Using lipid profiling to better characterize metabolic differences in apolipoprotein E (APOE) genotype among community-dwelling older Black men.

Apolipoprotein E (APOE) allelic variation is associated with differences in overall circulating lipids and risks of major health outcomes. Lipid profiling provides the opportunity for a more detailed description of lipids that differ by APOE, to potentially inform therapeutic targets for mitigating higher morbidity and mortality associated with certain APOE genotypes. Here, we sought to identify lipids, lipid-like molecules, and important mediators of fatty acid metabolism that differ by APOE among 278 Black men ages 70-81. Using liquid chromatography-mass spectrometry methods, 222 plasma metabolites classified as lipids, lipid-like molecules, or essential in fatty acid metabolism were detected. We applied principal factor analyses to calculate a factor score for each main lipid category. APOE was categorized as ε4 carriers (n = 83; ε3ε4 or ε4ε4), ε2 carriers (n = 58; ε2ε3 or ε2ε2), or ε3 homozygotes (n = 137; ε3ε3). Using analysis of variance, the monoacylglycerol factor, cholesterol ester factor, the factor for triacylglycerols that consist mostly of polyunsaturated fatty acids, sphingosine, and free carnitine significantly differed by APOE (p < 0.05, false discovery rate < 0.30). The monoacylglycerol factor, cholesterol ester factor, and sphingosine were lower, whereas the factor for triacylglycerols that consisted mostly of polyunsaturated fatty acids was higher among ε2 carriers than remaining participants. Free carnitine was lower among ε4 carriers than ε3 homozygotes. Lower monoacylglycerols and cholesteryl esters and higher triacylglycerols that consist mostly of polyunsaturated fatty acids may be protective metabolic characteristics of APOE ε2 carriers, whereas lower carnitine may reflect altered mitochondrial functioning among ε4 carriers in this cohort of older Black men.

Beneficial Effects of Akkermansia muciniphila Are Not Associated with Major Changes in the Circulating Endocannabinoidome but Linked to Higher Mono-Palmitoyl-Glycerol Levels as New PPARα Agonists.

Akkermansia muciniphila is considered as one of the next-generation beneficial bacteria in the context of obesity and associated metabolic disorders. Although a first proof-of-concept of its beneficial effects has been established in the context of metabolic syndrome in humans, mechanisms are not yet fully understood. This study aimed at deciphering whether the bacterium exerts its beneficial properties through the modulation of the endocannabinoidome (eCBome). Circulating levels of 25 endogenous endocannabinoid-related lipids were quantified by liquid chromatography with tandem mass spectrometry (LC-MS/MS) in the plasma of overweight or obese individuals before and after a 3 months intervention consisting of the daily ingestion of either alive or pasteurized A. muciniphila. Results from multivariate analyses suggested that the beneficial effects of A. muciniphila were not linked to an overall modification of the eCBome. However, subsequent univariate analysis showed that the decrease in 1-Palmitoyl-glycerol (1-PG) and 2-Palmitoyl-glycerol (2-PG), two eCBome lipids, observed in the placebo group was significantly counteracted by the alive bacterium, and to a lower extent by the pasteurized form. We also discovered that 1- and 2-PG are endogenous activators of peroxisome proliferator-activated receptor alpha (PPARα). We hypothesize that PPARα activation by mono-palmitoyl-glycerols may underlie part of the beneficial metabolic effects induced by A. muciniphila in human metabolic syndrome.

The mitochondrial-derived peptide MOTS-c is a regulator of plasma metabolites and enhances insulin sensitivity.

MOTS-c is an exercise mimetic and improves insulin sensitivity in aged and diet-induced obese mice. Although plasma markers are good markers for the metabolic condition, whether MOTS-c changes plasma markers in diet-induced obese mice has not been examined. Here, we used an unbiased metabolomics approach to examine the effect of MOTS-c on plasma markers of metabolic dysfunction. We found that three pathways - sphingolipid metabolism, monoacylglycerol metabolism, and dicarboxylate metabolism - were reduced in MOTS-c-injected mice. Interestingly, these pathways are upregulated in obese and T2D models. MOTS-c improves insulin sensitivity and increases beta-oxidation to prevent fat accumulation in DIO mice through these pathways. These results provide us a better understanding of the mechanism of how MOTS-c improves insulin sensitivity and reduces the body weight and fatty liver and opens a new venue for further study.

Boronic Derivatization of Monoacylglycerol and Monitoring in Biofluids.

Monoacylglycerols (MAGs) are active mediators involved in multiple biological processes closely related to the pathological development of diabetes, obesity, and cancers. Sensitive and unambiguous detection of MAGs is thus essential; however, previous methods are both indirect and labor-intensive. Herein, we developed a straightforward approach by derivatization of MAGs with 3-nitrophenylboronic acid (3-NPB) for sensitive and selective analysis in cell lysates, tissues, and serums by mass spectrometry (MS). Reaction occurring between boronic acid and cis-diol moiety of MAGs blocked the formation of multiple adduct ions and tuned MAGs to negatively charged carrying species. In addition, the characteristic isotopic distribution of boron specialized the presence of modified MAGs in MS and led to distinctive identification. To eliminate endogenous interferences, we further introduced isotopic labeled d4-NPB equivalently premixed with d0-NPB to perform MAG derivatization, which resulted in rapid identification of modified MAGs in biofluids by displaying doublet peak characteristics. A comparative quantification approach was thereafter evoluted to reveal the amount variation of MAGs by d0-NPB and d4-NPB separately derivatized in different pathological tissue and serum samples. The presently developed NPB-based derivatization approach is expected to be essential in the metabolic study of MAG-related diseases.

Metabolic disease and ABHD6 alter the circulating bis(monoacylglycerol)phosphate profile in mice and humans.

Bis(monoacylglycerol)phosphate (BMP) is a phospholipid that is crucial for lipid degradation and sorting in acidic organelles. Genetic and drug-induced lysosomal storage disorders (LSDs) are associated with increased BMP concentrations in tissues and in the circulation. Data on BMP in disorders other than LSDs, however, are scarce, and key enzymes regulating BMP metabolism remain elusive. Here, we demonstrate that common metabolic disorders and the intracellular BMP hydrolase α/ß-hydrolase domain-containing 6 (ABHD6) affect BMP metabolism in mice and humans. In mice, dietary lipid overload strongly affects BMP concentration and FA composition in the liver and plasma, similar to what has been observed in LSDs. Notably, distinct changes in the BMP FA profile enable a clear distinction between lipid overload and drug-induced LSDs. Global deletion of ABHD6 increases circulating BMP concentrations but does not cause LSDs. In humans, nonalcoholic fatty liver disease and liver cirrhosis affect the serum BMP FA composition and concentration. Furthermore, we identified a patient with a loss-of-function mutation in the ABHD6 gene, leading to an altered circulating BMP profile. In conclusion, our results suggest that common metabolic diseases and ABHD6 affect BMP metabolism in mice and humans.

Plasma concentrations of oleoylethanolamide in a primary care sample of depressed patients are increased in those treated with selective serotonin reuptake inhibitor-type antidepressants.

Oleoylethanolamide (OEA) is a non-cannabinoid acylethanolamide with multiple physiological roles that has been proposed to have antidepressant-like activity in preclinical models. OEA shares biosynthetic pathways with anandamide (AEA) a transmitter involved in affective disorders and anxiety in humans. However, although the participation of OEA in depression has been proposed, both, the contribution of OEA to the depressive phenotype and the effect of antidepressant therapy on circulating levels of this and related non-cannabinoid acylethanolamides in humans are basically unknown. The main objective of this study is to compare the plasma concentrations of OEA and related acylethanolamides in a sample of primary care patients with depression (n = 69) with those of healthy non-depressed patients (n = 47). At the time of admission to the study, 22 patients were under selective serotonin reuptake inhibitor (SSRI) antidepressant treatment and 47 patients were not receiving any type of intervention. In addition, plasma concentrations of the endocannabinoid 2-AG and two related monoacylglycerols were monitored. Plasma OEA concentrations were found to be elevated in depressed patients and to correlate with somatic symptoms of depression. Plasma concentrations of both, AEA and 2-AG, were found to be elevated also in depressed patients. Further analysis demonstrated that the elevation observed in the plasma concentrations of both, OEA and 2-AG, was associated to SSRI antidepressant therapy at the time of recruitment. Further clinical research is needed to understand whether SSRI-induced elevations in OEA levels contribute to the response to SSRI in depressed patients as described in preclinical models.

Comparison of the Incorporation of DHA in Circulatory and Neural Tissue When Provided as Triacylglycerol (TAG), Monoacylglycerol (MAG) or Phospholipids (PL) Provides New Insight into Fatty Acid Bioavailability.

Phospholipids (PL) or partial acylglycerols such as sn-1(3)-monoacylglycerol (MAG) are potent dietary carriers of long-chain polyunsaturated fatty acids (LC-PUFA) and have been reported to provide superior bioavailability when compared to conventional triacylglycerol (TAG). The main objective of the present study was to compare the incorporation of docosahexaenoic acid (DHA) in plasma, erythrocytes, retina and brain tissues in adult rats when provided as PL (PL-DHA) and MAG (MAG-DHA). Conventional dietary DHA oil containing TAG (TAG-DHA) as well as control chow diet were used to evaluate the potency of the two alternative DHA carriers over a 60-day feeding period. Fatty acid profiles were determined in erythrocytes and plasma lipids at time 0, 7, 14, 28, 35 and 49 days of the experimental period and in retina, cortex, hypothalamus, and hippocampus at 60 days. The assessment of the longitudinal evolution of DHA in erythrocyte and plasma lipids suggest that PL-DHA and MAG-DHA are efficient carriers of dietary DHA when compared to conventional DHA oil (TAG-DHA). Under these experimental conditions, both PL-DHA and MAG-DHA led to higher incorporations of DHA erythrocytes lipids compared to TAG-DHA group. After 60 days of supplementation, statistically significant increase in DHA level incorporated in neural tissues analyzed were observed in the DHA groups compared with the control. The mechanism explaining hypothetically the difference observed in circulatory lipids is discussed.

Systems characterization of differential plasma metabolome perturbations following thrombotic and non-thrombotic myocardial infarction.

Myocardial infarction (MI) is an acute event characterized by myocardial necrosis. Thrombotic MI is caused by spontaneous atherosclerotic plaque disruption that results in a coronary thrombus; non-thrombotic MI occurs secondary to oxygen supply-demand mismatch. We sought to characterize the differential metabolic perturbations associated with these subtypes utilizing a systems approach. Subjects presenting with thrombotic MI, non-thrombotic MI and stable coronary artery disease (CAD) were included. Whole blood was collected at two acute time-points and at a time-point representing the quiescent stable disease state. Plasma metabolites were analyzed by untargeted UPLC-MS/MS and GC-MS. A weighted network was constructed, and modules were determined from the resulting topology. To determine perturbed modules, an enrichment analysis for metabolites that demonstrated between-group differences in temporal change across the disease state transition was then conducted. BIOLOGICAL SIGNIFICANCE: We report evidence of metabolic perturbations of acute MI and determine perturbations specific to thrombotic MI. Specifically, a module characterized by elevated glucocorticoid steroid metabolites following acute MI showed greatest perturbation following thrombotic MI. Modules characterized by elevated pregnenolone metabolites, monoacylglycerols, and acylcarnitines were perturbed following acute MI. A module characterized by a decrease in plasma amino acids following thrombotic MI was differentially perturbed between MI subtypes.

Analysis of Serum Metabolites to Diagnose Bicuspid Aortic Valve.

Bicuspid aortic valve (BAV) is the most common congenital heart disease. The current study aims to construct a diagnostic model based on metabolic profiling as a non-invasive tool for BAV screening. Blood serum samples were prepared from an estimation group and a validation group, each consisting of 30 BAV patients and 20 healthy individuals, and analyzed by liquid chromatography-mass spectrometry (LC-MS). In total, 2213 metabolites were detected and 41 were considered different. A model for predicting BAV in the estimation group was constructed using the concentration levels of monoglyceride (MG) (18:2) and glycerophospho-N-oleoyl ethanolamine (GNOE). A novel model named Zhongshan (ZS) was developed to amplify the association between BAV and the two metabolites. The area under curve (AUC) of ZS for BAV prediction was 0.900 (0.782-0.967) and was superior to all single-metabolite models when applied to the estimation group. Using optimized cutoff (-0.1634), ZS model had a sensitivity score of 76.7%, specificity score of 90.0%, positive predictive value of 80% and negative predictive value of 85.0% for the validation group. These results support the use of serum-based metabolomics profiling method as a complementary tool for BAV screening in large populations.

Ozone Exposure Increases Circulating Stress Hormones and Lipid Metabolites in Humans.

RATIONALE: Air pollution has been associated with increased prevalence of type 2 diabetes; however, the mechanisms remain unknown. We have shown that acute ozone exposure in rats induces release of stress hormones, hyperglycemia, leptinemia, and glucose intolerance that are associated with global changes in peripheral glucose, lipid, and amino acid metabolism. OBJECTIVES: To examine ozone-induced metabolic derangement in humans using serum metabolomic assessment, establish human-to-rodent coherence, and identify novel nonprotein biomarkers. METHODS: Serum samples were obtained from a crossover clinical study that included two clinic visits (n = 24 each) where each subject was blindly exposed in the morning to either filtered air or 0.3 parts per million ozone for 2 hours during 15-minute on-off exercise. Serum samples collected within 1 hour after exposure were assessed for changes in metabolites using a metabolomic approach. MEASUREMENTS AND MAIN RESULTS: Metabolomic analysis revealed that ozone exposure markedly increased serum cortisol and corticosterone together with increases in monoacylglycerol, glycerol, and medium- and long-chain free fatty acids, reflective of lipid mobilization and catabolism. Additionally, ozone exposure increased serum lysolipids, potentially originating from membrane lipid breakdown. Ozone exposure also increased circulating mitochondrial ß-oxidation-derived metabolites, such as acylcarnitines, together with increases in the ketone body 3-hydroxybutyrate. These changes suggested saturation of ß-oxidation by ozone in exercising humans. CONCLUSIONS: As in rodents, acute ozone exposure increased stress hormones and globally altered peripheral lipid metabolism in humans, likely through activation of a neurohormonally mediated stress response pathway. The metabolomic assessment revealed new biomarkers and allowed for establishment of rodent-to-human coherence. Clinical trial registered with www.clinicaltrials.gov (NCT 01492517).

Targeted Lipidomics of Fontal Cortex and Plasma Diacylglycerols (DAG) in Mild Cognitive Impairment and Alzheimer's Disease: Validation of DAG Accumulation Early in the Pathophysiology of Alzheimer's Disease.

Previous studies have demonstrated augmented levels of diacylglycerols (DAG) in the frontal cortex and plasma of Alzheimer's disease (AD) patients. We extended these findings from non-targeted lipidomics studies to design a lipidomics platform to interrogate DAGs and monoacylglycerols (MAG) in the frontal cortex and plasma of MCI subjects. Control subjects included both aged normal controls and controls with normal cognition, but AD pathology at autopsy, individuals termed non-demented AD neuropathology. DAGs with saturated, unsaturated, and polyunsaturated fatty acid substituents were found to be elevated in MCI frontal cortex and plasma. Tandem mass spectrometry of the DAGs did not reveal any differences in the distributions of the fatty acid substitutions between MCI and control subjects. While triacylglycerols were not altered in MCI subjects there were increases in MAG levels both in the frontal cortex and plasma. In toto, increased levels of DAGs and MAGs appear to occur early in AD pathophysiology and require both further validation in a larger patient cohort and elucidation of the lipidomics alteration(s) that lead to the accumulation of DAGs in MCI subjects.

Global deletion of MGL in mice delays lipid absorption and alters energy homeostasis and diet-induced obesity.

Monoacylglycerol lipase (MGL) is a ubiquitously expressed enzyme that catalyzes the hydrolysis of monoacylglycerols (MGs) to yield FFAs and glycerol. MGL contributes to energy homeostasis through the mobilization of fat stores and also via the degradation of the endocannabinoid 2-arachidonoyl glycerol. To further examine the role of MG metabolism in energy homeostasis, MGL(-/-) mice were fed either a 10% (kilocalories) low-fat diet (LFD) or a 45% (kilocalories) high-fat diet (HFD) for 12 weeks. Profound increases of MG species in the MGL(-/-) mice compared with WT control mice were found. Weight gain over the 12 weeks was blunted in both diet groups. MGL(-/-) mice were leaner than WT mice at both baseline and after 12 weeks of LFD feeding. Circulating lipids were decreased in HFD-fed MGL(-/-) mice, as were the levels of several plasma peptides involved in glucose homeostasis and energy balance. Interestingly, MGL(-/-) mice had markedly reduced intestinal TG secretion following an oral fat challenge, suggesting delayed lipid absorption. Overall, the results indicate that global MGL deletion leads to systemic changes that produce a leaner phenotype and an improved serum metabolic profile.

Large-scale metabolomic profiling identifies novel biomarkers for incident coronary heart disease.

Analyses of circulating metabolites in large prospective epidemiological studies could lead to improved prediction and better biological understanding of coronary heart disease (CHD). We performed a mass spectrometry-based non-targeted metabolomics study for association with incident CHD events in 1,028 individuals (131 events; 10 y. median follow-up) with validation in 1,670 individuals (282 events; 3.9 y. median follow-up). Four metabolites were replicated and independent of main cardiovascular risk factors [lysophosphatidylcholine 18∶1 (hazard ratio [HR] per standard deviation [SD] increment = 0.77, P-value<0.001), lysophosphatidylcholine 18∶2 (HR = 0.81, P-value<0.001), monoglyceride 18∶2 (MG 18∶2; HR = 1.18, P-value = 0.011) and sphingomyelin 28∶1 (HR = 0.85, P-value = 0.015)]. Together they contributed to moderate improvements in discrimination and re-classification in addition to traditional risk factors (C-statistic: 0.76 vs. 0.75; NRI: 9.2%). MG 18∶2 was associated with CHD independently of triglycerides. Lysophosphatidylcholines were negatively associated with body mass index, C-reactive protein and with less evidence of subclinical cardiovascular disease in additional 970 participants; a reverse pattern was observed for MG 18∶2. MG 18∶2 showed an enrichment (P-value = 0.002) of significant associations with CHD-associated SNPs (P-value = 1.2×10-7 for association with rs964184 in the ZNF259/APOA5 region) and a weak, but positive causal effect (odds ratio = 1.05 per SD increment in MG 18∶2, P-value = 0.05) on CHD, as suggested by Mendelian randomization analysis. In conclusion, we identified four lipid-related metabolites with evidence for clinical utility, as well as a causal role in CHD development.

Di-22:6-bis(monoacylglycerol)phosphate: A clinical biomarker of drug-induced phospholipidosis for drug development and safety assessment.

The inability to routinely monitor drug-induced phospholipidosis (DIPL) presents a challenge in pharmaceutical drug development and in the clinic. Several nonclinical studies have shown di-docosahexaenoyl (22:6) bis(monoacylglycerol) phosphate (di-22:6-BMP) to be a reliable biomarker of tissue DIPL that can be monitored in the plasma/serum and urine. The aim of this study was to show the relevance of di-22:6-BMP as a DIPL biomarker for drug development and safety assessment in humans. DIPL shares many similarities with the inherited lysosomal storage disorder Niemann-Pick type C (NPC) disease. DIPL and NPC result in similar changes in lysosomal function and cholesterol status that lead to the accumulation of multi-lamellar bodies (myeloid bodies) in cells and tissues. To validate di-22:6-BMP as a biomarker of DIPL for clinical studies, NPC patients and healthy donors were classified by receiver operator curve analysis based on urinary di-22:6-BMP concentrations. By showing 96.7-specificity and 100-sensitivity to identify NPC disease, di-22:6-BMP can be used to assess DIPL in human studies. The mean concentration of di-22:6-BMP in the urine of NPC patients was 51.4-fold (p ≤ 0.05) above the healthy baseline range. Additionally, baseline levels of di-22:6-BMP were assessed in healthy non-medicated laboratory animals (rats, mice, dogs, and monkeys) and human subjects to define normal reference ranges for nonclinical/clinical studies. The baseline ranges of di-22:6-BMP in the plasma, serum, and urine of humans and laboratory animals were species dependent. The results of this study support the role of di-22:6-BMP as a biomarker of DIPL for pharmaceutical drug development and health care settings.

Estimation of reference intervals of five endocannabinoids and endocannabinoid related compounds in human plasma by two dimensional-LC/MS/MS.

The elucidation of the role of endocannabinoids in physiological and pathological conditions and the transferability of the importance of these mediators from basic evidence into clinical practice is still hampered by the indefiniteness of their circulating reference intervals. In this work, we developed and validated a two-dimensional LC/MS/MS method for the simultaneous measurement of plasma endocannabinoids and related compounds such as arachidonoyl-ethanolamide, palmitoyl-ethanolamide, and oleoyl-ethanolamide, belonging to the N-acyl-ethanolamide (NAE) family, and 2-arachidonoyl-glycerol and its inactive isomer 1-arachidonoyl-glycerol from the monoacyl-glycerol (MAG) family. We found that several pitfalls in the endocannabinoid measurement may occur, from blood withdrawal to plasma processing. Plasma extraction with toluene followed by on-line purification was chosen, allowing high-throughput and reliability. We estimated gender-specific reference intervals on 121 healthy normal weight subjects fulfilling rigorous anthropometric and hematic criteria. We observed no gender differences for NAEs, whereas significantly higher MAG levels were found in males compared with females. MAGs also significantly correlated with triglycerides. NAEs increased with age in females, and arachidonoyl-ethanolamide correlated with adiposity and metabolic parameters in females. This work paves the way to the establishment of definitive reference intervals for circulating endocannabinoids to help physicians move from the speculative research field into the clinical field.

The bioavailability of eicosapentaenoic acid from reconstituted triglyceride fish oil is higher than that obtained from the triglyceride and monoglyceride forms.

Omega 3 fatty acids have healthcare benefits, but their absorption characteristics are not well defined, particularly for strategies to improve their bioavailability. We performed a double blind study comparing the bioavailability of 20% eicosapentaenoic acid in 4.5 grams of: natural triglyceride, reconstituted triglyceride, enzymatically synthesized triglyceride, monoglyceride and diglyceride. Seven healthy volunteers were given the supplements on five occasions while repeated measurements of eicosapentaenoic acid were taken to calculate the area under the curve for the next 24 hours. There was a significant difference between the mean of calculated area under the curve of eicosapentaenoic acid from reconstituted triglyceride (30.2) and that of the enzymatically synthesized triglyceride (11.9) and monoglyceride (13.4), z=-2.36 and -2.19, respectively, p<0.05. In summary, eicosapentaenoic acid bioavailability of chemically reconstituted triglycerides was better than that obtained from enzymatically synthesized triglyceride and monoglyceride.

Accelerated clearance of plasma triglycerides and free fatty acids in omega3-depleted rats.

The present study aims mainly at exploring the effects of a severe depletion in polyunsaturated long-chain omega3 fatty acids upon the fate of circulating lipids. The plasma concentration and fatty acid pattern of triglycerides, diglycerides, free fatty acids, and phospholipids were measured in omega3-depleted and control rats injected intravenously one hour before sacrifice with either saline, a control medium-chain triglyceride:olive oil emulsion or a medium-chain triglyceride:fish oil emulsion recently found to rapidly increase the phospholipid content of C20:5omega3 and C22:6omega3 in different cell types. The estimated fractional removal rate of the injected triglycerides and the clearance of free fatty acids from circulation were both higher in omega3-depleted rats than in control animals. The injection of the lipid emulsions apparently inhibited intracellular lipolysis, this being least pronounced in omega3-depleted rats. The increased clearance of circulating triglycerides and unesterified fatty acids in omega3-depleted rats may favor the cellular accumulation of lipids. In turn, such an accumulation and the lesser regulatory inhibition of tissular lipolysis may match the increased clearance of circulating unesterified fatty acids and, hence, account for the lack of any significant difference in plasma unesterified fatty acid concentration between these and control animals.