Early-life stress and dietary fatty acids impact the brain lipid/oxylipin profile into adulthood, basally and in response to LPS.

Brain lipid dysregulation is a hallmark of depression and Alzheimer's disease, also marked by chronic inflammation. Early-life stress (ELS) and dietary intake of polyunsaturated fatty acids (PUFAs) are risk factors for these pathologies and are known to impact inflammatory processes. However, if these early-life factors alter brain lipid homeostasis on the long-term and thereby contribute to this risk remains to be elucidated. We have recently shown that an early diet enriched in omega(ω)-3 PUFAs protected against the long-term negative effects of ELS on cognition and neuroinflammation. Here, we aim to understand if modulation of brain lipid and oxylipin profiles contributes to the detrimental effects of ELS and the protective ones of the diet. We therefore studied if and how ELS and early dietary PUFAs modulate the brain lipid and oxylipin profile, basally as well as in response to an inflammatory challenge, to unmask possible latent effects. Male mice were exposed to ELS via the limited bedding and nesting paradigm, received an early diet with high or low ω6/ω3 ratio (HRD and LRD) and were injected with saline or lipopolysaccharide (LPS) in adulthood. Twenty-four hours later plasma cytokines (Multiplex) and hypothalamic lipids and oxylipins (liquid chromatography tandem mass spectrometry) were measured. ELS exacerbated the LPS-induced increase in IL-6, CXCL1 and CCL2. Both ELS and diet affected the lipid/oxylipin profile long-term. For example, ELS increased diacylglycerol and LRD reduced triacylglycerol, free fatty acids and ceramides. Importantly, the ELS-induced alterations were strongly influenced by the early diet. For example, the ELS-induced decrease in eicosapentaenoic acid was reversed when fed LRD. Similarly, the majority of the LPS-induced alterations were distinct for control and ELS exposed mice and unique for mice fed with LRD or HRD. LPS decreased ceramides and lysophosphotidylcholine, increased hexosylceramides and prostaglandin E2, reduced triacylglycerol species and ω6-derived oxylipins only in mice fed LRD and ELS reduced the LPS-induced increase in phosphatidylcholine. These data give further insights into the alterations in brain lipids and oxylipins that might contribute to the detrimental effects of ELS, to the protective ones of LRD and the possible early-origin of brain lipid dyshomeostasis characterizing ELS-related psychopathologies.

Effect of soya phosphatidylcholine and possible underlying mechanism on ischemia/reperfusion injury in isolated perfused rat heart: an experimental and computational study.

This study was designed to assess the effect of soya phosphatidylcholine (SPC) against ischemia/reperfusion (I/R) injury and the possible underlying mechanism using experimental and computational studies. I/R injury was induced by global ischemia for 30 min followed by reperfusion for 120 min. The perfusion of the SPC was performed for 10 min before inducing global ischemia. In the mechanistic study, the involvement of specific cellular pathways was identified using various inhibitors such as ATP-dependent potassium channel (KATP) inhibitor (glibenclamide), protein kinase C (PKC) inhibitor (chelerythrine), non-selective nitric oxide synthase inhibitor (L-NAME), and endothelium remover (Triton X-100). The computational study of various ligands was performed on toll-like receptor 4 (TLR4) protein using AutoDock version 4.0. SPC (100 µM) significantly decreased the levels of cardiac damage markers and %infarction compared with the vehicle control (VC). Furthermore, cardiodynamics (indices of left ventricular contraction (dp/dtmax), indices of left ventricular relaxation (dp/dtmin), coronary flow, and antioxidant enzyme levels were significantly improved as compared with VC. This protective effect was attenuated by glibenclamide, chelerythrine, and Triton X-100, but it was not attenuated by L-NAME. The computational study showed a significant bonding affinity of SPC to the TLR4-MD2 complex. Thus, SPC reduced myocardial I/R injury in isolated perfused rat hearts, which might be governed by the KATP channel, PKC, endothelium response, and TLR4-MyD88 signaling pathway.

Novel pharmacological effects of lecithinized superoxide dismutase on ischemia/reperfusion injury in the kidneys of mice.

Renal ischemia/reperfusion (I/R) injury is a major clinical problem because it can cause acute kidney injury (AKI) or lead to the transition from AKI to chronic kidney disease (CKD). Oxidative stress, which involves the production of reactive oxygen species (ROS), plays an important role in the development and exacerbation of I/R-induced kidney injury. However, we have previously reported that lecithinized superoxide dismutase (PC-SOD), a SOD derivative with high tissue affinity and high stability in plasma, has beneficial effects in various disease models because of its inhibitory effect on ROS production. Therefore, we aimed to determine the effects of intravenous PC-SOD administration in a mouse model of renal injury induced by I/R. PC-SOD markedly ameliorated the I/R-induced increases in markers of renal damage (urea nitrogen, creatinine, neutrophil gelatinase-associated lipocalin, and interleukin-6) and tubular necrosis 48 h after the intervention. We also found that PC-SOD significantly ameliorated the I/R-induced increase in ROS production, using an ex vivo imaging system. Furthermore, PC-SOD inhibited the increases in expression of markers of fibrosis (α-smooth muscle actin and collagen 1A1) 96 h after, and renal fibrosis 25 days after I/R was induced. Finally, we found that PC-SOD ameliorated the I/R-induced AKI in mice with high-fat diet-induced prediabetes. These results suggest that PC-SOD inhibits AKI and the transition from AKI to CKD through the inhibition of ROS production. Therefore, we believe that PC-SOD may represent an effective therapeutic agent for I/R-induced renal injury.

Gut microbiota-generated metabolite, trimethylamine-N-oxide, and subclinical myocardial damage: a multicenter study from Thailand.

Plasma Trimethylamine-N-oxide (TMAO), a gut microbiota metabolite from dietary phosphatidylcholine, is mechanistically linked to cardiovascular disease (CVD) and adverse cardiovascular events. We aimed to examine the relationship between plasma TMAO levels and subclinical myocardial damage using high-sensitivity cardiac troponin-T (hs-cTnT). We studied 134 patients for whom TMAO data were available from the Cohort Of patients at a high Risk of Cardiovascular Events-Thailand (CORE-Thailand) registry, including 123 (92%) patients with established atherosclerotic disease and 11 (8%) with multiple risk factors. Plasma TMAO was measured by NMR spectroscopy. In our study cohort (mean age 64 ± 8.9 years; 61% men), median TMAO was 3.81 µM (interquartile range [IQR] 2.89-5.50 µM), and median hs-cTnT was 15.65 ng/L (IQR 10.17-26.67). Older patients and those with diabetic or hypertension were more likely to have higher TMAO levels. Plasma TMAO levels correlated with those of hs-cTnT (r = 0.54; p < 0.0001) and were significantly higher in patients with subclinical myocardial damage (hs-cTnT ≥ 14 ng/L; 4.48 µM vs 2.98 µM p < 0.0001). After adjusting for traditional risk factors, elevated TMAO levels remained independently associated with subclinical myocardial damage (adjusted odds ratio [OR]: 1.58; 95% CI 1.24-2.08; p = 0.0007). This study demonstrated that plasma TMAO was an independent predictor for subclinical myocardial damage in this study population.

HDL Improves Cholesterol and Glucose Homeostasis and Reduces Atherosclerosis in Diabetes-Associated Atherosclerosis.

BACKGROUND AND AIMS: Apolipoprotein A-I (ApoA-I), the main component of high-density lipoprotein (HDL), not only promotes reverse cholesterol transport (RCT) in atherosclerosis but also increases insulin secretion in pancreatic ß-cells, suggesting that interventions which raise HDL levels may be beneficial in diabetes-associated cardiovascular disease (CVD). Previously, we showed that TNF-related apoptosis-inducing ligand (TRAIL) deletion in Apolipoprotein Eknockout (Apoe-/- ) mice results in diabetes-accelerated atherosclerosis in response to a "Western" diet. Here, we sought to identify whether reconstituted HDL (rHDL) could improve features of diabetes-associated CVD in Trail-/-Apoe-/- mice. METHODS AND RESULTS: Trail-/-Apoe-/- and Apoe-/- mice on a "Western" diet for 12 weeks received 3 weekly infusions of either PBS (vehicle) or rHDL (containing ApoA-I (20 mg/kg) and 1-palmitoyl-2-linoleoyl phosphatidylcholine). Administration of rHDL reduced total plasma cholesterol, triglyceride, and glucose levels in Trail-/-Apoe-/- but not in Apoe-/- mice, with no change in weight gain observed. rHDL treatment also improved glucose clearance in response to insulin and glucose tolerance tests. Immunohistological analysis of pancreata revealed increased insulin expression/production and a reduction in macrophage infiltration in mice with TRAIL deletion. Furthermore, atherosclerotic plaque size in Trail-/-Apoe-/- mice was significantly reduced associating with increased expression of the M2 macrophage marker CD206, suggesting HDL's involvement in the polarization of macrophages. rHDL also increased vascular mRNA expression of RCT transporters, ABCA1 and ABCG1, in Trail-/-Apoe-/- but not in Apoe-/- mice. Conclusions. rHDL improves features of diabetes-associated atherosclerosis in mice. These findings support the therapeutic potential of rHDL in the treatment of atherosclerosis and associated diabetic complications. More studies are warranted to understand rHDL's mechanism of action.

Dietary Choline Supplements, but Not Eggs, Raise Fasting TMAO Levels in Participants with Normal Renal Function: A Randomized Clinical Trial.

BACKGROUND: Choline is a dietary precursor to the gut microbial generation of the prothrombotic and proatherogenic metabolite trimethylamine-N-oxide (TMAO). Eggs are rich in choline, yet the impact of habitual egg consumption on TMAO levels and platelet function in human subjects remains unclear. METHODS: Healthy volunteers (41% male, 81% Caucasian, median age 28 years) with normal renal function (estimated glomerular filtration rate >60) were recruited and assigned to 1 of 5 daily interventions for 4 weeks: 1) hardboiled eggs (n = 18); 2) choline bitartrate supplements (n = 20); 3) hardboiled eggs + choline bitartrate supplements (n = 16); 4) egg whites + choline bitartrate supplements (n = 18); 5) phosphatidylcholine supplements (n = 10). Fasting blood and urine samples were collected for quantification of TMAO, its precursors, and platelet aggregometry. RESULTS: Participants' plasma TMAO levels increased significantly in all 3 intervention arms containing choline bitartrate (all P < .0001), but daily ingestion of 4 large eggs (P = .28) or phosphatidylcholine supplements (P = .27) failed to increase plasma TMAO levels. Platelet reactivity also significantly increased in the 3 intervention arms containing choline bitartrate (all P < .01), but not with eggs (P = .10) or phosphatidylcholine supplements (P = .79). CONCLUSIONS: Despite high choline content in egg yolks, healthy participants consuming 4 eggs daily showed no significant increase in TMAO or platelet reactivity. However, choline bitartrate supplements providing comparable total choline raised both TMAO and platelet reactivity, demonstrating that the form and source of dietary choline differentially contributes to systemic TMAO levels and platelet responsiveness.

Effects of Dietary Supplementation with EPA-enriched Phosphatidylcholine and Phosphatidylethanolamine on Glycerophospholipid Profile in Cerebral Cortex of SAMP8 Mice fed with High-fat Diet.

The destruction of lipid homeostasis is associated with nervous system diseases such as Alzheimer's disease (AD). It has been reported that dietary EPA-enriched phosphatidylcholine (EPA-PC) and phosphatidylethanolamine (EPA-PE) could improve brain function. However, it was unclear that whether EPA-PC and EPA-PE intervention could change the lipid composition of cerebral cortex in AD mice. All the senescence-accelerated mouse-prone 8 (SAMP8) mice were fed with a high-fat diet for 8 weeks. After another 8 weeks of intervention with EPA-PC and EPA-PE (1%, w/w), the cerebral cortex lipid levels were determined by lipidomics. Results demonstrated that dietary supplementation with EPA-PE and EPA PC for 8 weeks significantly increased the amount of choline plasmalogen (pPC) and Lyso phosphatidylethanolamine (LPE) in the cerebral cortex of SAMP8 mice fed with high fat diet. Meanwhile, administration with EPA-PE and EPA-PC could significantly decrease the level of docosapentaenoic acid (DPA)-containing phosphatidylserine (PS) as well as increase the levels of arachidonic acid (AA)-containing phosphatidylethanolamine and PS in cerebral cortex. EPA-PE and EPA-PC could restore the lipid homeostasis of dementia mice to a certain degree, which might provide a potential novel therapy strategy and direction of dietary intervention in patients with cognitive impairment.

DHA/EPA-Enriched Phosphatidylcholine Suppresses Tumor Growth and Metastasis via Activating Peroxisome Proliferator-Activated Receptor γ in Lewis Lung Cancer Mice.

In the present study, the antitumor effects of docosahexaenoic acid-phosphatidylcholine (DHA-PC) and eicosapentanoic acid-phosphatidylcholine (EPA-PC) in Lewis lung cancer mice were investigated. As observed, DHA-PC and EPA-PC obviously inhibited the transplanted tumor growth and the positive expression of Ki67. The metastatic nodules and hematoxylin and eosin (HE) staining of the lung indicated that DHA-PC and EPA-PC suppressed lung metastasis. PPARγ has a key role in cell survival, which may be a target for cancer therapy. Further mechanism research indicated that DHA-PC and EPA-PC significantly enhanced the levels of PPARγ and subsequently downregulated the NF-κB pathway. DHA-PC and EPA-PC accelerate cancer cell apoptosis by decreasing NF-κB-mediated antiapoptotic factors Bcl-2 and Bcl-XL, thereby inhibiting tumor growth. In addition, DHA-PC and EPA-PC significantly decreased the levels of NF-κB-mediated matrix metallopeptidase 9 (MMP9) and heparanase (HPA), which block the extracellular matrix (ECM) degradation, thereby suppressing lung metastasis. These findings suggested that DHA-PC and EPA-PC could be used as food supplements and/or functional ingredients for cancer patients.

Phosphatidylcholine Ameliorates LPS-Induced Systemic Inflammation and Cognitive Impairments via Mediating the Gut-Brain Axis Balance.

Systemic inflammation will cause an imbalance in the steady state of the gut-brain axis. Phosphatidylcholine (PC) is a phospholipid found in egg yolk that has anti-inflammatory and antioxidant properties. The present research proved that PC supplementation (60 mg/kg body weight) for 35 days prevented inflammatory responses and behavioral disturbances in lipopolysaccharide (LPS)-induced mice. PC could regulate the expression of neurotrophic factors and synaptic proteins, which effectively alleviated the nerve damage and synaptic dysfunction caused by LPS. In addition, PC supplementation ameliorated gut barrier damage, altered gut genes, and improved gut health by modulating the cell adhesion molecule (CAM) pathway. Furthermore, PC remodeled the gut microbiome structure in the mice of the LPS group by increasing the relative abundance of Rikenellaceae and Lachnospiraceae. PC also increased short-chain fatty acid (SCFA) production in LPS-induced mice, which in turn ameliorated brain inflammatory responses. In conclusion, PC supplementation may be a nutritional strategy for the prevention of systemic inflammation via the gut-brain axis.

A TLR7/8 Agonist-Including DOEPC-Based Cationic Liposome Formulation Mediates Its Adjuvanticity Through the Sustained Recruitment of Highly Activated Monocytes in a Type I IFN-Independent but NF-κB-Dependent Manner.

Novel adjuvants, such as Toll-like receptors (TLRs) agonists, are needed for the development of new formulations able to circumvent limitations of current vaccines. Among TLRs, TLR7/8 agonists represent promising candidates, as they are well described to enhance antigen-specific antibody responses and skew immunity toward T helper (TH) 1 responses. We find here that the incorporation of the synthetic TLR7/8 ligand 3M-052 in a cationic DOEPC-based liposome formulation shifts immunity toward TH1 responses and elicits strong and long-lasting germinal center and follicular T helper cell responses in adult mice. This reflects the prolonged recruitment of innate cells toward the site of immunization and homing of activated antigen-loaded monocytes and monocyte-derived dendritic cells toward draining lymph nodes. We further show that this adjuvanticity is independent of type I IFN but NF-κB-dependent. Overall, our data identify TLR7/8 agonists incorporated in liposomes as promising and effective adjuvants to enhance TH1 and germinal center responses.

Comparative study of DHA-enriched phosphatidylcholine and EPA-enriched phosphatidylcholine on ameliorating high bone turnover via regulation of the osteogenesis-related Wnt/ß-catenin pathway in ovariectomized mice.

Here, we compared the effects of marine DHA-enriched phosphatidylcholine (DHA-PC) and EPA-enriched phosphatidylcholine (EPA-PC) on high bone turnover in a model of osteoporosis induced by bilateral ovariectomy in vivo, and further investigated the possible protective mechanisms. Meanwhile, DHA-PC and EPA-PC clearly ameliorated the microstructure of the trabecular bone and accelerated bone mineral apposition rate, additionally increasing bone mineral density and biomechanical properties of the bone. Furthermore, gene and protein expression levels suggest that DHA-PC and EPA-PC inhibited overactive osteogenesis via down-regulation of the expression of the osteogenesis-related Wnt/ß-catenin signaling pathway. In conclusion, DHA-PC and EPA-PC reduced excessive osteogenesis via normalization of Wnt/ß-catenin expression. These results may contribute to the elucidation of the anti-osteoporotic properties of DHA-PC and EPA-PC and further develop their potential application value as a functional food.

A phospholipid-based formulation for the treatment of airway inflammation in chronic respiratory diseases.

Inflammation, the major hallmark of all chronic respiratory diseases is generally managed by inhaled corticosteroids. However, long term high dose treatment can result in significant side effects. Hence, there is a medical need for non-steroidal anti-inflammatory therapies to address airway inflammation. Phospholipids have been shown to reduce inflammation in several inflammatory conditions; however, their clinical translation has been limited to liposomal formulations traditionally used as drug carriers and their biological activity has not been investigated. Here we report the first application of empty liposomes as an anti-inflammatory treatment in airway inflammation. In the current study, liposomes (UTS-001) were prepared from cholesterol and a synthetic phospholipid (DOPC). The formulation was characterised in terms of size, charge, polydispersity index, morphology and stability as colloidal suspension and freeze-dried nanoparticles. Time-dependant uptake of UTS-001 in airway epithelial cells was observed which was inhibited by nystatin demonstrating that the uptake is via the caveolae pathway. In-vitro, in primary nasal epithelial cells, UTS-001 treatment successfully attenuated IL-6 levels following TNF-α stimulation. Consistent with the in-vitro findings, in-vivo, in the ovalbumin model of allergic airway inflammation, UTS-001 significantly reduced total immune cell counts in bronchoalveolar lavage fluid and reduced airway hyperresponsiveness in response to increasing doses of methacholine challenge. Therefore, our results establish UTS-001 as a potential anti-inflammatory treatment that may be useful as a therapeutic for lung inflammatory diseases.

Immediate inhibition of spinal secretory phospholipase A2 prevents the pain and elevated spinal neuronal hyperexcitability and neuroimmune regulatory genes that develop with nerve root compression.

Cervical nerve root injury induces a host of inflammatory mediators in the spinal cord that initiate and maintain neuronal hyperexcitability and pain. Secretory phospholipase A2 (sPLA2) is an enzyme that has been implicated as a mediator of pain onset and maintenance in inflammation and neural injury. Although sPLA2 modulates nociception and excitatory neuronal signaling in vitro, its effects on neuronal activity and central sensitization early after painful nerve root injury are unknown. This study investigated whether inhibiting spinal sPLA2 at the time of nerve root compression (NRC) modulates the pain, dorsal horn hyperexcitability, and spinal genes involved in glutamate signaling, nociception, and inflammation that are seen early after injury. Rats underwent a painful C7 NRC injury with immediate intrathecal administration of the sPLA2 inhibitor thioetheramide-phosphorlycholine. Additional groups underwent either injury alone or sham surgery. One day after injury, behavioral sensitivity, spinal neuronal excitability, and spinal cord gene expression for glutamate receptors (mGluR5 and NR1) and transporters (GLT1 and EAAC1), the neuropeptide substance P, and pro-inflammatory cytokines (TNFα, IL1α, and IL1ß) were assessed. Treatment with the sPLA2 inhibitor prevented mechanical allodynia, attenuated neuronal hyperexcitability in the spinal dorsal horn, restored the proportion of spinal neurons classified as wide dynamic range, and reduced genes for mGluR5, substance P, IL1α, and IL1ß to sham levels. These findings indicate spinal regulation of central sensitization after painful neuropathy and suggest that spinal sPLA2 is implicated in those early spinal mechanisms of neuronal excitability, perhaps via glutamate signaling, neurotransmitters, or inflammatory cascades.

Exogenous natural EPA-enriched phosphatidylcholine and phosphatidylethanolamine ameliorate lipid accumulation and insulin resistance via activation of PPARα/γ in mice.

Nuclear receptor peroxisome proliferator-activated receptors (PPARs) play an important role in the regulation of glucose homeostasis and lipid metabolism. Here, in a protein-lipid overlay assay, we show that EPA-enriched phosphatidylcholine (EPA-PC) and phosphatidylethanolamine (EPA-PE), isolated from sea cucumber, bind to PPARα/PPARγ. An established dual-luciferase reporter gene assay system in NIH3T3 cells showed the exert agonistic activity of EPA-PC and EPA-PE with respect to the transcription of PPARα and PPARγ. The treatments of EPA-PC and EPA-PE induced PPARα-mediated fatty acid oxidation in mouse hepatocytes and liver. In a preadipocytes differentiation assay, EPA-PC and EPA-PE promoted the differentiation of preadipocytes to differentiated adipocytes and upregulated the expression of lipid metabolic target genes of the PPARγ and inhibited the phosphorylation of PPARγ at Ser273. We further examined the effects of EPA-PC and EPA-PE on high-fat high-sucrose diet (HFSD) induced insulin resistance and found that insulin resistance as well as abnormal lipid accumulation was ameliorated by EPA-PC and EPA-PE.

A Phospholipase-A Activity in Soluble Leishmania Antigens Causes Instability of Liposomes.

AIM: This study aimed to investigate the existence of phospholipase-A (PLA) activity in Soluble L. major Antigens (SLA) because of no reports for it so far. Liposomes were used as sensors to evaluate PLA activity. OBJECTIVES: Liposomal SLA consisting of Egg Phosphatidylcholine (EPC) or Sphingomyelin (SM) were prepared by two different methods in different pH or temperatures and characterized by Dynamic Light Scattering (DLS) and Thin Layer Chromatography (TLC). METHODS: Lipid hydrolysis led to the disruption of EPC liposomal SLA in both methods but the Film Method (FM) produced more stable liposomes than the Detergent Removal Method (DRM). RESULT: The preparation of EPC liposomal SLA at pH 6 via FM protected liposomes from hydrolysis to some extent for a short time. EPC liposomes but not SM liposomes were disrupted in the presence of SLA. CONCLUSION: Therefore, a phospholipid without ester bond such as SM should be utilized in liposome formulations containing PLA as an encapsulating protein.

The exogenous natural phospholipids, EPA-PC and EPA-PE, contribute to ameliorate inflammation and promote macrophage polarization.

Dietary intake of sea cucumber phospholipids, a rich source of eicosapentaenoic acid in the form of phospholipids (EPA-PLs), has been shown to improve obesity and related disorders. However, whether dietary eicosapentaenoic acid in the form of phosphatidylcholine and phosphatidylethanolamine (EPA-PC and EPA-PE, respectively) shows anti-inflammatory efficacy and its underlying mechanism has scarcely been investigated to date. Thus, the purpose of this study was to determine if EPA-PC and EPA-PE improve chronic inflammation and alter the interaction between macrophages and adipocytes. We found that EPA-PC and EPA-PE reduced the elevated levels of serum TNF-α, IL-6 and MCP1 and attenuated macrophage infiltration in the liver and iWAT of an HFSD-induced inflammatory model. Importantly, EPA-PC and EPA-PE promoted macrophage polarization in white adipose tissue. Furthermore, this effect on macrophage polarization was also observed in a 3T3L1 and Raw 264.7 Transwell co-culture system, which suggests that EPA-PC and EPA-PE attenuate chronic inflammation by promoting the M2-dominant polarization of macrophages in vitro. Our experiments in vitro illustrated that EPA-PC and EPA-PE attenuated the phosphorylation of p65 NFκB in Raw264.7 macrophages and increased PPARγ expression in 3T3-L1 adipocytes during the co-culture, which may contribute to the improvement in adipose inflammation. Thus, dietary eicosapentaenoic acid in the form of phosphatidylcholine and phosphatidylethanolamine may be a therapeutic strategy for chronic inflammation in obese adipose tissue.

Effect of Choline Forms and Gut Microbiota Composition on Trimethylamine-N-Oxide Response in Healthy Men.

BACKGROUND: Trimethylamine-N-oxide (TMAO), a choline-derived gut microbiota-dependent metabolite, is a newly recognized risk marker for cardiovascular disease. We sought to determine: (1) TMAO response to meals containing free versus lipid-soluble choline and (2) effects of gut microbiome on TMAO response. METHODS: In a randomized, controlled, double-blinded, crossover study, healthy men (n = 37) were provided meals containing 600 mg choline either as choline bitartrate or phosphatidylcholine, or no choline control. RESULTS: Choline bitartrate yielded three-times greater plasma TMAO AUC (p = 0.01) and 2.5-times greater urinary TMAO change from baseline (p = 0.01) compared to no choline and phosphatidylcholine. Gut microbiota composition differed (permutational multivariate analysis of variance, PERMANOVA; p = 0.01) between high-TMAO producers (with ≥40% increase in urinary TMAO response to choline bitartrate) and low-TMAO producers (with <40% increase in TMAO response). High-TMAO producers had more abundant lineages of Clostridium from Ruminococcaceae and Lachnospiraceae compared to low-TMAO producers (analysis of composition of microbiomes, ANCOM; p < 0.05). CONCLUSION: Given that phosphatidylcholine is the major form of choline in food, the absence of TMAO elevation with phosphatidylcholine counters arguments that phosphatidylcholine should be avoided due to TMAO-producing characteristics. Further, development of individualized dietary recommendations based on the gut microbiome may be effective in reducing disease risk.

Development of an HPLC-MS/MS Method for the Determination of Silybin in Human Plasma, Urine and Breast Tissue.

Silybin is a flavonolignan extracted from Silybum marianum with chemopreventive activity against various cancers, including breast. This study was designed to develop an HPLC-MS/MS method for the determination of silybin in human plasma, urine and breast tissue in early breast cancer patients undergoing Siliphos® supplementation, an oral silybin-phosphatidylcholine complex. The determination of silybin was carried out by liquid-liquid extraction with methyl-tert-butyl ether (MTBE); total silybin concentration was determined by treating the samples with ß-glucuronidase, while for the determination of free silybin, the hydrolytic step was omitted. Naringenin and naproxen were selected as internal standards. The detection of the analyte was carried out by mass spectrometry and by chromatography. The HPLC-MS/MS method was evaluated in terms of selectivity, linearity, limit of quantification, precision and accuracy, and carryover. The method proved to be selective, linear, precise and accurate for the determination of silybin. To the best of our knowledge, this presents the first analytical method with the capacity to quantify the major bioactive components of milk thistle in three different biological matrices with a lower limit of quantification of 0.5 ng/mL for plasma. Silybin phosphatidylcholine, taken orally, can deliver high blood concentrations of silybin, which selectively accumulates in breast tumor tissue.

Effect of phosphatidylcholine on the stability and lipolysis of nanoemulsion drug delivery systems.

Phosphatidylcholines (PCs) have been widely used in pharmaceutical research. Unfortunately, our understanding of how PCs influence the in vivo lipolysis process of drug delivery systems is still limited. In this study, PCs with fatty acid chains of varying lengths and saturability were used as emulsifiers to prepare curcumin-loaded nanoemulsions (Cur-NEs). The differences in particle size as well as drug and free fatty acid release during the lipolysis process were studied in a simulated blood environment. Furthermore, the pharmacokinetics and antitumor efficacy of Cur-NEs were evaluated in mice. The prepared 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)-stabilized Cur-NEs showed similar particle size and stability during storage but exhibited different lipolysis behaviors in vitro and in vivo. Due to the gel state of DPPC in the physiological environment, DPPC-stabilized Cur-NEs had low binding affinity with proteins and maintained their integrity in plasma, leading to sustained drug release, prolonged circulation time and enhanced antitumor efficacy in 4T1 tumor-bearing mice. In contrast, DOPC and DSPC-stabilized Cur-NEs were prone to coalescence in the plasma, resulting in rapid drug release and elimination from circulation. Our findings demonstrated that proper use of PCs is beneficial for obtaining desired transport behavior and drug therapeutic effects, providing guiding principles for rational design of nanodelivery systems.

Reconstituted High-density Lipoprotein Therapy Improves Survival in Mouse Models of Sepsis.

BACKGROUND: High-density lipoproteins exert pleiotropic effects including antiinflammatory, antiapoptotic, and lipopolysaccharide-neutralizing properties. The authors assessed the effects of reconstituted high-density lipoproteins (CSL-111) intravenous injection in different models of sepsis. METHODS: Ten-week-old C57BL/6 mice were subjected to sepsis by cecal ligation and puncture or intraperitoneal injection of Escherichia coli or Pseudomonas aeruginosa pneumonia. CSL-111 or saline solution was administrated 2 h after the sepsis. Primary outcome was survival. Secondary outcomes were plasma cell-free DNA and cytokine concentrations, histology, bacterial count, and biodistribution. RESULTS: Compared with saline, CSL-111 improved survival in cecal ligation and puncture and intraperitoneal models (13 of 16 [81%] survival rate vs. 6 of 16 [38%] in the cecal ligation and puncture model; P = 0.011; 4 of 10 [40%] vs. 0 of 10 [0%] in the intraperitoneal model; P = 0.011). Cell-free DNA concentration was lower in CSL-111 relative to saline groups (68 [24 to 123] pg/ml vs. 351 [333 to 683] pg/ml; P < 0.001). Mice injected with CSL-111 presented a decreased bacterial count at 24 h after the cecal ligation and puncture model both in plasma (200 [28 to 2,302] vs. 2,500 [953 to 3,636] colony-forming unit/ml; P = 0.021) and in the liver (1,359 [360 to 1,648] vs. 1,808 [1,464 to 2,720] colony-forming unit/ml; P = 0.031). In the pneumonia model, fewer bacteria accumulated in liver and lung of the CSL-111 group. CSL-111-injected mice had also less lung inflammation versus saline mice (CD68+ to total cells ratio: saline, 0.24 [0.22 to 0.27]; CSL-111, 0.07 [0.01 to 0.09]; P < 0.01). In all models, no difference was found for cytokine concentration. Indium bacterial labeling underlined a potential hepatic bacterial clearance possibly promoted by high-density lipoprotein uptake. CONCLUSIONS: CSL-111 infusion improved survival in different experimental mouse models of sepsis. It reduced inflammation in both plasma and organs and decreased bacterial count. These results emphasized the key role for high-density lipoproteins in endothelial and organ protection, but also in lipopolysaccharide/bacteria clearance. This suggests an opportunity to explore the therapeutic potential of high-density lipoproteins in septic conditions.