Profiling Intact Glycosphingolipids with Automated Structural Annotation and Quantitation from Human Samples with Nanoflow Liquid Chromatography Mass Spectrometry.

Sphingolipids are an essential subset of bioactive lipids found in most eukaryotic cells that contribute to membrane biophysical properties and are involved in cellular differentiation, recognition, and mediating interactions. The described nanoHPLC-ESI-Q/ToF methodology utilizes known biosynthetic pathways, accurate mass detection, optimized collision-induced disassociation, and a robust nanoflow chromatographic separation for the analysis of intact sphingolipids found in human tissue, cells, and serum. The methodology was developed and validated with an emphasis on addressing the common issues experienced in profiling these amphipathic lipids, which are part of the glycocalyx and lipidome. The high sensitivity obtained using nanorange flow rates with robust chromatographic reproducibility over a wide range of concentrations and injection volumes results in confident identifications for profiling these low-abundant biomolecules.

Regio-Specific N-Glycome and N-Glycoproteome Map of the Elderly Human Brain With and Without Alzheimer's Disease.

The proteins in the cell membrane of the brain are modified by glycans in highly interactive regions. The glycans and glycoproteins are involved in cell-cell interactions that are of fundamental importance to the brain. In this study, the comprehensive N-glycome and N-glycoproteome of the brain were determined in 11 functional brain regions, some of them known to be affected with the progression of Alzheimer's disease. N-glycans throughout the regions were generally highly branched and highly sialofucosylated. Regional variations were also found with regard to the glycan types including high mannose and complex-type structures. Glycoproteomic analysis identified the proteins that differed in glycosylation in the various regions. To obtain the broader representation of glycan compositions, four subjects with two in their 70s and two in their 90s representing two Alzheimer's disease subjects, one hippocampal sclerosis subject, and one subject with no cognitive impairment were analyzed. The four subjects were all glycomically mapped across 11 brain regions. Marked differences in the glycomic and glycoproteomic profiles were observed between the samples.

The role of FUT8-catalyzed core fucosylation in Alzheimer's amyloid-ß oligomer-induced activation of human microglia.

Fucosylation, especially core fucosylation of N-glycans catalyzed by α1-6 fucosyltransferase (fucosyltransferase 8 or FUT8), plays an important role in regulating the peripheral immune system and inflammation. However, its role in microglial activation is poorly understood. Here we used human induced pluripotent stem cells-derived microglia (hiMG) as a model to study the role of FUT8-catalyzed core fucosylation in amyloid-ß oligomer (AßO)-induced microglial activation, in view of its significant relevance to the pathogenesis of Alzheimer's disease (AD). HiMG responded to AßO and lipopolysaccharides (LPS) with a pattern of pro-inflammatory activation as well as enhanced core fucosylation and FUT8 expression within 24 h. Furthermore, we found increased FUT8 expression in both human AD brains and microglia isolated from 5xFAD mice, a model of AD-like cerebral amyloidosis. Inhibition of fucosylation in AßO-stimulated hiMG reduced the induction of pro-inflammatory cytokines, suppressed the activation of p38MAPK, and rectified phagocytic deficits. Specific inhibition of FUT8 by siRNA-mediated knockdown also reduced AßO-induced pro-inflammatory cytokines. We further showed that p53 binds to the two consensus binding sites in the Fut8 promoter, and that p53 knockdown abolished FUT8 overexpression in AßO-activated hiMG. Taken together, our evidence supports that FUT8-catalyzed core fucosylation is a signaling pathway required for AßO-induced microglia activation and that FUT8 is a component of the p53 signaling cascade regulating microglial behavior. Because microglia are a key driver of AD pathogenesis, our results suggest that microglial FUT8 could be an anti-inflammatory therapeutic target for AD.

Precision Nutrition and Cardiovascular Disease Risk Reduction: the Promise of High-Density Lipoproteins.

PURPOSE OF REVIEW: Emerging evidence supports the promise of precision nutritional approaches for cardiovascular disease (CVD) prevention. Here, we discuss current findings from precision nutrition trials and studies reporting substantial inter-individual variability in responses to diets and dietary components relevant to CVD outcomes. We highlight examples where early precision nutrition research already points to actionable intervention targets tailored to an individual's biology and lifestyle. Finally, we make the case for high-density lipoproteins (HDL) as a compelling next generation target for precision nutrition aimed at CVD prevention. HDL possesses complex structural features including diverse protein components, lipids, size distribution, extensive glycosylation, and interacts with the gut microbiome, all of which influence HDL's anti-inflammatory, antioxidant, and cholesterol efflux properties. Elucidating the nuances of HDL structure and function at an individual level may unlock personalized dietary and lifestyle strategies to optimize HDL-mediated atheroprotection and reduce CVD risk. RECENT FINDINGS: Recent human studies have demonstrated that HDL particles are key players in the reduction of CVD risk. Our review highlights the role of HDL and the importance of personalized therapeutic approaches to improve their potential for reducing CVD risk. Factors such as diet, genetics, glycosylation, and gut microbiome interactions can modulate HDL structure and function at the individual level. We emphasize that fractionating HDL into size-based subclasses and measuring particle concentration are necessary to understand HDL biology and for developing the next generation of diagnostics and biomarkers. These discoveries underscore the need to move beyond a one-size-fits-all approach to HDL management. Precision nutrition strategies that account for personalized metabolic, genetic, and lifestyle data hold promise for optimizing HDL therapies and function to mitigate CVD risk more potently. While human studies show HDL play a key role in reducing CVD risk, recent findings indicate that factors such as diet, genetics, glycosylation, and gut microbes modulate HDL function at the individual level, underscoring the need for precision nutrition strategies that account for personalized variability to optimize HDL's potential for mitigating CVD risk.

HDL Function across the Lifespan: From Childhood, to Pregnancy, to Old Age.

The function of high-density lipoprotein (HDL) particles has emerged as a promising therapeutic target and the measurement of HDL function is a promising diagnostic across several disease states. The vast majority of research on HDL functional biology has focused on adult participants with underlying chronic diseases, whereas limited research has investigated the role of HDL in childhood, pregnancy, and old age. Yet, it is apparent that functional HDL is essential at all life stages for maintaining health. In this review, we discuss current data regarding the role of HDL during childhood, pregnancy and in the elderly, how disturbances in HDL may lead to adverse health outcomes, and knowledge gaps in the role of HDL across these life stages.

Cholesterol, Amyloid Beta, Fructose, and LPS Influence ROS and ATP Concentrations and the Phagocytic Capacity of HMC3 Human Microglia Cell Line.

Research has found that genes specific to microglia are among the strongest risk factors for Alzheimer's disease (AD) and that microglia are critically involved in the etiology of AD. Thus, microglia are an important therapeutic target for novel approaches to the treatment of AD. High-throughput in vitro models to screen molecules for their effectiveness in reversing the pathogenic, pro-inflammatory microglia phenotype are needed. In this study, we used a multi-stimulant approach to test the usefulness of the human microglia cell 3 (HMC3) cell line, immortalized from a human fetal brain-derived primary microglia culture, in duplicating critical aspects of the dysfunctional microglia phenotype. HMC3 microglia were treated with cholesterol (Chol), amyloid beta oligomers (AßO), lipopolysaccharide (LPS), and fructose individually and in combination. HMC3 microglia demonstrated changes in morphology consistent with activation when treated with the combination of Chol + AßO + fructose + LPS. Multiple treatments increased the cellular content of Chol and cholesteryl esters (CE), but only the combination treatment of Chol + AßO + fructose + LPS increased mitochondrial Chol content. Microglia treated with combinations containing Chol + AßO had lower apolipoprotein E (ApoE) secretion, with the combination of Chol + AßO + fructose + LPS having the strongest effect. Combination treatment with Chol + AßO + fructose + LPS also induced APOE and TNF-α expression, reduced ATP production, increased reactive oxygen species (ROS) concentration, and reduced phagocytosis events. These findings suggest that HMC3 microglia treated with the combination of Chol + AßO + fructose + LPS may be a useful high-throughput screening model amenable to testing on 96-well plates to test potential therapeutics to improve microglial function in the context of AD.

Human Milk Oligosaccharide Compositions Illustrate Global Variations in Early Nutrition.

BACKGROUND: Human milk oligosaccharides (HMOs) are an abundant class of compounds found in human milk and have been linked to the development of the infant, and specifically the brain, immune system, and gut microbiome. OBJECTIVES: Advanced analytical methods were used to obtain relative quantitation of many structures in approximately 2000 samples from over 1000 mothers in urban, semirural, and rural sites across geographically diverse countries. METHODS: LC-MS-based analytical methods were used to profile the compounds with broad structural coverage and quantitative information. The profiles revealed their structural heterogeneity and their potential biological roles. Comparisons of HMO compositions were made between mothers of different age groups, lactation periods, infant sexes, and residing geographical locations. RESULTS: A common behavior found among all sites was a decrease in HMO abundances during lactation until approximately postnatal month 6, where they remained relatively constant. The greatest variations in structural abundances were associated with the presence of α(1,2)-fucosylated species. Genomic analyses of the mothers were not performed; instead, milk was phenotyped according to the abundances of α(1,2)-fucosylated structures. Mothers from the South American sites tended to have higher proportions of phenotypic secretors [mothers with relatively high concentrations of α(1,2)-fucosylated structures] in their populations compared to the rest of the globe, with Bolivia at ∼100% secretors, Peru at ∼97%, Brazil at ∼90%, and Argentina at ∼85%. Conversely, the cohort sampled in Africa manifested the lowest proportion of secretors (South Africa ∼ 63%, the Gambia ∼ 64%, and Malawi ∼ 75%). Furthermore, we compared total abundances of HMOs in secretors compared with nonsecretors and found that nonsecretors have lower abundances of HMOs compared to secretors, regardless of geographical location. We also observed compositional differences of the 50+ most abundant HMOs between milk types and geographical locations. CONCLUSIONS: This study represents the largest structural HMO study to date and reveals the general behavior of HMOs during lactation among different populations.

Proceedings of the Ninth HDL (High-Density Lipoprotein) Workshop: Focus on Cardiovascular Disease.

The HDL (high-density lipoprotein) Workshop was established in 2009 as a forum for candid discussions among academic basic scientists, clinical investigators, and industry researchers about the role of HDL in cardiovascular disease. This ninth HDL Workshop was held on May 16 to 17, 2019 in Boston, MA, and included outstanding oral presentations from established and emerging investigators. The Workshop featured 5 sessions with topics that tackled the role of HDL in the vasculature, its structural complexity, its role in health and disease states, and its interaction with the intestinal microbiome. The highlight of the program was awarding the Jack Oram Award to the distinguished professor emeritus G.S. Getz from the University of Chicago. The tenth HDL Workshop will be held on May 2020 in Chicago and will continue the focus on intellectually stimulating presentations by established and emerging investigators on novel roles of HDL in cardiovascular and noncardiovascular health and disease states.

Site-Specific Glycoprofiles of HDL-Associated ApoE are Correlated with HDL Functional Capacity and Unaffected by Short-Term Diet.

Since high-density lipoprotein (HDL) glycoprofiles are associated with HDL functional capacity, we set out to determine whether diet can alter the glycoprofiles of key HDL-associated proteins, including ApoE, a potent driver of chronic disease risk. Ten healthy subjects consumed a fast food (FF) and a Mediterranean (Med) diet for 4 days in randomized order, with a 4-day wash-out between treatments. A multiple reaction monitoring method was used to characterize the site-specific glycoprofiles of HDL proteins, and HDL functional capacity was analyzed. We describe for the first time that ApoE has 7 mucin-type O-glycosylation sites, which were not affected by short-term diet. The glycoprofiles of other HDL-associated proteins were also unaffected, except that a disialylated ApoC-III glycan was enriched after Med diet, and a nonsialylated ApoC-III glycan was enriched after FF diet. Twenty-five individual glycopeptides were significantly correlated with cholesterol efflux capacity and 21 glycopeptides were correlated with immunomodulatory capacity. Results from this study indicate that the glycoprofiles of HDL-associated proteins including ApoE are correlated with HDL functional capacity but generally unaffected by diet in the short term, except ApoC-III sialylation. These results suggest that HDL protein glycoprofiles are affected by both acute and long-term factors and may be useful for biomarker discovery.

The HDL lipidome is widely remodeled by fast food versus Mediterranean diet in 4 days.

INTRODUCTION: HDL is associated with increased longevity and protection from multiple chronic diseases. The major HDL protein ApoA-I has a half-life of about 4 days, however, the effects of diet on the composition of HDL particles at this time scale have not been studied. OBJECTIVES: The objective of this study is to investigate the short term dietary effect on HDL lipidomic composition. METHODS: In this randomized order cross-over study, ten healthy subjects consumed a Mediterranean (Med) and a fast food (FF) diet for 4 days, with a 4-day wash-out between treatments. Lipidomic composition was analyzed in isolated HDL fractions by an untargeted LC-MS method with 15 internal standards. RESULTS: HDL phosphatidylethanolamine (PE) content was increased by FF diet, and 41 out of 170 lipid species were differentially affected by diet. Saturated fatty acids (FAs) and odd chain FA were enriched after FF diet, while very-long chain FA and unsaturated FA were enriched after Med diet. The composition of phosphatidylcholine (PC), triacylglycerol (TG) and cholesteryl ester (CE) were significantly altered to reflect the FA composition of the diet whereas the composition of sphingomyelin (SM) and ceramides were generally unaffected. CONCLUSION: Results from this study indicate that the HDL lipidome is widely remodeled within 4 days of diet change and that certain lipid classes are more sensitive markers of diet whereas other lipid classes are better indicators of non-dietary factors.

Targeted Measurements of O- and N-Glycopeptides Show That Proteins in High Density Lipoprotein Particles Are Enriched with Specific Glycosylation Compared to Plasma.

High density lipoprotein (HDL) particles are believed to be protective due to their inverse correlation with the prevalence of cardiovascular diseases. However, recent studies show that in some conditions such as heart disease and diabetes, HDL particles can become dysfunctional. Great attention has been directed toward HDL particle composition because the relative abundances of HDL constituents determine HDL's functional properties. A key factor to consider when studying the structure and composition of plasma particles is the protein glycosylation. Here, we profile the O- and N-linked glycosylation of HDL associated-proteins including the truncated form of Apo CIII and their glycan heterogeneity in a site-specific manner. Apolipoprotein CIII, fetuin A, and alpha 1 antitrypsin are glycoproteins associated with lipoproteins and are implicated in many cardiovascular and other disease conditions. A targeted method (UHPLC-QQQ) was used to measure the glycoprotein concentrations and site-specific glycovariations of the proteins in human plasma and compared with HDL particles isolated from the same plasma samples. The proteins found in the plasma are differentially glycosylated compared to those isolated in HDL. The results of this study suggest that glycosylation may play a role in protein partitioning in the blood, with possible functional implications.

NMR-based metabolite profiling of human milk: A pilot study of methods for investigating compositional changes during lactation.

Low-molecular-weight metabolites in human milk are gaining increasing interest in studies of infant nutrition. In the present study, the milk metabolome from a single mother was explored at different stages of lactation. Metabolites were extracted from sample aliquots using either methanol/water (MeOH/H2O) extraction or ultrafiltration. Nuclear magnetic resonance (NMR) spectroscopy was used for metabolite identification and quantification, and multi- and univariate statistical data analyses were used to detect changes over time of lactation. Compared to MeOH/H2O extraction, ultrafiltration more efficiently reduced the interference from lipid and protein resonances, thereby enabling the identification and quantification of 36 metabolites. The human milk metabolomes at the early (9-24 days after delivery) and late (31-87 days after delivery) stages of lactation were distinctly different according to multi- and univariate statistics. The late lactation stage was characterized by significantly elevated concentrations of lactose, choline, alanine, glutamate, and glutamine, as well as by reduced levels of citrate, phosphocholine, glycerophosphocholine, and N-acetylglucosamine. Our results indicate that there are significant compositional changes of the human milk metabolome also in different phases of the matured lactation stage. These findings complement temporal studies on the colostrum and transitional metabolome in providing a better understanding of the nutritional variations received by an infant.

Oxylipins, endocannabinoids, and related compounds in human milk: Levels and effects of storage conditions.

The presence of fatty acid derived oxylipins, endocannabinoids and related compounds in human milk may be of importance to the infant. Presently, clinically relevant protocols for storing and handling human milk that minimize error and variability in oxylipin and endocannabinoid concentrations are lacking. In this study, we compared the individual and combined effects of the following storage conditions on the stability of these fatty acid metabolites in human milk: state (fresh or frozen), storage temperature (4 °C, -20 °C or -80 °C), and duration (1 day, 1 week or 3 months). Thirteen endocannabinoids and related compounds, as well as 37 oxylipins were analyzed simultaneously by liquid chromatography coupled to tandem mass spectrometry. Twelve endocannabinoids and related compounds (2-111 nM) and 31 oxylipins (1.2 pM-1242 nM) were detected, with highest levels being found for 2-arachidonoylglycerol and 17(R)hydroxydocosahexaenoic acid, respectively. The concentrations of most endocannabinoid-related compounds and oxylipins were dependent on storage condition, and especially storage at 4 °C introduced significant variability. Our findings suggest that human milk samples should be analyzed immediately after, or within one day of collection (if stored at 4 °C). Storage at -80 °C is required for long-term preservation, and storage at -20 °C is acceptable for no more than one week. These findings provide a protocol for investigating the oxylipin and endocannabinoid metabolome in human milk, useful for future milk-related clinical studies.

Combined High-Density Lipoprotein Proteomic and Glycomic Profiles in Patients at Risk for Coronary Artery Disease.

OBJECTIVES: To test whether recently developed methods for comprehensive profiling of the high-density lipoprotein (HDL) glycome combined with the HDL proteome can distinguish individuals with coronary artery disease (CAD) from those without. METHODS: Twenty subjects at risk for CAD, who underwent diagnostic coronary arteriography, were analyzed. Ten subjects had CAD, and ten did not. HDL was extracted from fasting plasma samples by ultracentrifugation, followed by shotgun proteomic, glycomic, and ganglioside analyses using LC-MS. CAD vs non-CAD subjects' data were compared using univariate and multivariate statistics. RESULTS: Principal components analysis showed a clear separation of CAD and non-CAD subjects, confirming that combined HDL proteomic and glycomic profiles distinguished at-risk subjects with atherosclerosis from those without. CAD patients had lower HDL apolipoprotein content (specifically ApoA-I, A-II, and E, p < 0.05), and lower serum amyloid A2 (SAA2, p = 0.020) and SAA4 (p = 0.007) but higher sialylated glycans (p < 0.05). CONCLUSION: Combined proteomic and glycomic profiling of isolated HDL was tested as a novel analytical approach for developing biomarkers of disease. In this pilot study we found that HDL proteome and glycome distinguished between individuals who had CAD from those who did not within a group of individuals equally at risk for heart disease.

Glycomic analysis of high density lipoprotein shows a highly sialylated particle.

Many of the functional proteins and lipids in high density lipoprotein (HDL) particles are potentially glycosylated, yet very little is known about the glycoconjugates of HDL. In this study, HDL was isolated from plasma by sequential micro-ultracentrifugation, followed by glycoprotein and glycolipid analysis. N-Glycans, glycopeptides, and gangliosides were extracted and purified followed by analysis with nano-HPLC Chip quadrupole time of flight mass spectrometry and MS/MS. HDL particles were found to be highly sialylated. Most of the N-glycans (∼90%) from HDL glycoproteins were sialylated with one or two neuraminic acids (Neu5Ac). The most abundant N-glycan was a biantennary complex type glycan with two sialic acids (Hexose5HexNAc4Neu5Ac2) and was found in multiple glycoproteins using site-specific glycosylation analysis. The observed O-glycans were all sialylated, and most contained a core 1 structure with two Neu5Acs, including those that were associated with apolipoprotein CIII (ApoC-III) and fetuin A. GM3 (monosialoganglioside, NeuAc2-3Gal1-4Glc-Cer) and GD3 (disialoganglioside, NeuAc2-8NeuAc2-3Gal1-4Glc-Cer) were the major gangliosides in HDL. A 60% GM3 and 40% GD3 distribution was observed. Both GM3 and GD3 were composed of heterogeneous ceramide lipid tails, including d18:1/16:0 and d18:1/23:0. This report describes for the first time a glycomic approach for analyzing HDL, highlighting that HDL are highly sialylated particles.

Human milk glycobiome and its impact on the infant gastrointestinal microbiota.

Human milk contains an unexpected abundance and diversity of complex oligosaccharides apparently indigestible by the developing infant and instead targeted to its cognate gastrointestinal microbiota. Recent advances in mass spectrometry-based tools have provided a view of the oligosaccharide structures produced in milk across stages of lactation and among human mothers. One postulated function for these oligosaccharides is to enrich a specific "healthy" microbiota containing bifidobacteria, a genus commonly observed in the feces of breast-fed infants. Isolated culture studies indeed show selective growth of infant-borne bifidobacteria on milk oligosaccharides or core components therein. Parallel glycoprofiling documented that numerous Bifidobacterium longum subsp. infantis strains preferentially consume small mass oligosaccharides that are abundant early in the lactation cycle. Genome sequencing of numerous B. longum subsp. infantis strains shows a bias toward genes required to use mammalian-derived carbohydrates by comparison with adult-borne bifidobacteria. This intriguing strategy of mammalian lactation to selectively nourish genetically compatible bacteria in infants with a complex array of free oligosaccharides serves as a model of how to influence the human supraorganismal system, which includes the gastrointestinal microbiota.

Elevated lipopolysaccharide binding protein in Alzheimer's disease patients with APOE3/E3 but not APOE3/E4 genotype.

Introduction: The role of lipopolysaccharide binding protein (LBP), an inflammation marker of bacterial translocation from the gastrointestinal tract, in Alzheimer's disease (AD) is not clearly understood. Methods: In this study the concentrations of LBP were measured in n = 79 individuals: 20 apolipoprotein E (APOE)3/E3 carriers with and 20 without AD dementia, and 19 APOE3/E4 carriers with and 20 without AD dementia. LBP was found to be enriched in the 1.21-1.25 g/mL density fraction of plasma, which has previously been shown to be enriched in intestinally derived high-density lipoproteins (HDL). LBP concentrations were measured by ELISA. Results: LBP was significantly increased within the 1.21-1.25 g/mL density fraction of plasma in APOE3/E3 AD patients compared to controls, but not APOE3/E4 patients. LBP was positively correlated with Clinical Dementia Rating (CDR) and exhibited an inverse relationship with Verbal Memory Score (VMS). Discussion: These results underscore the potential contribution of gut permeability to bacterial toxins, measured as LBP, as an inflammatory mediator in the development of AD, particularly in individuals with the APOE3/E3 genotype, who are genetically at 4-12-fold lower risk of AD than individuals who express APOE4.

N-Glycan profile of the cell membrane as a probe for lipopolysaccharide-induced microglial neuroinflammation uncovers the effects of common fatty acid supplementation.

Altered N-glycosylation of proteins on the cell membrane is associated with several neurodegenerative diseases. Microglia are an ideal model for studying glycosylation and neuroinflammation, but whether aberrant N-glycosylation in microglia can be restored by diet remains unknown. Herein, we profiled the N-glycome, proteome, and glycoproteome of the human microglia following lipopolysaccharide (LPS) induction to probe the impact of dietary and gut microbe-derived fatty acids-oleic acid, lauric acid, palmitic acid, valeric acid, butyric acid, isobutyric acid, and propionic acid-on neuroinflammation using liquid chromatography-tandem mass spectrometry. LPS changed N-glycosylation in the microglial glycocalyx altering high mannose and sialofucosylated N-glycans, suggesting the dysregulation of mannosidases, fucosyltransferases, and sialyltransferases. The results were consistent as we observed the restoration effect of the fatty acids, especially oleic acid, on the LPS-treated microglia, specifically on the high mannose and sialofucosylated glycoforms of translocon-associated proteins, SSRA and SSRB along with the cell surface proteins, CD63 and CD166. In addition, proteomic analysis and in silico modeling substantiated the potential of fatty acids in reverting the effects of LPS on microglial N-glycosylation. Our results showed that N-glycosylation is likely affected by diet by restoring alterations following LPS challenge, which may then influence the disease state.

Lutein and Zeaxanthin Enhance, Whereas Oxidation, Fructosylation, and Low pH Damage High-Density Lipoprotein Biological Functionality.

High-density lipoproteins (HDLs) are key regulators of cellular cholesterol homeostasis but are functionally altered in many chronic diseases. The factors that cause HDL functional loss in chronic disease are not fully understood. It is also unknown what roles antioxidant carotenoids play in protecting HDL against functional loss. The aim of this study was to measure how various disease-associated chemical factors including exposure to (1) Cu2+ ions, (2) hypochlorous acid (HOCL), (3) hydrogen peroxide (H2O2), (4) sialidase, (5) glycosidase, (6) high glucose, (7) high fructose, and (8) acidic pH, and the carotenoid antioxidants (9) lutein and (10) zeaxanthin affect HDL functionality. We hypothesized that some of the modifications would have stronger impacts on HDL particle structure and function than others and that lutein and zeaxanthin would improve HDL function. HDL samples were isolated from generally healthy human plasma and incubated with the corresponding treatments listed above. Cholesterol efflux capacity (CEC), lecithin-cholesterol acyl transferase (LCAT) activity, and paraoxonase-1 (PON1) activity were measured in order to determine changes in HDL functionality. Median HDL particle diameter was increased by acidic pH treatment and reduced by HOCl, high glucose, high fructose, N-glycosidase, and lutein treatments. Acidic pH, oxidation, and fructosylation all reduced HDL CEC, whereas lutein, zeaxanthin, and sialidase treatment improved HDL CEC. LCAT activity was reduced by acidic pH, oxidation, high fructose treatments, and lutein. PON1 activity was reduced by sialidase, glycosidase, H2O2, and fructose and improved by zeaxanthin and lutein treatment. These results show that exposure to oxidizing agents, high fructose, and low pH directly impairs HDL functionality related to cholesterol efflux and particle maturation, whereas deglycosylation impairs HDL antioxidant capacity. On the other hand, the antioxidants lutein and zeaxanthin improve or preserve both HDL cholesterol efflux and antioxidant activity but have no effect on particle maturation.

Human fasting modulates macrophage function and upregulates multiple bioactive metabolites that extend lifespan in Caenorhabditis elegans: a pilot clinical study.

BACKGROUND: Periodic prolonged fasting (PF) extends lifespan in model organisms and ameliorates multiple disease states both clinically and experimentally owing, in part, to its ability to modulate the immune system. However, the relationship between metabolic factors, immunity, and longevity during PF remains poorly characterized especially in humans. OBJECTIVE: This study aimed to observe the effects of PF in human subjects on the clinical and experimental markers of metabolic and immune health and uncover underlying plasma-borne factors that may be responsible for these effects. METHODS: In this rigorously controlled pilot study (ClinicalTrial.gov identifier, NCT03487679), 20 young males and females participated in a 3-d study protocol including assessments of 4 distinct metabolic states: 1) overnight fasted baseline state, 2) 2-h postprandial fed state, 3) 36-h fasted state, and 4) final 2-h postprandial re-fed state 12 h after the 36-h fasting period. Clinical and experimental markers of immune and metabolic health were assessed for each state along with comprehensive metabolomic profiling of participant plasma. Bioactive metabolites identified to be upregulated in circulation after 36 h of fasting were then assessed for their ability to mimic the effects of fasting in isolated human macrophage as well as the ability to extend lifespan in Caenorhabditis elegans. RESULTS: We showed that PF robustly altered the plasma metabolome and conferred beneficial immunomodulatory effects on human macrophages. We also identified 4 bioactive metabolites that were upregulated during PF (spermidine, 1-methylnicotinamide, palmitoylethanolamide, and oleoylethanolamide) that could replicate these immunomodulatory effects. Furthermore, we found that these metabolites and their combination significantly extended the median lifespan of C. elegans by as much as 96%. CONCLUSIONS: The results of this study reveal multiple functionalities and immunological pathways affected by PF in humans, identify candidates for the development of fasting mimetic compounds, and uncover targets for investigation in longevity research.