Neuronal SIRT3 Deletion Predisposes to Female-Specific Alterations in Cellular Metabolism, Memory, and Network Excitability.

Mitochondrial dysfunction is an early event in the pathogenesis of neurologic disorders and aging. Sirtuin 3 (SIRT3) regulates mitochondrial function in response to the cellular environment through the reversible deacetylation of proteins involved in metabolism and reactive oxygen species detoxification. As the primary mitochondrial deacetylase, germline, or peripheral tissue-specific deletion of SIRT3 produces mitochondrial hyperacetylation and the accelerated development of age-related diseases. Given the unique metabolic demands of neurons, the role of SIRT3 in the brain is only beginning to emerge. Using mass spectrometry-based acetylomics, high-resolution respirometry, video-EEG, and cognition testing, we report targeted deletion of SIRT3 from select neurons in the cortex and hippocampus produces altered neuronal excitability and metabolic dysfunction in female mice. Targeted deletion of SIRT3 from neuronal helix-loop-helix 1 (NEX)-expressing neurons resulted in mitochondrial hyperacetylation, female-specific superoxide dismutase-2 (SOD2) modification, increased steady-state superoxide levels, metabolic reprogramming, altered neuronal excitability, and working spatial memory deficits. Inducible neuronal deletion of SIRT3 likewise produced female-specific deficits in spatial working memory. Together, the data demonstrate that deletion of SIRT3 from forebrain neurons selectively predisposes female mice to deficits in mitochondrial and cognitive function.SIGNIFICANCE STATEMENT Mitochondrial SIRT3 is an enzyme shown to regulate energy metabolism and antioxidant function, by direct deacetylation of proteins. In this study, we show that neuronal SIRT3 deficiency renders female mice selectively vulnerable to impairment in redox and metabolic function, spatial memory, and neuronal excitability. The observed sex-specific effects on cognition and neuronal excitability in female SIRT3-deficient mice suggest that mitochondrial dysfunction may be one factor underlying comorbid neuronal diseases, such as Alzheimer's disease and epilepsy. Furthermore, the data suggest that SIRT3 dysfunction may predispose females to age-related metabolic and cognitive impairment.

Consuming Mushrooms When Adopting a Healthy Mediterranean-Style Dietary Pattern Does Not Influence Short-Term Changes of Most Cardiometabolic Disease Risk Factors in Healthy Middle-Aged and Older Adults.

BACKGROUND: Mushrooms are a nutritious food, though knowledge of the effects of mushroom consumption on cardiometabolic risk factors is limited and inconsistent. OBJECTIVE: We assessed the effects of consuming mushrooms as part of a healthy United States Mediterranean-style dietary pattern (MED) on traditional and emerging cardiometabolic disease (CMD) risk factors. We hypothesized that adopting a MED diet with mushrooms would lead to greater improvements in multiple CMD risk factors. METHODS: Using a randomized, parallel study design, 60 adults (36 females, 24 males; aged 46 ± 12 y; body mass index 28.3 ± 2.84 kg/m2, mean ± standard deviation) without diagnosed CMD morbidities consumed a MED diet (all foods provided) without (control with breadcrumbs) or with 84 g/d of Agaricus bisporus (White Button, 4 d/wk) and Pleurotus ostreatus (Oyster, 3 d/wk) mushrooms for 8 wk. Fasting baseline and postintervention outcome measurements were traditional CMD risk factors, including blood pressure and fasting serum lipids, lipoproteins, glucose, and insulin. Exploratory CMD-related outcomes included lipoprotein particle sizes and indexes of inflammation. RESULTS: Adopting the MED-mushroom diet compared with the MED-control diet without mushrooms improved fasting serum glucose (change from baseline -2.9 ± 1.18 compared with 0.6 ± 1.10 mg/dL; time × group P = 0.034). Adopting the MED diet, independent of mushroom consumption, reduced serum total cholesterol (-10.2 ± 3.77 mg/dL; time P = 0.0001). Concomitantly, there was a reduction in high-density lipoprotein (HDL) cholesterol, buoyant HDL2b, and apolipoprotein A1, and an increase in lipoprotein(a) concentrations (main effect of time P < 0.05 for all). There were no changes in other measured CMD risk factors. CONCLUSIONS: Consuming a Mediterranean-style healthy dietary pattern with 1 serving/d of whole Agaricus bisporus and Pleurotus ostreatus mushrooms improved fasting serum glucose but did not influence other established or emerging CMD risk factors among middle-aged and older adults classified as overweight or obese but with clinically normal cardiometabolic health. TRIAL REGISTRATION NUMBER: https://www. CLINICALTRIALS: gov/study/NCT04259229?term=NCT04259229&rank=1.

Salmon Food-Specific Compounds and Their Metabolites Increase in Human Plasma and Are Associated with Cardiometabolic Health Indicators Following a Mediterranean-Style Diet Intervention.

BACKGROUND: Nutrimetabolomics allows for the comprehensive analysis of foods and human biospecimens to identify biomarkers of intake and begin to probe their associations with health. Salmon contains hundreds of compounds that may provide cardiometabolic benefits. OBJECTIVES: We used untargeted metabolomics to identify salmon food-specific compounds (FSCs) and their predicted metabolites that were found in plasma after a salmon-containing Mediterranean-style (MED) diet intervention. Associations between changes in salmon FSCs and changes in cardiometabolic health indicators (CHIs) were also explored. METHODS: For this secondary analysis of a randomized, crossover, controlled feeding trial, 41 participants consumed MED diets with 2 servings of salmon per week for 2 5-wk periods. CHIs were assessed, and fasting plasma was collected pre- and postintervention. Plasma, salmon, and 99 MED foods were analyzed using liquid chromatography-mass spectrometry-based metabolomics. Compounds were characterized as salmon FSCs if detected in all salmon replicates but none of the other foods. Metabolites of salmon FSCs were predicted using machine learning. For salmon FSCs and metabolites found in plasma, linear mixed-effect models were used to assess change from pre- to postintervention and associations with changes in CHIs. RESULTS: Relative to the other 99 MED foods, there were 508 salmon FSCs with 237 unique metabolites. A total of 143 salmon FSCs and 106 metabolites were detected in plasma. Forty-eight salmon FSCs and 30 metabolites increased after the intervention (false discovery rate <0.05). Increases in 2 annotated salmon FSCs and 2 metabolites were associated with improvements in CHIs, including total cholesterol, low-density lipoprotein cholesterol, triglycerides, and apolipoprotein B. CONCLUSIONS: A data-driven nutrimetabolomics strategy identified salmon FSCs and their predicted metabolites that were detectable in plasma and changed after consumption of a salmon-containing MED diet. Findings support this approach for the discovery of compounds in foods that may serve, upon further validation, as biomarkers or act as bioactive components influential to health. The trials supporting this work were registered at NCT02573129 (Mediterranean-style diet intervention) and NCT05500976 (ongoing clinical trial).

Docosahexaenoic Acid Controls Pulmonary Macrophage Lipid Raft Size and Inflammation.

BACKGROUND: Docosahexaenoic acid (DHA) controls the biophysical organization of plasma membrane sphingolipid/cholesterol-enriched lipid rafts to exert anti-inflammatory effects, particularly in lymphocytes. However, the impact of DHA on the spatial arrangement of alveolar macrophage lipid rafts and inflammation is unknown. OBJECTIVES: The primary objective was to determine how DHA controls lipid raft organization and function of alveolar macrophages. As proof-of-concept, we also investigated DHA's anti-inflammatory effects on select pulmonary inflammatory markers with a murine influenza model. METHODS: MH-S cells, an alveolar macrophage line, were treated with 50 µM DHA or vehicle control and were used to study plasma membrane molecular organization with fluorescence-based methods. Biomimetic membranes and coarse grain molecular dynamic (MD) simulations were employed to investigate how DHA mechanistically controls lipid raft size. qRT-PCR, mass spectrometry, and ELISAs were used to quantify downstream inflammatory signaling transcripts, oxylipins, and cytokines, respectively. Lungs from DHA-fed influenza-infected mice were analyzed for specific inflammatory markers. RESULTS: DHA increased the size of lipid rafts while decreasing the molecular packing of the MH-S plasma membrane. Adding a DHA-containing phospholipid to a biomimetic lipid raft-containing membrane led to condensing, which was reversed with the removal of cholesterol. MD simulations revealed DHA nucleated lipid rafts by driving cholesterol and sphingomyelin into rafts. Downstream of the plasma membrane, DHA lowered the concentration of select inflammatory transcripts, oxylipins, and IL-6 secretion. DHA lowered pulmonary Il6 and Tnf-α mRNA expression and increased anti-inflammatory oxylipins of influenza-infected mice. CONCLUSIONS: The data suggest a model in which the localization of DHA acyl chains to nonrafts is driving sphingomyelin and cholesterol molecules into larger lipid rafts, which may serve as a trigger to impede signaling and lower inflammation. These findings also identify alveolar macrophages as a target of DHA and underscore the anti-inflammatory properties of DHA for lung inflammation.

Current Practices in LC-MS Untargeted Metabolomics: A Scoping Review on the Use of Pooled Quality Control Samples.

Untargeted metabolomics is an analytical approach with numerous applications serving as an effective metabolic phenotyping platform to characterize small molecules within a biological system. Data quality can be challenging to evaluate and demonstrate in metabolomics experiments. This has driven the use of pooled quality control (QC) samples for monitoring and, if necessary, correcting for analytical variance introduced during sample preparation and data acquisition stages. Described herein is a scoping literature review detailing the use of pooled QC samples in published untargeted liquid chromatography-mass spectrometry (LC-MS) based metabolomics studies. A literature query was performed, the list of papers was filtered, and suitable articles were randomly sampled. In total, 109 papers were each reviewed by at least five reviewers, answering predefined questions surrounding the use of pooled quality control samples. The results of the review indicate that use of pooled QC samples has been relatively widely adopted by the metabolomics community and that it is used at a similar frequency across biological taxa and sample types in both small- and large-scale studies. However, while many studies generated and analyzed pooled QC samples, relatively few reported the use of pooled QC samples to improve data quality. This demonstrates a clear opportunity for the field to more frequently utilize pooled QC samples for quality reporting, feature filtering, analytical drift correction, and metabolite annotation. Additionally, our survey approach enabled us to assess the ambiguity in the reporting of the methods used to describe the generation and use of pooled QC samples. This analysis indicates that many details of the QC framework are missing or unclear, limiting the reader's ability to determine which QC steps have been taken. Collectively, these results capture the current state of pooled QC sample usage and highlight existing strengths and deficiencies as they are applied in untargeted LC-MS metabolomics.

Lung Microbiota and Metabolites Collectively Associate with Clinical Outcomes in Milder Stage Chronic Obstructive Pulmonary Disease.

Rationale: Chronic obstructive pulmonary disease (COPD) is variable in its development. Lung microbiota and metabolites collectively may impact COPD pathophysiology, but relationships to clinical outcomes in milder disease are unclear. Objectives: Identify components of the lung microbiome and metabolome collectively associated with clinical markers in milder stage COPD. Methods: We analyzed paired microbiome and metabolomic data previously characterized from bronchoalveolar lavage fluid in 137 participants in the SPIROMICS (Subpopulations and Intermediate Outcome Measures in COPD Study), or (GOLD [Global Initiative for Chronic Obstructive Lung Disease Stage 0-2). Datasets used included 1) bacterial 16S rRNA gene sequencing; 2) untargeted metabolomics of the hydrophobic fraction, largely comprising lipids; and 3) targeted metabolomics for a panel of hydrophilic compounds previously implicated in mucoinflammation. We applied an integrative approach to select features and model 14 individual clinical variables representative of known associations with COPD trajectory (lung function, symptoms, and exacerbations). Measurements and Main Results: The majority of clinical measures associated with the lung microbiome and metabolome collectively in overall models (classification accuracies, >50%, P < 0.05 vs. chance). Lower lung function, COPD diagnosis, and greater symptoms associated positively with Streptococcus, Neisseria, and Veillonella, together with compounds from several classes (glycosphingolipids, glycerophospholipids, polyamines and xanthine, an adenosine metabolite). In contrast, several Prevotella members, together with adenosine, 5'-methylthioadenosine, sialic acid, tyrosine, and glutathione, associated with better lung function, absence of COPD, or less symptoms. Significant correlations were observed between specific metabolites and bacteria (Padj < 0.05). Conclusions: Components of the lung microbiome and metabolome in combination relate to outcome measures in milder COPD, highlighting their potential collaborative roles in disease pathogenesis.

Lipid mediators are detectable in the nasal epithelium and differ by asthma status in female subjects.

BACKGROUND: Lipid mediators, bioactive products of polyunsaturated fatty acid metabolism, contribute to inflammation initiation and resolution in allergic diseases; however, their presence in lung-related biosamples has not been fully described. OBJECTIVE: We aimed to quantify lipid mediators in the nasal airway epithelium and characterize preliminary associations with asthma. METHODS: Using liquid chromatography-mass spectrometry, we conducted a pilot study to quantify 56 lipid mediators from nasal epithelial samples collected from 11 female participants of an outpatient asthma clinic and community controls (aged 30-55 years). We examined the presence of each compound using descriptive statistics to test whether lipid mediators could distinguish subjects with asthma (n = 8) from control subjects (n = 3) using linear regression and partial least squares discriminant analysis. RESULTS: Fifteen lipid mediators were detectable in all samples, including resolvin (Rv) D5 (RvD5), with the highest median concentrations (in pg/µg protein) of 13-HODE (126.481), 15-HETE (32.869), and 13-OxoODE (13.251). From linear regression adjusted for age, prostaglandin E2 (PGE2) had a trend (P < .1) for higher concentrations in patients with severe asthma compared to controls (mean difference, 0.95; 95% confidence interval, -0.04 to 1.95). Asthma patients had higher scores on principal component 3 compared to controls (mean difference, 2.42; 95% confidence interval, 0.89 to 3.96), which represented lower levels of proresolving 15-HEPE, 19,20-DiHDPA, RvD5, 14-HDHA, 17-HDHA, and 13-HOTrE. Most of these compounds were best at discriminating asthma cases from controls in partial least squares discriminant analysis. CONCLUSION: Lipid mediators are detectable in the nasal epithelium, and their levels distinguish asthma cases from controls.

Discovering metabolite quantitative trait loci in asthma using an isolated population.

BACKGROUND: Integration of metabolomics with genetics may advance understanding of disease pathogenesis but has been underused in asthma genetic studies. OBJECTIVE: We sought to discover new genetic effects in asthma and to characterize the molecular consequences of asthma genetic risk through integration with the metabolome in a homogeneous population. METHODS: From fasting serum samples collected on 348 Tangier Island residents, we quantified 2612 compounds using untargeted metabolomics. Genotyping was performed using Illumina's MEGA array imputed to the TOPMed reference panel. To prioritize metabolites for genome-wide association analysis, we performed a metabolome-wide association study with asthma, selecting asthma-associated metabolites with heritability q value less than 0.01 for genome-wide association analysis. We also tested the association between all metabolites and 8451 candidate asthma single nucleotide polymorphisms previously associated with asthma in the UK Biobank. We followed up significant associations by characterizing shared genetic signal for metabolites and asthma using colocalization analysis. For detailed Methods, please see this article's Online Repository at www.jacionline.org. RESULTS: A total of 60 metabolites were associated with asthma (P < .01), including 40 heritable metabolites tested in genome-wide association analysis. We observed a strong association peak for the endocannabinoid linoleoyl ethanolamide on chromosome 6 in VNN1 (P < 2.7 × 10-9). We found strong evidence (colocalization posterior probability >75%) for a shared causal variant between 3 metabolites and asthma, including the polyamine acisoga and variants in LPP, and derivative leukotriene B4 and intergenic variants in chr10p14. CONCLUSIONS: We identified novel metabolite quantitative trait loci with asthma associations. Identification and characterization of these genetically driven metabolites may provide insight into the functional consequences of genetic risk factors for asthma.

Inflammation and Metabolism of Influenza-Stimulated Peripheral Blood Mononuclear Cells From Adults With Obesity Following Bariatric Surgery.

BACKGROUND: Obesity dysregulates immunity to influenza infection. Therefore, there is a critical need to investigate how obesity impairs immunity and to establish therapeutic approaches that mitigate the impact of increased adiposity. One mechanism by which obesity may alter immune responses is through changes in cellular metabolism. METHODS: We studied inflammation and cellular metabolism of peripheral blood mononuclear cells (PBMCs) isolated from individuals with obesity relative to lean controls. We also investigated if impairments to PBMC metabolism were reversible upon short-term weight loss following bariatric surgery. RESULTS: Obesity was associated with systemic inflammation and poor inflammation resolution. Unstimulated PBMCs from participants with obesity had lower oxidative metabolism and adenosine triphosphate (ATP) production compared to PBMCs from lean controls. PBMC secretome analyses showed that ex vivo stimulation with A/Cal/7/2009 H1N1 influenza led to a notable increase in IL-6 with obesity. Short-term weight loss via bariatric surgery improved biomarkers of systemic metabolism but did not improve markers of inflammation resolution, PBMC metabolism, or the PBMC secretome. CONCLUSIONS: These results show that obesity drives a signature of impaired PBMC metabolism, which may be due to persistent inflammation. PBMC metabolism was not reversed after short-term weight loss despite improvements in measures of systemic metabolism.

Microbiome, Metabolism, and Immunoregulation of Asthma: An American Thoracic Society and National Institute of Allergy and Infectious Diseases Workshop Report.

This report presents the proceedings from a workshop titled "Microbiome, Metabolism and Immunoregulation of Asthma" that was held virtually May 13 and 14, 2021. The workshop was jointly sponsored by the American Thoracic Society (Assembly on Allergy, Immunology, and Inflammation) and the National Institute of Allergy and Infectious Diseases. It convened an interdisciplinary group of experts with backgrounds in asthma immunology, microbiome science, metabolomics, computational biology, and translational pulmonary research. The main purpose was to identify key scientific gaps and needs to further advance research on microbial and metabolic mechanisms that may contribute to variable immune responses and disease heterogeneity in asthma. Discussions were structured around several topics, including 1) immune and microbial mechanisms of asthma pathogenesis in murine models, 2) the role of microbes in pediatric asthma exacerbations, 3) dysregulated metabolic pathways in asthma associated with obesity, 4) metabolism effects on macrophage function in adipose tissue and the lungs, 5) computational approaches to dissect microbiome-metabolite links, and 6) potential confounders of microbiome-disease associations in human studies. This report summarizes the major points of discussion, which included identification of specific knowledge gaps, challenges, and suggested directions for future research. These include questions surrounding mechanisms by which microbiota and metabolites shape host health versus an allergic or asthmatic state; direct and indirect influences of other biological factors, exposures, and comorbidities on these interactions; and ongoing technical and analytical gaps for clinical translation.

Obesity reprograms the pulmonary polyunsaturated fatty acid-derived lipidome, transcriptome, and gene-oxylipin networks.

Obesity exacerbates inflammation upon lung injury; however, the mechanisms by which obesity primes pulmonary dysregulation prior to external injury are not well studied. Herein, we tested the hypothesis that obesity dysregulates pulmonary PUFA metabolism that is central to inflammation initiation and resolution. We first show that a high-fat diet (HFD) administered to C57BL/6J mice increased the relative abundance of pulmonary PUFA-containing triglycerides and the concentration of PUFA-derived oxylipins (particularly prostaglandins and hydroxyeicosatetraenoic acids), independent of an increase in total pulmonary PUFAs, prior to onset of pulmonary inflammation. Experiments with a genetic model of obesity (ob/ob) generally recapitulated the effects of the HFD on the pulmonary oxylipin signature. Subsequent pulmonary next-generation RNA sequencing identified complex and unique transcriptional regulation with the HFD. We found the HFD increased pathways related to glycerophospholipid metabolism and immunity, including a unique elevation in B cell differentiation and signaling. Furthermore, we conducted computational integration of lipidomic with transcriptomic data. These analyses identified novel HFD-driven networks between glycerophospholipid metabolism and B cell receptor signaling with specific PUFA-derived pulmonary oxylipins. Finally, we confirmed the hypothesis by demonstrating that the concentration of pulmonary oxylipins, in addition to inflammatory markers, were generally increased in mice consuming a HFD upon ozone-induced acute lung injury. Collectively, these data show that a HFD dysregulates pulmonary PUFA metabolism prior to external lung injury, which may be a mechanism by which obesity primes the lungs to respond poorly to infectious and/or inflammatory challenges.

Multivalent lipid targeting by the calcium-independent C2A domain of synaptotagmin-like protein 4/granuphilin.

Synaptotagmin-like protein 4 (Slp-4), also known as granuphilin, is a Rab effector responsible for docking secretory vesicles to the plasma membrane before exocytosis. Slp-4 binds vesicular Rab proteins via an N-terminal Slp homology domain, interacts with plasma membrane SNARE complex proteins via a central linker region, and contains tandem C-terminal C2 domains (C2A and C2B) with affinity for phosphatidylinositol-(4,5)-bisphosphate (PIP2). The Slp-4 C2A domain binds with low nanomolar apparent affinity to PIP2 in lipid vesicles that also contain background anionic lipids such as phosphatidylserine but much weaker when either the background anionic lipids or PIP2 is removed. Through computational and experimental approaches, we show that this high-affinity membrane binding arises from concerted interaction at multiple sites on the C2A domain. In addition to a conserved PIP2-selective lysine cluster, a larger cationic surface surrounding the cluster contributes substantially to the affinity for physiologically relevant lipid compositions. Although the K398A mutation in the lysine cluster blocks PIP2 binding, this mutated protein domain retains the ability to bind physiological membranes in both a liposome-binding assay and MIN6 cells. Molecular dynamics simulations indicate several conformationally flexible loops that contribute to the nonspecific cationic surface. We also identify and characterize a covalently modified variant that arises through reactivity of the PIP2-binding lysine cluster with endogenous bacterial compounds and binds weakly to membranes. Overall, multivalent lipid binding by the Slp-4 C2A domain provides selective recognition and high-affinity docking of large dense core secretory vesicles to the plasma membrane.

Lower citrate synthase activity, mitochondrial complex expression, and fewer oxidative myofibers characterize skeletal muscle from growth-restricted fetal sheep.

Skeletal muscle from the late gestation sheep fetus with intrauterine growth restriction (IUGR) has evidence of reduced oxidative metabolism. Using a sheep model of placental insufficiency and IUGR, we tested the hypothesis that by late gestation, IUGR fetal skeletal muscle has reduced capacity for oxidative phosphorylation because of intrinsic deficits in mitochondrial respiration. We measured mitochondrial respiration in permeabilized muscle fibers from biceps femoris (BF) and soleus (SOL) from control and IUGR fetal sheep. Using muscles including BF, SOL, tibialis anterior (TA), and flexor digitorum superficialis (FDS), we measured citrate synthase (CS) activity, mitochondrial complex subunit abundance, fiber type distribution, and gene expression of regulators of mitochondrial biosynthesis. Ex vivo mitochondrial respiration was similar in control and IUGR muscle. However, CS activity was lower in IUGR BF and TA, indicating lower mitochondrial content, and protein expression of individual mitochondrial complex subunits was lower in IUGR TA and BF in a muscle-specific pattern. IUGR TA, BF, and FDS also had lower expression of type I oxidative fibers. Fiber-type shifts that support glycolytic instead of oxidative metabolism may be advantageous for the IUGR fetus in a hypoxic and nutrient-deficient environment, whereas these adaptions may be maladaptive in postnatal life.

Chromogranin A is an autoantigen in type 1 diabetes.

Autoreactive CD4(+) T cells are involved in the pathogenesis of many autoimmune diseases, but the antigens that stimulate their responses have been difficult to identify and in most cases are not well defined. In the nonobese diabetic (NOD) mouse model of type 1 diabetes, we have identified the peptide WE14 from chromogranin A (ChgA) as the antigen for highly diabetogenic CD4(+) T cell clones. Peptide truncation and extension analysis shows that WE14 bound to the NOD mouse major histocompatibility complex class II molecule I-A(g7) in an atypical manner, occupying only the carboxy-terminal half of the I-A(g7) peptide-binding groove. This finding extends the list of T cell antigens in type 1 diabetes and supports the idea that autoreactive T cells respond to unusually presented self peptides.

Resolvin E1 derived from eicosapentaenoic acid prevents hyperinsulinemia and hyperglycemia in a host genetic manner.

Eicosapentaenoic acid (EPA) has garnered attention after the success of the REDUCE-IT trial, which contradicted previous conclusions on EPA for cardiovascular disease risk. Here we first investigated EPA's preventative role on hyperglycemia and hyperinsulinemia. EPA ethyl esters prevented obesity-induced glucose intolerance, hyperinsulinemia, and hyperglycemia in C57BL/6J mice. Supporting NHANES analyses showed that fasting glucose levels of obese adults were inversely related to EPA intake. We next investigated how EPA improved murine hyperinsulinemia and hyperglycemia. EPA overturned the obesity-driven decrement in the concentration of 18-hydroxyeicosapentaenoic acid (18-HEPE) in white adipose tissue and liver. Treatment of obese inbred mice with RvE1, the downstream immunoresolvant metabolite of 18-HEPE, but not 18-HEPE itself, reversed hyperinsulinemia and hyperglycemia through the G-protein coupled receptor ERV1/ChemR23. To translate the findings, we determined if the effects of RvE1 were dependent on host genetics. RvE1's effects on hyperinsulinemia and hyperglycemia were divergent in diversity outbred mice that model human genetic variation. Secondary SNP analyses further confirmed extensive genetic variation in human RvE1/EPA-metabolizing genes. Collectively, the data suggest EPA prevents hyperinsulinemia and hyperglycemia, in part, through RvE1's activation of ERV1/ChemR23 in a host genetic manner. The studies underscore the need for personalized administration of RvE1 based on genetic/metabolic enzyme profiles.

Comparing the effects of an exposure to a polycyclic aromatic hydrocarbon mixture versus individual polycyclic aromatic hydrocarbons during monocyte to macrophage differentiation: Mixture exposure results in altered immune metrics.

Polycyclic aromatic hydrocarbons (PAHs) are generated by the incomplete combustion of carbon. Exposures correlate with systemic immune dysfunction and overall immune suppression. Real-world exposures to PAHs are almost always encountered as mixtures; however, research overwhelmingly centers on isolated exposures to a single PAH, benzo[a]pyrene (B[a]P). Here, a human monocyte line (U937) was exposed to B[a]P, benz[a]anthracene (B[a]A), or a mixture of six PAHs (6-MIX) to assess the differential toxicity on monocytes. Further, monocytes were exposed to PAHs with and without CYP1A1 inhibitors during macrophage differentiation to delineate PAH exposure and PAH metabolism-driven alterations to the immune response. U937 monocytes exposed to B[a]P, B[a]A, or 6-MIX had higher levels of cellular health and growth not observed following equimolar exposures to other individual PAHs. PAH exposures during differentiation did not alter monocyte-derived macrophage (MDM) numbers; however, B[a]A and 6-MIX exposures significantly altered M1/M2 polarization in a CYP1A1-dependent manner. U937-MDM adherence was differentially suppressed by all three PAH treatments with 6-MIX exposed U937-MDM having significantly more adhesion than U937-MDM exposed to either individual PAH. Finally, 6-MIX exposures during differentiation reduced U937-MDM endocytic function significantly less than B[a]A exposed cells. Exposure to a unique PAH mixture during U937-MDM differentiation resulted in mixture-specific alterations of pro-inflammatory markers compared to individual PAH exposures. While subtle, these differences highlight the probability that using a model PAH, B[a]P, may not accurately reflect the effects of PAH mixture exposures. Therefore, future studies should include various PAH mixtures that encompass probable real-world PAH exposures for the endpoints under investigation.

Comparison of reported versus measured content of over-the-counter and prescription prenatal multivitamins and minerals.

OBJECTIVE: To examine the measured contents of over-the-counter (OTC) and prescription (Rx) prenatal multivitamins and minerals (PMVMs) and compare the findings with the amounts reported on the nutrition labels. The findings were subsequently examined on the basis of cost and ability to adequately supplement dietary intake during pregnancy on the basis of The National Academies' dietary reference intakes (DRIs) and tolerable upper intake levels. METHODS: This was an observational convenience sample of OTC and Rx PMVMs available through online retailers and retail pharmacies. The amounts of folic acid, vitamin B6, vitamin C, and choline were measured in triplicate using mass spectrometry. RESULTS: Twenty OTC and 16 Rx PMVMs were evaluated. The average measured quantities of the vitamins were not statistically different from the mean reported quantities for OTC and Rx PMVMs. When a standard diet was combined with the labeled nutrition information, 95% of the OTC PMVMs and 88% of the Rx PMVMs met the DRIs for folic acid and vitamins B6 and C. When a standard diet was combined with the actual measured PMVM quantities, 79% of the OTC PMVMs and 82% of the Rx PMVMs met the DRIs for folic acid and vitamins B6 and C. The measured choline content, with and without diet considerations, did not meet the DRIs. No statistically significant difference was found for the adequacy of supplementation between the OTC and Rx PMVMs on the basis of cost. CONCLUSION: On the basis of a comparison of the measured and reported values for folic acid, vitamin C, vitamin B6, and choline, it seems that either OTC or Rx PMVMs at low or high cost can be recommended to supplement diets and meet the DRIs during pregnancy for these vitamins.

Defining decreased protein succinylation of failing human cardiac myofibrils in ischemic cardiomyopathy.

Succinylation is a post-translational modification of protein lysine residues with succinyl groups derived from succinyl CoA. Succinylation is considered a significant post-translational modification with the potential to impact protein function which is highly conserved across numerous species. The role of succinylation in the heart, especially in heart failure and myofibril mechanics, remains largely unexplored. Mechanical parameters were measured in myofibrils isolated from failing hearts of ischemic cardiomyopathy patients and non-failing donor controls. We employed mass spectrometry to quantify differential protein expression in myofibrils from failing ischemic cardiomyopathy hearts compared to non-failing hearts. In addition, we combined peptide enrichment by immunoprecipitation with liquid chromatography tandem mass spectrometry to quantitatively analyze succinylated lysine residues in these myofibrils. Several key parameters of sarcomeric mechanical interactions were altered in myofibrils isolated from failing ischemic cardiomyopathy hearts, including lower resting tension and a faster rate of activation. Of the 100 differentially expressed proteins, 46 showed increased expression in ischemic heart failure, while 54 demonstrated decreased expression in ischemic heart failure. Our quantitative succinylome analysis identified a total of 572 unique succinylated lysine sites located on 181 proteins, with 307 significantly changed succinylation events. We found that 297 succinyl-Lys demonstrated decreased succinylation on 104 proteins, while 10 residues demonstrated increased succinylation on 4 proteins. Investigating succinyl CoA generation, enzyme activity assays demonstrated that α-ketoglutarate dehydrogenase and succinate dehydrogenase activities were significantly decreased in ischemic heart failure. An activity assay for succinyl CoA synthetase demonstrated a significant increase in ischemic heart failure. Taken together, our findings support the hypothesis that succinyl CoA production is decreased and succinyl CoA turnover is increased in ischemic heart failure, potentially resulting in an overall decrease in the mitochondrial succinyl CoA pool, which may contribute to decreased myofibril protein succinylation in heart failure.

Improved quantification of lipid mediators in plasma and tissues by liquid chromatography tandem mass spectrometry demonstrates mouse strain specific differences.

A liquid chromatography tandem mass spectrometry-based method for the quantitation of 39 lipid mediators in four sample types and in two mouse strains is described. The method builds upon existing methodologies for analysis of lipid mediators by A) utilizing a bead homogenization step for tissue samples; this eliminates the need for homogenization glassware and improves homogenization consistency, B) optimizing the isolation and purification of lipid mediators with polymeric reverse phase SPE columns with lower sorbent masses; this results in lower solvent elution volumes without loss of recovery and C) utilizing an on-column enrichment method to improve analyte focusing before chromatographic separation. The method is linear from 0.25-250 pg on column for low level lipid mediators and from 5-5000 pg on column for high level lipid mediators. The addition of a methyl formate elution step to a previously published method dramatically improved precision and recovery for the cysteinyl leukotrienes. Accuracy and precision for 4 different sample types including human plasma, mouse lung, mouse spleen and mouse liver is demonstrated. Liver samples had extremely high levels of a tentatively identified bile acid which interfered with quantitation of resolvin E1, 11B-prostaglandin F2a and thromboxane A2. Results from 2 different tissue sources from untreated mice (C57BL/6 versus BALB/c) showed dramatically different concentrations of lipid mediators.

Maternal Amino Acid Profiles to Distinguish Constitutionally Small versus Growth-Restricted Fetuses Defined by Doppler Ultrasound: A Pilot Study.

OBJECTIVE: Fetuses measuring below the 10th percentile for gestational age may be either constitutionally small for gestational age (SGA) or have pathologic fetal growth restriction (FGR). FGR is associated with adverse outcomes; however, identification of low-risk SGA cases is difficult. We performed a pilot study evaluating maternal markers of pathologic FGR, hypothesizing there are distinct amino acid signatures that might be used for diagnosis and development of new interventions. STUDY DESIGN: This was a cohort study of healthy women with sonographic fetal estimated fetal weight <5th percentile divided into two groups based upon umbilical artery (UmA) Doppler studies or uterine artery (UtA) Doppler studies. We collected maternal blood samples prior to delivery and used ion pair reverse phase liquid chromatography-mass spectrometry or gas chromatography-mass spectrometry to assess 44 amino acids. RESULTS: Among 14 women included, five had abnormal UmA, and three had abnormal UtA Doppler results. Those with abnormal UmA showed elevated ornithine. Those with abnormal UtA had lower dimethylglycine, isoleucine, methionine, phenylalanine, and 1-methylhistidine. CONCLUSION: We found several amino acids that might identify pregnancies affected by pathologic FGR. These findings support the feasibility of future larger studies to identify maternal metabolic approaches to accurately stratify risk for small fetuses.