhibition and apoptosis in prostate cancer PC-3 cells, which express high levels of cannabinoid receptor types 1 and 2 (CB1 and CB2). In this study, we investigated the role of CB2 receptor in the anti-proliferative action of cannabinoids and the signal transduction triggered by receptor ligation.Methods:The human prostate cancer cell lines, namely PC-3, DU-145 and LNCaP, were used for this study. Cell proliferation was measured using MTT proliferation assay, [3H]-thymidine incorporation assay and cell-cycle study by flow cytometry. Ceramide quantification was performed using the DAG kinase method. The CB2 receptor was silenced with specific small interfering RNA, and was blocked pharmacologically with SR 144528. In vivo studies were conducted by the induction of prostate xenograft tumours in nude mice.Results:We found that the anandamide analogue, R(+)-Methanandamide (MET), as well as JWH-015, a synthetic CB2 agonist, exerted anti-proliferative effects in PC-3 cells. R(+)-Methanandamide- and JWH-015-induced cell death was rescued by treatment with the CB2 receptor antagonist, SR 144528. Downregulation of CB2 expression reversed the effects of JWH-015, confirming the involvement of CB2 in the pro-apoptotic effect of cannabinoids. Further analysing the mechanism of JWH-015-induced cell growth inhibition, we found that JWH-015 triggered a de novo synthesis of ceramide, which was involved in cannabinoid-induced cell death, insofar as blocking ceramide synthesis with Fumonisin B1 reduced cell death. Signalling pathways activated by JWH-015 included JNK (c-Jun N-terminal kinase) activation and Akt inhibition. In vivo treatment with JWH-015 caused a significant reduction in tumour growth in mice.Conclusions:This study defines the involvement of CB2-mediated signalling in the in vivo and in vitro growth inhibition of prostate cancer cells and suggests that CB2 agonists have potential therapeutic interest and deserve to be explored in the management of prostate cancer.

Characterization and quantification of ceramides in the nonlesional skin of canine patients with atopic dermatitis compared with controls

As in humans, there is mounting evidence in support of an abnormal skin barrier contributing to the pathogenesis of canine atopic dermatitis (AD). Studies in people with AD have associated an abnormal skin barrier with deficiencies in ceramides, which represent important components of the stratum corneum (SC) intercellular lipid lamellae. Therefore, the goal of this study was to determine if the SC of dogs with AD is deficient in ceramides compared to normal dogs. Samples of SC were obtained from nonlesional skin of the caudal abdomen of 14 patients with AD and 14 age-, breed- and sex-matched healthy controls using a cyanoacrylate stripping procedure, and the subclass and relative amount of ceramides were assessed blindly by thin layer chromatography. Paired t-tests using R statistical computer software revealed the percentage amounts of ceramides 1 and 9 were significantly lower in nonlesional skin of AD dogs compared to controls (P= 0.034 and P= 0.047, respectively), and the cholesterol percentage amount was significantly higher in AD dogs than in controls (P= 0.016). Furthermore, the cholesterol/ceramide ratio was significantly higher in the AD group with respect to controls (P= 0.014). These findings suggest that decreased amounts of ceramides in the skin of dogs with AD may be involved in the impaired barrier function of their skin.

Induction of apoptosis by novel synthesized acylamides of human lymphocytes

To investigate a pathway to apoptosis which may involve ceramides and to elucidate the minimum structure which leads to apoptosis, we synthesized several novel acylamides. Although the four synthesized compounds were different in structure from C2-ceramide, they caused Jurkat cells to undergo apoptosis. The most effective of them was N-myristoyl- d-alaninol ( d-MA), as shown by DNA fragmentation (detected with propidium iodide) and a decrease in the mitochondrial transmembrane potential (ΔΨ m) (detected with rhodamine 123). Nevertheless, peripheral blood leukocytes exhibited no change after d-MA exposure, like after C2-ceramide or anti-Fas antibody treatment. The DNA fragmentation and ΔΨ m caused by d-MA were blocked by a caspase-3 specific inhibitor as in the case of anti-Fas antibody stimulation. Quantification of ceramides by metabolic labeling with [ 14C]palmitic acid and HPTLC showed no increases in the ceramide levels on stimulation with d-MA, C2-ceramide or anti-Fas antibodies. Furthermore, d-MA had an apoptosis-inducing effect on an anti-Fas-resistant subline of Jurkat cells. These data suggest that d-MA may cause apoptosis of Jurkat cells without distinct ceramide formation and that this apoptotic pathway is very comparable, i.e. not identical, to that induced by anti-Fas antibodies.

Identification of ceramides in human cells using liquid chromatography with detection by atmospheric pressure chemical ionization-mass spectrometry.

Ceramides are intermediates in the biosynthesis of membrane sphingolipids. These biomolecules are also important as second messengers in signal transduction pathways controlling cell growth. We have developed two reversed-phase high pressure liquid chromatography (RPHPLC) techniques for identification and quantification of ceramides from mammalian cells. One method was based on atmospheric pressure chemical ionization-mass spectrometry (APCI-MS) detection of ceramides and had the advantage of requiring minimal sample preparation, yielding significant structural information, and affording high sensitivity. The second method relied on perbenzoylation of the ceramides and detection at 230 nm. The predominant ceramides detected in the human leukemic HL-60 cell were N-(palmitoyl)-sphingosine, N-(nervonyl)-sphingosine, and N-(lignoceroyl)-sphingosine. When selected ion monitoring was used with RPHPLC/APCI-MS, approximately 2.2 pmol N-(palmitoyl)-sphingosine and 1.7 pmol N-(nervonyl)-sphingosine were observed in an extract from 40,000 HL-60 cells. Perbenzoylation with benzoyl chloride permitted RPHPLC separation and 230 nm UV absorbance detection of the trisbenzoyl derivatives of sphingosine, N-(palmitoyl)-sphingosine, N-(nervonyl)-sphingosine, and N-(lignoceroyl)-sphingosine in the HL-60 cells. These results demonstrate the utility of utilizing two different methods coupled with APCI-MS for the quantification and identification of ceramides in biological samples.