Dihydrosphingomyelin Research Articles

Abstract 2699: FF-10850, a dihydrosphingomyelin (DHSM)-based novel liposomal topotecan, achieved favorable efficacy and safety in combination with platinum, PARP and immune checkpoint inhibitors

Abstract Introduction: Topotecan is approved as a monotherapy for ovarian and small cell lung cancers. Although topotecan is also approved in combination with cisplatin for cervical cancer, reductions of dose and administration frequency are required due to safety issues. Similar observations have been reported in combination with other anti-cancer agents. We previously reported on a dihydrosphingomyelin (DHSM)-based novel liposomal topotecan, FF-10850 which has been in a Phase I clinical study for solid tumors including ovarian cancer and Merkel cell carcinoma (NCT04047251). FF-10850 demonstrated superior efficacy and safety to non-liposomal topotecan via preferential tumor accumulation and efficient payload release mediated by both tumor-associated macrophages and ammonia produced by glutaminolysis in the tumor microenvironment. In the present study, we examined if FF-10850 could enhance efficacy in combination with platinum, PARP, and immune checkpoint inhibitors in mice models, leveraging its preferential accumulation in the tumor microenvironment. Methods: FF-10850 in combination with carboplatin or olaparib were evaluated in a platinum-sensitive A2780 and a BRCA2-deficient Capan-1 xenograft model, respectively. A CT26 syngeneic model was used for evaluation of FF-10850 in combination with anti-PD-1 antibody. To confirm establishment of tumor-specific immunity with the FF-10850 and anti-PD-1 antibody combination, re-inoculation of CT26 cells and subsequent inoculation of 4T1 cells was performed in mice that achieved durable complete regressions with the prior combination treatment. Results: FF-10850 in combination with carboplatin or olaparib significantly improved efficacy compared to each monotherapy without increased body weight loss. Olaparib also attenuated tumor regrowth in a maintenance setting after the completion of FF-10850 monotherapy. Although only 1 or 0 of 8 mice showed complete regression with FF-10850 or anti-PD-1 antibody monotherapy, 6 of 8 mice achieved durable complete regressions with the combination treatment. Furthermore, these mice successfully rejected re-inoculated CT26 cells without subsequent rejection of irrelevant 4T1 cells. Conclusion: FF-10850 demonstrated favorable efficacy and safety in combination with carboplatin, olaparib or anti-PD-1 antibody, likely related to preferential tumor drug exposure. The combination of FF-10850 with anti-PD-1 antibody efficiently established tumor-specific immunity and long-lasting tumor suppression. Collectively, these observations strongly support further investigations of FF-10850 and improved efficacy in combination with various anti-cancer agents. Citation Format: Ken Okada, Toshifumi Kimura, Noriyuki Kasagi, Mikinaga Mori, Susumu Shimoyama. FF-10850, a dihydrosphingomyelin (DHSM)-based novel liposomal topotecan, achieved favorable efficacy and safety in combination with platinum, PARP and immune checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2699.

Optimization of dihydrosphingomyelin/cholesterol mol ratio in topotecan-loaded liposomes to enhance drug retention and plasma half-life by understanding physicochemical and thermodynamic properties of the lipid membrane

Robust retention of drugs in liposomes is one of the most critical factors for liposomal drug formulation. It improves loading efficiency of the active pharmaceutical ingredient (API), stability during long-term storage, and half-life of the drug in the body. This study deductively optimized the lipid composition of topotecan-loaded liposome using dihydrosphingomyelin (DHSM), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (DSPE PEG) for intravenous injection by thermodynamic and fluidity analyses of the liposome membrane. High efficiency in topotecan loading was achieved in the range of the liquid-ordered phase (egg-derived DHSM/Chol molar ratio 70/25% or a higher Chol ratio with PEG constant at 5 mol%) as predicted by thermodynamic analysis. The egg-derived DHSM/Chol/PEG mol ratio that minimizes topotecan leakage was estimated to be 57/38/5 via electron spin resonance (ESR) for fluidity analysis. The optimized liposome significantly prolonged the half-life of topotecan in blood compared to non-liposomal topotecan. To minimize the risk of virus contamination and ensure easier quality control for clinical application, synthetic DHSM was used in the optimized formulation of topotecan encapsulated liposome instead of egg-derived DHSM (named FF-10850). The physicochemical and pharmacokinetic profiles of FF-10850 were equivalent to those of the liposome prepared using egg-derived DHSM and >99% drug retention was maintained for over 24 months under refrigeration, indicating stable encapsulation and prolonged blood circulation.

PMON209 Ceramides Predict Metabolic Response to Metformin in Women with PCOS

Abstract Polycystic ovarian syndrome (PCOS) is a complex disorder in which metabolic abnormalities are associated with reproductive dysfunction. Women with PCOS have been shown to have increased ceramide concentrations with the obese, insulin resistant phenotype having even higher levels. Ceramides modulate insulin signaling, promoting insulin resistance, and are markers of diabetes risk and cardiovascular mortality. Previous studies demonstrate that treating metabolic abnormalities of PCOS with metformin improved glucose effectiveness, caused weight loss, and restored ovulation in 61% of women after 12 weeks. However, these metabolic improvements did not explain improved ovulation experienced by some women. Hypothesis We hypothesized that in women with PCOS, baseline ceramide, diacylglycerol (DAG) and triacylyglycerol (TAG) concentrations are associated with an improved metabolic response to metformin.Subjects: Women (n=29), aged 29±5 years and diagnosed with PCOS by the NIH criteria were studied. Methods Individuals underwent an intravenous glucose tolerance test (IVGTT) and hCG-stimulation before and after 12-weeks of treatment with metformin (1500 mg per day). Metabolic responders were defined by improved glucose effectiveness after metformin treatment. Results Among predominantly white women with an average body mass index 29.4±6.1 kg/m2, 12-weeks of metformin resulted in weight loss (-1.7±2.6 kg, p<0.01) and a reduction in BMI (-0.6±0.9 kg/m2, p<0.01) with no change in HbA1c (5.5%±0.3 vs 5.5%±0.3, p=0.7). The concentrations of ceramides with acyl chain length at 22 and 24 (C22 and C24), total ceramides, dihydroceramides (C16: 0), C18: 2, DAG, dihydrosphingomyelin (DHSM), and TAG decreased after metformin treatment (p<0.05). Baseline total dihydroceramides (C16: 0) and DHSM were lower in metabolic responders compared to metabolic non-responders (C16: 0 172±11 vs 232±14 pmol/ml, p<0.01; DHSM 36650±2642 vs 48842±3379 pmol/ml, p<0.01). Total C16: 0 concentrations of <204 pmol/ml was 83% sensitive (AUC 0.79, p<0.01) and total DHSM concentrations of <51705 pmol/ml was 100% sensitive (AUC 0.73, p=0.02) in predicting improved metabolic response to metformin, as measured by IVGTT. After adjusting for multiple comparisons, baseline DAG and TAG concentrations were associated with baseline ß-cell function (DAG r2=0.47, p<0.01; TAG r2=0.32, p<0.01), basal insulin concentrations (DAG r2=0.48, p<0.01; TAG r2=0.33, p<0.01) and decreased insulin sensitivity (DAG r2= 0.45, p<0.01; TAG r2=0.3,p<0.01). Baseline ceramide concentrations were not associated with improved ovulation. Conclusion Ceramide concentrations predict metabolic disruption and lower levels are associated with a beneficial metabolic response to metformin in women with PCOS. Based on the known association between higher ceramide levels and type 2 diabetes, the data suggest that metformin improves metabolic parameters in women with mild metabolic derangements but not in those with severe insulin resistance. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

The Use of Ceramides to Predict Metabolic Response to Metformin in Women With PCOS.

ContextPolycystic ovarian syndrome (PCOS) is a complex disorder in which metabolic abnormalities are associated with reproductive dysfunction. Women with PCOS have increased ceramide concentrations. Previous studies demonstrated that treating metabolic abnormalities of PCOS with metformin improved glucose effectiveness after 12 weeks.ObjectiveWe evaluated whether, in women with PCOS, lower baseline ceramide, diacylglycerol (DAG), and triacylglycerol (TAG) concentrations were associated with improved metabolic response to metformin.MethodsWomen (n = 29), aged 29 ± 5 years and diagnosed with PCOS by the NIH criteria underwent an intravenous glucose tolerance test (IVGTT) before and after 12-week treatment with metformin (1500 mg per day). Metabolic responders were defined by improved glucose effectiveness, specifically, the ability of glucose to stimulate uptake and suppress production, after metformin treatment.ResultsTwelve weeks of metformin resulted in weight loss (−1.7 ± 2.6 kg, P < 0.01) and a reduction in BMI (−0.6 ± 0.9 kg/m2, P < 0.01) with no change in HbA1c. The concentrations of Cer(d18:1/22:0), Cer(d18:1/24:0), total ceramides, total Cer(d16:0), total Cer(d18:2), DAG, dihydrosphingomyelin (DHSM), and TAG decreased after metformin treatment (P < 0.05). Baseline total Cer(d16:0) concentration <204.1 pmol/mL was 82% sensitive (AUC 0.72, P = 0.03) and total DHSM concentration <32237 pmol/mL was 100% specific (AUC 0.73, P = 0.03) in predicting improved metabolic response to metformin, as measured by IVGTT.ConclusionLower total Cer(16:0) and DHSM concentrations are associated with a beneficial metabolic response to metformin in women with PCOS. Based on the known association between higher ceramide levels and type 2 diabetes, the data suggest that metformin improves metabolic parameters in women with mild metabolic derangements.

Alterations of Sphingolipid and Phospholipid Pathways and Ornithine Level in the Plasma as Biomarkers of Parkinson's Disease.

The biomarkers of Parkinson’s disease (PD) remain to be investigated. This work aimed to identify blood biomarkers for PD using targeted metabolomics analysis. We quantified the plasma levels of 255 metabolites in 92 PD patients and 60 healthy controls (HC). PD patients were sub-grouped into early (Hoehn–Yahr stage ≤ 2, n = 72) and advanced (Hoehn–Yahr stage > 2, n = 20) stages. Fifty-nine phospholipids, 3 fatty acids, 3 amino acids, and 7 biogenic amines, demonstrated significant alterations in PD patients. Six of them, dihydro sphingomyelin (SM) 24:0, 22:0, 20:0, phosphatidylethanolamine-plasmalogen (PEp) 38:6, and phosphatidylcholine 38:5 and 36:6, demonstrated lowest levels in PD patients in the advanced stage, followed by those in the early stage and HC. By contrast, the level of ornithine was highest in PD patients at the advanced stage, followed by those at the early stage and HC. These biomarker candidates demonstrated significant correlations with scores of motor disability, cognitive dysfunction, depression, and quality of daily life. The support vector machine algorithm using α-synuclein, dihydro SM 24:0, and PEp 38:6 demonstrated good ability to separate PD from HC (AUC: 0.820). This metabolomic analysis demonstrates new plasma biomarker candidates for PD and supports their role in participating PD pathogenesis and monitoring disease progression.

Sphingolipid Profiling Reveals Different Extent of Ceramide Accumulation in Bovine Retroperitoneal and Subcutaneous Adipose Tissues.

Sphingolipids are bioactive lipids that can modulate insulin sensitivity, cellular differentiation, and apoptosis in a tissue-specific manner. However, their comparative profiles in bovine retroperitoneal (RPAT) and subcutaneous adipose tissue (SCAT) are currently unknown. We aimed to characterize the sphingolipid profiles using a targeted lipidomics approach and to assess whether potentially related sphingolipid pathways are different between SCAT and RPAT. Holstein bulls (n = 6) were slaughtered, and SCAT and RPAT samples were collected for sphingolipid profiling. A total of 70 sphingolipid species were detected and quantified by UPLC-MS/MS in multiple reaction monitoring (MRM) mode, including ceramide (Cer), dihydroceramide (DHCer), sphingomyelin (SM), dihydrosphingomyelin (DHSM), ceramide-1-phosphate (C1P), sphingosine-1-phosphate (S1P), galactosylceramide (GalCer), glucosylceramide (GluCer), lactosylceramide (LacCer), sphinganine (DHSph), and sphingosine (Sph). Our results showed that sphingolipids of the de novo synthesis pathway, such as DHSph, DHCer, and Cer, were more concentrated in RPAT than in SCAT. Sphingolipids of the salvage pathway and the sphingomyelinase pathway, such as Sph, S1P, C1P, glycosphingolipid, and SM, were more concentrated in SCAT. Our results indicate that RPAT had a greater extent of ceramide accumulation, thereby increasing the concentration of further sphingolipid intermediates in the de novo synthesis pathway. This distinctive sphingolipid distribution pattern in RPAT and SCAT can potentially explain the tissue-specific activity in insulin sensitivity, proinflammation, and oxidative stress in RPAT and SCAT.

Phosphatidylcholine bearing 6,6-dideuterated oleic acid: A useful solid-state 2H NMR probe for investigating membrane properties

Lipid organization has been at the center of research on lipid rafts. Dioleoylphosphatidylcholine (DOPC) is a typical unsaturated lipid. Very few studies have reported its thermodynamics in raft-like membranes. Herein, we have developed a highly efficient synthetic method for [C6-2H2] oleic acid, and newly synthesized [C6-2H2] DOPC. In raft-like oriented bilayers, [C6-2H2] DOPC shows clear phase separation and characteristic phase behavior at various temperature. It has been successfully utilized for the comparison of membrane properties between sphingomyelin (SM) and dihydrosphingomyelin (DHSM) membranes.

Early and late HIV-1 membrane fusion events are impaired by sphinganine lipidated peptides that target the fusion site.

Lipid-conjugated peptides have advanced the understanding of membrane protein functions and the roles of lipids in the membrane milieu. These lipopeptides modulate various biological systems such as viral fusion. A single function has been suggested for the lipid, binding to the membrane and thus elevating the local concentration of the peptide at the target site. In the present paper, we challenged this argument by exploring in-depth the antiviral mechanism of lipopeptides, which comprise sphinganine, the lipid backbone of DHSM (dihydrosphingomyelin), and an HIV-1 envelope-derived peptide. Surprisingly, we discovered a partnership between the lipid and the peptide that impaired early membrane fusion events by reducing CD4 receptor lateral diffusion and HIV-1 fusion peptide-mediated lipid mixing. Moreover, only the joint function of sphinganine and its conjugate peptide disrupted HIV-1 fusion protein assembly and folding at the later fusion steps. Via imaging techniques we revealed for the first time the direct localization of these lipopeptides to the virus–cell and cell–cell contact sites. Overall, the findings of the present study may suggest lipid–protein interactions in various biological systems and may help uncover a role for elevated DHSM in HIV-1 and its target cell membranes.

A novel three serum phospholipid panel differentiates normal individuals from those with prostate cancer.

BackgroundThe results of prostate specific antigen (PSA) and digital rectal examination (DRE) screenings lead to both under and over treatment of prostate cancer (PCa). As such, there is an urgent need for the identification and evaluation of new markers for early diagnosis and disease prognosis. Studies have shown a link between PCa, lipids and lipid metabolism. Therefore, the aim of this study was to examine the concentrations and distribution of serum lipids in patients with PCa as compared with serum from controls.MethodUsing Electrospray ionization mass spectrometry (ESI-MS/MS) lipid profiling, we analyzed serum phospholipids from age-matched subjects who were either newly diagnosed with PCa or healthy (normal).ResultsWe found that cholester (CE), dihydrosphingomyelin (DSM), phosphatidylcholine (PC), egg phosphatidylcholine (ePC) and egg phoshphatidylethanolamine (ePE) are the 5 major lipid groups that varied between normal and cancer serums. ePC 38:5, PC 40:3, and PC 42:4 represent the lipids species most prevalent in PCa as compared with normal serum. Further analysis revealed that serum ePC 38:5 ≥0.015 nmoles, PC 40.3 ≤0.001 nmoles and PC 42:4 ≤0.0001 nmoles correlated with the absence of PCa at 94% prediction. Conversely, serum ePC 38:5 ≤0.015 nmoles, PC 40:3 ≥0.001 nmoles, and PC 42:4 ≥0.0001 nmoles correlated with the presence of PCa.ConclusionIn summary, we have demonstrated that ePC 38:5, PC 40:3, and PC 42:4 may serve as early predictive serum markers for the presence of PCa.

Characterization of the ordered phase formed by sphingomyelin analogues and cholesterol binary mixtures.

The influences of structural alterations of sphingomyelin (SM) on its interactions with cholesterol (chol) and on ordered phase formation were examined by density measurements and surface pressure vs. molecular area isotherm measurements. In addition, we quantitatively characterized the ordered phase formed in each SM and chol binary mixture on the basis of the molecular compressional modulus of SM ( ). Density measurements demonstrated that the ordered phase formation in threo-SM (tSM)/chol and dihydrosphingomyelin (DHSM)/chol binary bilayers shows similar chol concentration-dependency to that of natural erythro-SM (eSM)/chol bilayers; the ordered phase formation was completed in the presence of 25 mol% chol. In contrast, SM bearing a triple bond in the place of a double bond (tripleSM) required a greater concentration of chol to completely transform the bilayer into the ordered phase (at 40 mol% chol). Surface pressure vs. molecular area isotherms showed that the DHSM molecule ( = 290 mN/m) is more rigid than eSM ( = 240 mN/m) above 30 mol% chol (in the ordered phase), although these values are similar (140–150 mN/m) in the absence of chol (liquid condensed phase). Most likely, the DHSM/chol mixture forms a more ordered membrane than the eSM/chol mixture does. Moreover, in the absence of chol, the rigidity of the tripleSM molecule ( = 250 mN/m) is significantly higher as compared with that of the eSM molecule ( = 150 mN/m), which is probably due to the presence of a triple bond.

Plasma sphingomyelin is associated with cognitive progression in Alzheimer's Disease

Plasma sphingolipids have been shown to predict cognitive impairment and hippocampal volume loss, but there is little research in patients with Alzheimer’s disease dementia (AD). In this study we sought to determine whether plasma ceramides, dihydroceramides (DHCer), sphingomyelins (SM), or dihydrosphingomyelin (DHSM) levels and ratios of SM/ceramide or DHSM/DHCer were predictive of progression in AD. Probable AD patients (n=120) were enrolled in the Alzheimer’s Disease and Memory Disorders Center at Baylor College of Medicine. Plasma sphingolipids were assessed using ESI/MS/MS. Linear mixed effects models were used to examine the relation between baseline plasma sphingolipid levels and cross-sectional and longitudinal performance on the Mini-Mental State Exam (MMSE), Alzheimer’s Disease Assessment Scale-Cognitive Subscale (ADAS-Cog), and Clinical Dementia Rating-Sum of Boxes (CDR-Sum). Participants were followed a mean of 4.2 visits and 2.3 years. There were no crosssectional associations. In longitudinal analyses, high levels of DHCer and ceramide were associated with greater progression, but findings did not reach significance (p>0.05). In contrast, higher plasma levels of SM, DHSM, SM/ceramide and DHSM/DHCer ratios were associated with less progression on the MMSE and ADAS-Cog; the ratios were the strongest predictors of clinical progression. Compared to the lowest tertiles, the highest tertiles of DHSM/DHCer and SM/ ceramide ratios declined 1.35 points (p=0.001) and 1.19 (p=0.004) less per year on the MMSE and increased 3.18 points (p=0.001) and 2.42 (p=0.016) less per year on the ADAS-Cog. These results suggest that increased SM/ceramide and DHSM/DHCer ratios dose-dependently predict slower progression among AD patients and may be sensitive blood-based biomarkers for clinical progression.

Oxygen and cytokine-dependent changes in choline phospholipid saturation in hematopoietic progenitor cells detected by MALDI-TOF mass spectrometry

The adaptation of cells to a changing environment is normally accompanied by rapid and/or chronic remodeling of membrane lipids. In order to understand the role played by membrane lipid metabolism in such responses, it is necessary to characterize in more detail the changes in membrane composition occurring in response to defined stimuli. There has been intense interest in characterizing the “stem cell niche” in recent years and an emerging consensus that stem cells are located in regions of low oxygen tension and probably well-isolated from the blood supply. We report here the use of matrix-assisted laser desorption and ionization time-of-flight mass spectrometry to monitor changes in the composition and saturation degree of choline phospholipids of hematopoietic progenitor (FDCPmix) cells under standard nutrient-rich culture conditions and at low oxygen and low glucose concentrations. We found that the increase in proliferation rate driven by high concentrations of interleukin-3 (IL-3) is associated with a decrease in membrane phosphatidylcholine (PC) 18:0/20:4 and sphingomyelin (SM) together with an increase in PC 18:0/18:2 and dihydro SM. Furthermore, this effect is most pronounced under low oxygen and low glucose conditions, independent of cell proliferation rates.

Dihydrosphingomyelin Impairs HIV-1 infection by Rigidifying Liquid-Ordered Membrane Domains

The lateral organization of lipids in cell membranes is thought to regulate numerous cell processes. Most studies focus on the coexistence of two fluid phases, the liquid crystalline (ld) and the liquid-ordered (lo); the putative presence of gel domains (so) is not usually taken into account. We show that in phospholipid:sphingolipid:cholesterol mixtures, in which sphingomyelin (SM) promoted fluid lo domains, dihydrosphingomyelin (DHSM) tended to form rigid domains. Genetic and pharmacological blockade of the dihydroceramide desaturase (Des1), which replaced SM with DHSM in cultured cells, inhibited cell infection by replication-competent and deficient HIV-1. Increased DHSM levels gave rise to more rigid membranes, resistant to the insertion of the gp41 fusion peptide, thus inhibiting viral-cell membrane fusion. These results clarify the function of dihydrosphingolipids in biological membranes and identify Des1 as a potential target in HIV-1 infection.

Dihydrosphingomyelin Impairs HIV-1 Infection by Rigidifying Liquid-Ordered Membrane Domains

The lateral organization of lipids in cell membranes is thought to regulate numerous cell processes. Most studies focus on the coexistence of two fluid phases, the liquid crystalline (l(d)) and the liquid-ordered (l(o)); the putative presence of gel domains (s(o)) is not usually taken into account. We show that in phospholipid:sphingolipid:cholesterol mixtures, in which sphingomyelin (SM) promoted fluid l(o) domains, dihydrosphingomyelin (DHSM) tended to form rigid domains. Genetic and pharmacological blockade of the dihydroceramide desaturase (Des1), which replaced SM with DHSM in cultured cells, inhibited cell infection by replication-competent and -deficient HIV-1. Increased DHSM levels gave rise to more rigid membranes, resistant to the insertion of the gp41 fusion peptide, thus inhibiting viral-cell membrane fusion. These results clarify the function of dihydrosphingolipids in biological membranes and identify Des1 as a potential target in HIV-1 infection.

Genetic determinants of circulating sphingolipid concentrations in European populations.

Sphingolipids have essential roles as structural components of cell membranes and in cell signalling, and disruption of their metabolism causes several diseases, with diverse neurological, psychiatric, and metabolic consequences. Increasingly, variants within a few of the genes that encode enzymes involved in sphingolipid metabolism are being associated with complex disease phenotypes. Direct experimental evidence supports a role of specific sphingolipid species in several common complex chronic disease processes including atherosclerotic plaque formation, myocardial infarction (MI), cardiomyopathy, pancreatic β-cell failure, insulin resistance, and type 2 diabetes mellitus. Therefore, sphingolipids represent novel and important intermediate phenotypes for genetic analysis, yet little is known about the major genetic variants that influence their circulating levels in the general population. We performed a genome-wide association study (GWAS) between 318,237 single-nucleotide polymorphisms (SNPs) and levels of circulating sphingomyelin (SM), dihydrosphingomyelin (Dih-SM), ceramide (Cer), and glucosylceramide (GluCer) single lipid species (33 traits); and 43 matched metabolite ratios measured in 4,400 subjects from five diverse European populations. Associated variants (32) in five genomic regions were identified with genome-wide significant corrected p-values ranging down to 9.08×10−66. The strongest associations were observed in or near 7 genes functionally involved in ceramide biosynthesis and trafficking: SPTLC3, LASS4, SGPP1, ATP10D, and FADS1–3. Variants in 3 loci (ATP10D, FADS3, and SPTLC3) associate with MI in a series of three German MI studies. An additional 70 variants across 23 candidate genes involved in sphingolipid-metabolizing pathways also demonstrate association (p = 10−4 or less). Circulating concentrations of several key components in sphingolipid metabolism are thus under strong genetic control, and variants in these loci can be tested for a role in the development of common cardiovascular, metabolic, neurological, and psychiatric diseases.

Ab initio and DFT study of 31P‐NMR chemical shifts of sphingomyelin and dihydrosphingomyelin lipid molecule

AbstractOne of the phospholipids, sphingomyelin (SM, N‐acyl‐sphingosine‐1‐phosphorylcholine) is the most abundant component of mammalian membranes in brain, nervous tissues, and human ocular lens. It plays an important role for apoptosis, aging, and signal transduction. Recently, Yappert and coworkers have shown that human lens sphingomyelin and its hydrogenated derivative, dihydrosphingomyelin (DHSM) are interacted with Ca2+ ions to develop human cataracts. Previously, we have investigated conformational differences between an isolated SM/DHSM molecule and Ca2+‐coordinated form by using density functional theory (DFT) for geometry optimization and normal mode analysis. As a result, one of stable conformers of SMs has a hydrogen bonding between hydroxyl group and phosphate group, whereas another conformer has a hydrogen bonding between hydroxyl and phosphate amide group. In this study, 31P‐Nuclear Magnetic Resonance (NMR) shielding constants of the obtained conformers are investigated by using ab initio and DFT with NMR‐gauge invariant atomic orbitals (NMR‐GIAO) calculations. The experimental 31P‐NMR chemical shifts of SMs and DHSMs have significant small value around 0.1 ppm. We consider the relative conformational changes between SMs and DHSMs affect the slight deviations of 31P‐NMR chemical shifts, and discuss intramolecular hydrogen bondings and the solvent effect in relation to NMR experimental reference. © 2009 Wiley Periodicals, Inc. Int J Quantum Chem, 2009

Probing HIV-1 Membrane Liquid Order by Laurdan Staining Reveals Producer Cell-dependent Differences

Viruses acquire their envelope by budding from a host cell membrane, but viral lipid composition may differ from that of the budding membrane. We have previously reported that the HIV-1 membrane is highly enriched in cholesterol, sphingolipids, and other raft lipids, suggesting that the virus may bud from pre-existing or virus-induced lipid rafts. Here, we employed the environmentally sensitive fluorescent dye Laurdan to study the membrane lateral structure of HIV-1 derived from different cell lines. Differences in viral membrane order detected by Laurdan staining were shown by mass spectrometry to be due to differences in lipid composition. Isogenic viruses from two different cell lines were both strongly enriched in raft lipids and displayed a liquid-ordered membrane, but these effects were significantly more pronounced for HIV-1 from the T-cell line MT-4 compared with virus from 293T cells. Host-dependent differences in the lipidomes predominantly affected the ratio of sphingomyelins (including dihydrosphingomyelin) to phosphatidylcholine, whereas cholesterol contents were similar. Accordingly, treatment of infectious HIV-1 with the sphingomyelin-binding toxins Equinatoxin-II or lysenin showed differential inhibition of infectivity. Liposomes consisting of lipids that had been extracted from viral particles exhibited slightly less liquid order than the respective viral membranes, which is likely to be due to absence of membrane proteins and to loss of lipid asymmetry. Synthetic liposomes consisting of a quaternary lipid mixture emulating the viral lipids showed a liquid order similar to liposomes derived from virion lipids. Thus, Laurdan staining represents a rapid and quantitative method to probe viral membrane liquid order and may prove useful in the search for lipid active drugs.

Quantitative Analysis of Dairy Phospholipids by 31P NMR

Abstract31P NMR analysis of samples prepared in a sodium cholate detergent system was assessed as a method for the quantitative measurement of dairy phospholipids. Major phospholipid (PL) classes measured included: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), sphingomyelin (SM) and dihydrosphingomyelin (DHSM). The 31P NMR method was validated by comparison with a quantitative two‐dimensional thin‐layer chromatography (2D‐TLC) technique. The 2D‐TLC system was more sensitive, able to detect some minor compounds not observed by 31P NMR. However, 31P NMR is more suited to routine analysis, with sample analysis taking 36 min. The method was also more versatile and sample analysis was possible on high phospholipid containing materials without prior lipid extraction (e.g. buttermilk protein concentrate, beta serum liquid).

A DFT study of infrared spectrum of sphingomyelin lipid molecule with calcium cation

AbstractOne of the phospholipids, sphingomyelin (SM) is the most abundant component of mammalian membranes in brain and nervous tissues. It plays an important role for apoptosis, aging, and signal transduction with cations. Recently, Yappert and co‐workers have shown that human lens sphingomyelin and its hydrogenated derivative, dihydrosphingomyelin (DHSM) are interacted with Ca2+ ions to develop human cataracts. In this study, we investigate conformational differences between an isolated SM/DHSM molecule and Ca2+‐coordinated form by using density functional theory (DFT) calculations. B3LYP functional and 6‐31G(d,p) double‐ζ split‐valence basis set is used for geometry optimization and normal mode analysis. One of resultant conformers of SMs has hydrogen bonding between hydroxyl and phosphate group, whereas another conformer has hydrogen bonding between hydroxyl and amide group. The red shift of calculated vibrational frequencies of amide band due to Ca2+ is compared with experimental infrared spectrum. Finally, we discuss the Ca2+‐induced effects from conformational and spectroscopic point of view to compare the experimental results. © 2008 Wiley Periodicals, Inc. Int J Quantum Chem, 2008

Liquid chromatography with dual parallel mass spectrometry and 31P nuclear magnetic resonance spectroscopy for analysis of sphingomyelin and dihydrosphingomyelin : II. Bovine milk sphingolipids

Liquid chromatography coupled to atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) mass spectrometry (MS), in parallel, was used for simultaneous detection of bovine milk sphingolipids (BMS). APCI-MS mass spectra exhibited mostly ceramide-like fragment ions, [Cer-H 2O + H] + and [Cer-2H 2O + H] +, which were used to identify individual molecular species of BMS according to fatty acyl chain length:degree of unsaturation and long-chain base (LCB). ESI-MS was used to confirm the molecular weights of BMS species. Both sphingomyelin (SM) and dihydrosphingomyelin (DSM) molecular species were identified, with DSM species constituting 20% of BMS. Approximately 56 to 58% of DSM species contained a d16:0 LCB, while 34 to 37% contained a d18:0 LCB. Approximately 26 to 30% of SM species contained a d16:1 LCB, while 57 to 60% contained a d18:1 LCB. BMS species contained both odd and even carbon chain lengths. The most abundant DSM species contained a d16:0 LCB with a 22:0, 23:0 or 24:0 fatty acyl chain, while the most abundant SM species contained a d18:1 LCB with a 16:0 or 23:0 fatty acyl chain. 31P NMR spectroscopy was used to conclusively confirm that DSM is a dietary component in BMS.