Effects of a diet based on the Dietary Guidelines on vascular health and TMAO in women with cardiometabolic risk factors.

BACKGROUND AND AIMS: Recent evidence links trimethylamine oxide (TMAO) to endothelial dysfunction, an early indicator of cardiovascular disease. We aimed to determine whether short-term consumption of a diet patterned after the 2010 Dietary Guidelines for Americans (DGA) would affect endothelial function, plasma TMAO concentrations, and cardiovascular disease risk, differently than a typical American Diet (TAD). METHODS AND RESULTS: An 8-wk controlled feeding trial was conducted in overweight/obese women pre-screened for insulin resistance and/or dyslipidemia. Women were randomized to a DGA or TAD group (n = 22/group). At wk0 (pre-intervention) and wk8 (post-intervention) vascular age was calculated; endothelial function (reactive hyperemia index (RHI)) and augmentation index (AI@75) were measured using EndoPAT, and plasma TMAO was measured by LC-MS/MS. Vascular age was reduced in DGA at wk8 compared to wk0 but TAD wk8 was not different from wk0 (DGA wk0: 54.2 ± 4.0 vs. wk8: 50.5 ± 3.1 (p = 0.05), vs. TAD wk8: 47.7 ± 2.3). Plasma TMAO concentrations, RHI, and AI@75 were not different between groups or weeks. CONCLUSION: Consumption of a diet based on the 2010 Dietary Guidelines for Americans for 8 weeks did not improve endothelial function or reduce plasma TMAO. CLINICALTRIALS.GOV: NCT02298725.

Preanalytical Sample Handling Conditions and Their Effects on the Human Serum Metabolome in Epidemiologic Studies.

Many epidemiologic studies use metabolomics for discovery-based research. The degree to which sample handling may influence findings, however, is poorly understood. In 2016, serum samples from 13 volunteers from the US Department of Agriculture's Beltsville Human Nutrition Research Center were subjected to different clotting (30 minutes/120 minutes) and refrigeration (0 minutes/24 hours) conditions, as well as different numbers (0/1/4) and temperatures (ice/refrigerator/room temperature) of thaws. The median absolute percent difference (APD) between metabolite levels and correlations between levels across conditions were estimated for 628 metabolites. The potential for handling artifacts to induce false-positive associations was estimated using variable hypothetical scenarios in which 1%-100% of case samples had different handling than control samples. All handling conditions influenced metabolite levels. Across metabolites, the median APD when extending clotting time was 9.08%. When increasing the number of thaws from 0 to 4, the median APD was 10.05% for ice and 5.54% for room temperature. Metabolite levels were correlated highly across conditions (all r's ≥ 0.84), indicating that relative ranks were preserved. However, if handling varied even modestly by case status, our hypotheticals showed that results can be biased and can result in false-positive findings. Sample handling affects levels of metabolites, and special care should be taken to minimize effects. Shorter room-temperature thaws should be preferred over longer ice thaws, and handling should be meticulously matched by case status.

Aberrant fatty acid metabolism in skeletal muscle contributes to insulin resistance in zinc transporter 7 (znt7)-knockout mice.

ZnT7 (Slc30a7) is a widely expressed zinc transporter involved in sequestration of zinc into the Golgi apparatus and vesicular compartments. znt7-knockout (KO) mice are mildly zinc-deficient and lean. Despite their lean phenotype, adult male znt7-KO mice are prone to insulin resistance. We hypothesized that fat partitioning from adipose to nonadipose tissues causes insulin resistance in znt7-KO mice. Here, we used biological and biochemical methods, including fatty acid and oxylipin profiling, EM, immunohistochemistry, quantitative RT-PCR, and Western blot analysis, to identify the underlying mechanism of insulin resistance in znt7-KO mice. We found that insulin resistance in this model was primarily associated with increased intracellular fatty acid levels in the skeletal muscle, which promoted intracellular lipid accumulation and production of bioactive lipid mediators, such as 12,13-dihydroxyoctadecanoic acid (12,13-DiHOME) and 12-hydroxyeicosatetraenoic acid (12-HETE). The expression of fatty acid-binding protein 3 (Fabp3) was dramatically up-regulated in the znt7-KO muscle cells accompanied by increased expression of Cd36, Slc27a1, and Slc27a4, the three major fatty acid transporters in the skeletal muscle. We also demonstrated that znt7-KO muscle cells had increased fatty acid oxidative capacity, indicated by enlarged mitochondria and increased mRNA or protein expression of key enzymes involved in the fatty acid mitochondrial shuttle and ß-oxidation. We conclude that increased fatty acid uptake in the znt7-KO skeletal muscle is a key factor that contributes to the excessive intracellular lipid deposit and elevated production of bioactive lipid mediators. These mediators may play pivotal roles in oxidative stress and inflammation, leading to insulin resistance.

Umbilical cord blood metabolomics reveal distinct signatures of dyslipidemia prior to bronchopulmonary dysplasia and pulmonary hypertension.

Pulmonary hypertension (PH) is a common consequence of bronchopulmonary dysplasia (BPD) and remains a primary contributor to increased morbidity and mortality among preterm infants. Unfortunately, at the present time, there are no reliable early predictive markers for BPD-associated PH. Considering its health consequences, understanding in utero perturbations that lead to the development of BPD and BPD-associated PH and identifying early predictive markers is of utmost importance. As part of the discovery phase, we applied a multiplatform metabolomics approach consisting of untargeted and targeted methodologies to screen for metabolic perturbations in umbilical cord blood (UCB) plasma from preterm infants that did ( n = 21; cases) or did not ( n = 21; controls) develop subsequent PH. A total of 1,656 features were detected, of which 407 were annotated by metabolite structures. PH-associated metabolic perturbations were characterized by reductions in major choline-containing phospholipids, such as phosphatidylcholines and sphingomyelins, indicating altered lipid metabolism. The reduction in UCB abundances of major choline-containing phospholipids was confirmed in an independent validation cohort consisting of UCB plasmas from 10 cases and 10 controls matched for gestational age and BPD status. Subanalyses in the discovery cohort indicated that elevations in the oxylipins PGE1, PGE2, PGF2a, 9- and 13-HOTE, 9- and 13-HODE, and 9- and 13-KODE were positively associated with BPD presence and severity. This expansive evaluation of cord blood plasma identifies compounds reflecting dyslipidemia and suggests altered metabolite provision associated with metabolic immaturity that differentiate subjects, both by BPD severity and PH development.

Impact of post-collection freezing delay on the reliability of serum metabolomics in samples reflecting the California mid-term pregnancy biobank.

BACKGROUND: Population-based biorepositories are important resources, but sample handling can affect data quality. OBJECTIVE: Identify metabolites of value for clinical investigations despite extended postcollection freezing delays, using protocols representing a California mid-term pregnancy biobank. METHODS: Blood collected from non-pregnant healthy female volunteers (n = 20) underwent three handling protocols after 30 min clotting at room temperature: (1) ideal-samples frozen (- 80 °C) within 2 h of collection; (2) delayed freezing-samples held at room temperature for 3 days, then 4 °C for 9 days, the median times for biobank samples, and then frozen; (3) delayed freezing with freeze-thaw-the delayed freezing protocol with a freeze-thaw cycle simulating retrieved sample sub-aliquoting. Mass spectrometry-based untargeted metabolomic analyses of primary metabolism and complex lipids and targeted profiling of oxylipins, endocannabinoids, ceramides/sphingoid-bases, and bile acids were performed. Metabolite concentrations and intraclass correlation coefficients (ICC) were compared, with the ideal protocol as the reference. RESULTS: Sixty-two percent of 428 identified compounds had good to excellent ICCs, a metric of concordance between measurements of samples handled with the different protocols. Sphingomyelins, phosphatidylcholines, cholesteryl esters, triacylglycerols, bile acids and fatty acid diols were the least affected by non-ideal handling, while sugars, organic acids, amino acids, monoacylglycerols, lysophospholipids, N-acylethanolamides, polyunsaturated fatty acids, and numerous oxylipins were altered by delayed freezing. Freeze-thaw effects were assay-specific with lipids being most stable. CONCLUSIONS: Despite extended post-collection freezing delays characteristic of some biobanks of opportunistically collected clinical samples, numerous metabolomic compounds had both stable levels and good concordance.

Effects of stimulation technique, anatomical region, and time on human sweat lipid mediator profiles.

Few studies compare sampling protocol effect on sweat composition. Here we evaluate the impact of sweat stimulation mode and site of collection on lipid mediator composition. Sweat from healthy males (n=7) was collected weekly for three weeks from the volar forearm following either pilocarpine iontophoresis or exercise, and from the forearm, back and thigh following pilocarpine iontophoresis only. Sweat content of over 150 lipid mediators were measured by liquid chromatography-tandem mass spectrometry. Seventy lipid mediators were routinely detected, including prostanoids, alcohols, diols, epoxides, ketones, nitrolipids, N-acylethanolamides, monoacylglycerols, and ceramides. Detected lipid mediators appeared unaffected by sampling site, though the forearm was the most consistent source of sweat. Pilocarpine-induced sweat showed increased concentrations of most detected compounds. Moreover, lipid mediator concentrations and profiles were temporally stable over the study duration. Sweat therefore appears to be a consistent and anatomically-stable source of lipid mediators, but care must be taken in comparing results obtained from different stimulation techniques.

Sweat lipid mediator profiling: a noninvasive approach for cutaneous research.

Recent advances in analytical and sweat collection techniques provide new opportunities to identify noninvasive biomarkers for the study of skin inflammation and repair. This study aims to characterize the lipid mediator profile including oxygenated lipids, endocannabinoids, and ceramides/sphingoid bases in sweat and identify differences in these profiles between sweat collected from nonlesional sites on the unflared volar forearm of subjects with and without atopic dermatitis (AD). Adapting routine procedures developed for plasma analysis, over 100 lipid mediators were profiled using LC-MS/MS and 58 lipid mediators were detected in sweat. Lipid mediator concentrations were not affected by sampling or storage conditions. Increases in concentrations of C30-C40 [NS] and [NdS] ceramides, and C18:1 sphingosine, were observed in the sweat of study participants with AD despite no differences being observed in transepidermal water loss between study groups, and this effect was strongest in men (P < 0.05, one-way ANOVA with Tukey's post hoc HSD). No differences in oxylipins and endocannabinoids were observed between study groups. Sweat mediator profiling may therefore provide a noninvasive diagnostic for AD prior to the presentation of clinical signs.

Circulating levels of endocannabinoids and oxylipins altered by dietary lipids in older women are likely associated with previously identified gene targets.

Postmenopausal women (PMW) report marginal n-3 PUFA intakes and are at risk of chronic diseases associated with the skeletal, muscular, neuroendocrine, and cardiovascular systems. How n-3 PUFA affect the amounts of endocannabinoids (ECs) and oxylipins (OLs) of metabolic and physiologic importance in PMW is not clear. Based on our recent findings that dietary n-3 PUFA alter gene targets of the EC system and lower pro-inflammatory OL we proceeded to characterize these actions in blood of PMW. Our aim was to determine levels of the ECs, OLs, and global metabolites (GM) in white PMW (75±7y), randomized in a double-masked manner, from baseline to 6mo after receiving a fish oil supplement of n-3 PUFA (720mg 20:5n3+480mg 22:6n3/d, n=20) or placebo (1.8g oleic acid/d, n=20). ECs and OLs in serum were determined by UPLC-MS/MS and GM by GC-MS and LC-MS/MS. Plasma 20:5n3 and 22:6n3 levels increased in PMW given fish oil. EC n-6 acyl-ethanolamides, arachidonate-derived diols were decreased and 20:5n3 and 22:6n3 diols, epoxides, and alcohols were increased in PMW given fish oil. GM analysis revealed that n-3 PUFA supplementation increased renal steroid hormone and proteolytic metabolite levels in PMW. Herein, we confirm that gene targets of the EC system, previously found as modifiable by n-3 PUFA result in changes in the levels of ECs and OLs in PMW. This study shows phenotypic responses (in levels) to n-3 PUFA supplementation in PMW and increases of n-3 acyl-ethanolamide and n-3-derived OL of clinical considerations in aging.

The Human Serum Metabolome of Vitamin B-12 Deficiency and Repletion, and Associations with Neurological Function in Elderly Adults.

BACKGROUND: The specific metabolomic perturbations that occur in vitamin B-12 deficiency, and their associations with neurological function, are not well characterized. OBJECTIVE: We sought to characterize the human serum metabolome in subclinical vitamin B-12 deficiency and repletion. METHODS: A before-and-after treatment study provided 1 injection of 10 mg vitamin B-12 (with 100 mg pyridoxine and 100 mg thiamin) to 27 community-dwelling elderly Chileans (∼74 y old) with vitamin B-12 deficiency, as evaluated with serum vitamin B-12, total plasma homocysteine (tHcy), methylmalonic acid (MMA), and holotranscobalamin. The combined indicator of vitamin B-12 status (cB-12) was computed. Targeted metabolites [166 acylcarnitines, amino acids, sugars, glycerophospholipids, and sphingolipids (liquid chromatography-tandem mass spectrometry)], and untargeted metabolites [247 chemical entities (gas chromatography time-of-flight mass spectrometry)] were measured at baseline and 4 mo after treatment. A peripheral nerve score was developed. Differences before and after treatment were examined. For targeted metabolomics, the data from 18 individuals with adequate vitamin B-12 status (selected from the same population) were added to the before-and-after treatment data set. Network visualizations and metabolic pathways are illustrated. RESULTS: The injection increased serum vitamin B-12, holotranscobalamin, and cB-12 (P < 0.001), and reduced tHcy and serum MMA (P < 0.001). Metabolomic changes from before to after treatment included increases (P < 0.001) in acylcarnitines, plasmalogens, and other phospholipids, whereas proline and other intermediaries of one-carbon metabolism-that is, methionine and cysteine-were reduced (P < 0.001). Direct significant correlations (P < 0.05 after the false discovery rate procedure) were identified between acylcarnitines, plasmalogens, phospholipids, lyso-phospholipids, and sphingomyelins compared with vitamin B-12 status and nerve function. Multiple connections were identified with primary metabolites (e.g., an inverse relation between vitamin B-12 markers and tryptophan, tyrosine, and pyruvic, succinic, and citric acids, and a direct correlation between the nerve score and arginine). CONCLUSIONS: The human serum metabolome in vitamin B-12 deficiency and the changes that occur after supplementation are characterized. Metabolomics revealed connections between vitamin B-12 status and serum metabolic markers of mitochondrial function, myelin integrity, oxidative stress, and peripheral nerve function, including some previously implicated in Alzheimer and Parkinson diseases. This trial was registered at www.controlled-trials.com as ISRCTN02694183.

Frataxin deficiency increases cyclooxygenase 2 and prostaglandins in cell and animal models of Friedreich's ataxia.

An inherited deficiency of the mitochondrial protein frataxin causes Friedreich's ataxia (FRDA); the mechanism by which this deficiency triggers neuro- and cardio-degeneration is unclear. Microarrays of neural tissue of animal models of the disease showed decreases in antioxidant genes, and increases in inflammatory genes. Cyclooxygenase (COX)-derived oxylipins are important mediators of inflammation. We measured oxylipin levels using tandem mass spectrometry and ELISAs in multiple cell and animal models of FRDA. Mass spectrometry revealed increases in concentrations of prostaglandins, thromboxane B2, 15-HETE and 11-HETE in cerebellar samples of knockin knockout mice. One possible explanation for the elevated oxylipins is that frataxin deficiency results in increased COX activity. While constitutive COX1 was unchanged, inducible COX2 expression was elevated over 1.35-fold (P < 0.05) in two Friedreich's mouse models and Friedreich's lymphocytes. Consistent with higher COX2 expression, its activity was also increased by 58% over controls. COX2 expression is driven by multiple transcription factors, including activator protein 1 and cAMP response element-binding protein, both of which were elevated over 1.52-fold in cerebella. Taken together, the results support the hypothesis that reduced expression of frataxin leads to elevation of COX2-mediated oxylipin synthesis stimulated by increases in transcription factors that respond to increased reactive oxygen species. These findings support a neuroinflammatory mechanism in FRDA, which has both pathomechanistic and therapeutic implications.

Daily Cholecalciferol Supplementation during Pregnancy Alters Markers of Regulatory Immunity, Inflammation, and Clinical Outcomes in a Randomized Controlled Trial.

BACKGROUND: Vitamin D deficiency is widespread in pregnancy and has been associated with adverse health conditions in mothers and infants. Vitamin D supplementation in pregnancy may support the maintenance of pregnancy by its effects on innate and adaptive immunity. OBJECTIVE: We assessed the effects of vitamin D supplementation during pregnancy on vitamin D status and markers of immune function associated with adverse pregnancy outcomes. METHODS: We conducted a randomized, controlled, double-blind intervention of 2 doses of cholecalciferol (400 and 2000 IU/d) from <20 wk to delivery in 57 pregnant women. Vitamin D status, regulatory and inflammatory T cells, markers of innate immunity and systemic inflammation, and clinical outcomes including maternal blood pressure and birth weight were assessed at 26 and 36 wk of pregnancy. RESULTS: Supplementation with 2000 IU/d vitamin D had a greater effect on the change in vitamin D status over pregnancy (P < 0.0001) and the final value at 36 wk (P < 0.0001) than 400 IU/d, increasing serum 25-hydroxyvitamin D from 81.1 nmol/L at baseline to 116 nmol/L at 36 wk and from 69.6 nmol/L at baseline to 85.6 nmol/L at 36 wk, respectively. The 2000-IU/d group had 36% more interleukin-10+ regulatory CD4+ T cells at 36 wk than did the 400-IU/d group (P < 0.007). The daily intake of 2000 compared with 400 IU/d tended to dampen the pregnancy-related increase in diastolic blood pressure by 1.3-fold (P = 0.06) and increase birth weight by 8.6% (P = 0.06), but these differences were not statistically significant. CONCLUSIONS: Supplementation with 2000 IU/d is more effective at increasing vitamin D status in pregnant women than 400 IU/d and is associated with increased regulatory T cell immunity that may prevent adverse outcomes caused by excess inflammation. This trial was registered at clinicaltrials.gov as NCT01417351.

Dietary long-chain omega-3 fatty acids do not diminish eosinophilic pulmonary inflammation in mice.

Although the effects of fish oil supplements on airway inflammation in asthma have been studied with varying results, the independent effects of the fish oil components, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), administered separately, are untested. Here, we investigated airway inflammation and hyperresponsiveness using a mouse ovalbumin exposure model of asthma assessing the effects of consuming EPA (1.5% wt/wt), DHA (1.5% wt/wt), EPA plus DHA (0.75% each), or a control diet with no added omega-3 polyunsaturated fatty acids. Consuming these diets for 6 weeks resulted in erythrocyte membrane EPA contents (molar %) of 9.0 (± 0.6), 3.2 (± 0.2), 6.8 (± 0.5), and 0.01 (± 0.0)%; DHA contents were 6.8 (± 0.1), 15.6 (± 0.5), 12.3 (± 0.3), and 3.8 (± 0.2)%, respectively. The DHA group had the highest bronchoalveolar lavage (BAL) fluid eosinophil and IL-6 levels (P < 0.05). Similar trends were seen for macrophages, IL-4, and IL-13, whereas TNF-α was lower in omega-3 polyunsaturated fatty acid groups than the control (P < 0.05). The DHA group also had the highest airway resistance, which differed significantly from the EPA plus DHA group (P < 0.05), which had the lowest. Oxylipins were measured in plasma and BAL fluid, with DHA and EPA suppressing arachidonic acid-derived oxylipin production. DHA-derived oxylipins from the cytochrome P450 and 15-lipoxygenase pathways correlated significantly with BAL eosinophil levels. The proinflammatory effects of DHA suggest that the adverse effects of individual fatty acid formulations should be thoroughly considered before any use as therapeutic agents in asthma.

Habitual diets rich in dark-green vegetables are associated with an increased response to ω-3 fatty acid supplementation in Americans of African ancestry.

Although substantial variation exists in individual responses to omega-3 (ω-3) (n-3) fatty acid supplementation, the causes for differences in response are largely unknown. Here we investigated the associations between the efficacy of ω-3 fatty acid supplementation and a broad range of nutritional and clinical factors collected during a double-blind, placebo-controlled trial in participants of African ancestry, randomly assigned to receive either 2 g eicosapentaenoic acid (EPA) + 1 g docosahexaenoic acid (n = 41) or corn/soybean oil placebo (n = 42) supplements for 6 wk. Food-frequency questionnaires were administered, and changes in erythrocyte lipids, lipoproteins, and monocyte 5-lipoxygenase-dependent metabolism were measured before and after supplementation. Mixed-mode linear regression modeling identified high (n = 28) and low (n = 13) ω-3 fatty acid response groups on the basis of changes in erythrocyte EPA abundance (P < 0.001). Compliance was equivalent (∼88%), whereas decreases in plasma triglycerides and VLDL particle sizes and reductions in stimulated monocyte leukotriene B4 production were larger in the high-response group. Although total diet quality scores were similar, the low-response group showed lower estimated 2005 Healthy Eating Index subscores for dark-green and orange vegetables and legumes (P = 0.01) and a lower intake of vegetables (P = 0.02), particularly dark-green vegetables (P = 0.002). Because the findings reported here are associative in nature, prospective studies are needed to determine if dietary dark-green vegetables or nutrients contained in these foods can enhance the efficacy of ω-3 fatty acid supplements. This trial was registered at clinicaltrials.gov as NCT00536185.

Blood-based targeted metabolipidomics reveals altered omega fatty acid-derived lipid mediators in relapsing-remitting multiple sclerosis patients.

Unresolved and uncontrolled inflammation is considered a hallmark of pathogenesis in chronic inflammatory diseases like multiple sclerosis (MS), suggesting a defective resolution process. Inflammatory resolution is an active process partially mediated by endogenous metabolites of dietary polyunsaturated fatty acids (PUFA), collectively termed specialized pro-resolving lipid mediators (SPMs). Altered levels of resolution mediators have been reported in several inflammatory diseases and may partly explain impaired inflammatory resolution. Performing LC-MS/MS-based targeted lipid mediator profiling, we observed distinct changes in fatty acid metabolites in serum from 30 relapsing-remitting MS (RRMS) patients relative to 30 matched healthy subjects (HS). Robust linear regression revealed 12 altered lipid mediators after adjusting for confounders (p <0.05). Of these, 15d-PGJ2, PGE3, and LTB5 were increased in MS while PGF2a, 8,9-DiHETrE, 5,6-DiHETrE, 20-HETE, 15-HETE, 12-HETE, 12-HEPE, 14-HDoHE, and DHEA were decreased in MS compared to HS. In addition, 12,13-DiHOME and 12,13-DiHODE were positively correlated with expanded disability status scale values (EDSS). Using Partial Least Squares, we identified several lipid mediators with high VIP scores (VIP > 1: 32% - 52%) of which POEA, PGE3, DHEA, LTB5, and 12-HETE were top predictors for distinguishing between RRMS and HS (AUC =0.75) based on the XGBoost Classifier algorithm. Collectively, these findings suggest an imbalance between inflammation and resolution. Altogether, lipid mediators appear to have potential as diagnostic and prognostic biomarkers for RRMS.

Direct comparison of fatty acid ratios in single cellular lipid droplets as determined by comparative Raman spectroscopy and gas chromatography.

Cellular lipid droplets are the least studied and least understood cellular organelles in eukaryotic and prokaryotic cells. Despite a significant body of research studying the physiology of lipid droplets it has not yet been possible to fully determine the composition of individual cellular lipid droplets. In this paper we use Raman spectroscopy on single cellular lipid droplets and least-squares fitting of pure fatty acid spectra to determine the composition of individual lipid droplets in cells after treatment with different ratios of oleic and palmitic acid. We validate the results of the Raman spectroscopy-based single lipid droplet analysis with results obtained by gas chromatography analysis of millions of cells, and find that our approach can accurately predict the relative amount of a specific fatty acid in the lipid droplet. Based on these results we show that the fatty acid composition in individual lipid droplets is on average similar to that of all lipid droplets found in the sample. Furthermore, we expand this approach to the investigation of the lipid composition in single cellular peroxisomes. We determine the location of cellular peroxisomes based on two-photon excitation fluorescence (TPEF) imaging of peroxisomes labeled with the green fluorescent protein, and successive Raman spectroscopy of peroxisomes. We find that in some cases peroxisomes can produce a detectable CARS signal, and that the peroxisomal Raman spectra exhibit an oleic acid-like signature.

Basal omega-3 fatty acid status affects fatty acid and oxylipin responses to high-dose n3-HUFA in healthy volunteers.

A subject's baseline FA composition may influence the ability of dietary highly unsaturated omega-3 FAs (n3-HUFA) to change circulating profiles of esterified FAs and their oxygenated metabolites. This study evaluates the influence of basal n3-HUFA and n3-oxylipin status on the magnitude of response to n3-HUFA consumption. Blood was collected from fasting subjects (n = 30) before and after treatment (4 weeks; 11 ± 2 mg/kg/day n3-HUFA ethyl esters). Esterified FAs were quantified in erythrocytes, platelets, and plasma by GC-MS. Esterified oxylipins were quantified in plasma by LC-MS/MS. Treatment with n3-HUFAs increased n3-HUFAs and decreased n6-HUFAs in all reservoirs and increased plasma n3-oxylipins without significantly changing n6-oxylipin concentrations. As subject basal n3-HUFAs increased, treatment-associated changes decreased, and this behavior was reflected in the percentage of 20:5n3 + 22:6n3 in red blood cell membrane FAs (i.e., the omega-3 index). To maintain an omega-3 index of 8% and thus reduce cardiovascular disease risk, our analyses suggest a maintenance dose of 7 mg/kg/day n3-HUFA ethyl esters for a 70-kg individual. These results suggest that the basal n3 index may have clinical utility to establish efficacious therapeutic experimental feeding regimens and to evaluate the USDA Dietary Guidelines recommendations for n3-HUFA consumption.

Quantitative profiling of oxylipins through comprehensive LC-MS/MS analysis: application in cardiac surgery.

Oxylipins, including eicosanoids, affect a broad range of biological processes, such as the initiation and resolution of inflammation. These compounds, also referred to as lipid mediators, are (non-) enzymatically generated by oxidation of polyunsaturated fatty acids such as arachidonic acid (AA). A plethora of lipid mediators exist which makes the development of generic analytical methods challenging. Here we developed a robust and sensitive targeted analysis platform for oxylipins and applied it in a biological setting, using high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) operated in dynamic multiple reaction monitoring (dMRM). Besides the well-described AA metabolites, oxylipins derived from linoleic acid, dihomo-γ-linolenic acid, α-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid were included. Our comprehensive platform allows the quantitative evaluation of approximately 100 oxylipins down to low nanomolar levels. Applicability of the analytical platform was demonstrated by analyzing plasma samples of patients undergoing cardiac surgery. Altered levels of some of the oxylipins, especially in certain monohydroxy fatty acids such as 12-HETE and 12-HEPE, were observed in samples collected before and 24 h after cardiac surgery. These findings indicate that this generic oxylipin profiling platform can be applied broadly to study these highly bioactive compounds in relation to human disease.

ALOX5 gene variants affect eicosanoid production and response to fish oil supplementation.

The objective of this study was to determine whether 5-lipoxygenase (ALOX5) gene variants associated with cardiovascular disease affect eicosanoid production by monocytes. The study was a randomized, double-masked, parallel intervention trial with fish oil (5.0 g of fish oil daily, containing 2.0 g of eicosapentaenoic acid [EPA] and 1.0 g of docosahexaenoic acid [DHA]) or placebo oil (5.0 g of corn/soy mixture). A total of 116 subjects (68% female, 20-59 years old) of African American ancestry enrolled, and 98 subjects completed the study. Neither ALOX5 protein nor arachidonic acid-derived LTB4, LTD4, and LTE4 varied by genotype, but 5-hydroxyeicosatetraenoate (5-HETE), 6-trans-LTB4, 5-oxo-ETE, 15-HETE, and 5,15-diHETE levels were higher in subjects homozygous for the ALOX5 promoter allele containing five Sp1 element tandem repeats ("55" genotype) than in subjects with one deletion (d) (three or four repeats) and one common ("d5" genotype) allele or with two deletion ("dd") alleles. The EPA-derived metabolites 5-HEPE and 15-HEPE and the DHA-derived metabolite 17-HDoHE had similar associations with genotype and increased with supplementation; 5-HEPE and 15-HEPE increased, and 5-oxo-ETE decreased to a greater degree in the 55 than in the other genotypes. This differential eicosanoid response is consistent with the previously observed interaction of these variants with dietary intake of omega-3 fatty acids in predicting cardiovascular disease risk.

Effects of dynamic exercise on plasma arachidonic acid epoxides and diols in human volunteers.

Metabolites of the cytochrome P450 (CYP) pathway may contribute to vasodilation of the vasculature. However, it is not known whether exercise affects their circulating concentrations. The authors determined effects of exercise intensity and duration on plasma concentrations of epoxy and dihydroxy metabolites of arachidonic acid. Their goal was to delineate the threshold workload, optimal workload, and duration required to produce increases in plasma concentrations of these vasoactive substances. Healthy volunteers (N = 14) performed maximal exercise testing on a bicycle ergometer during Visit 1. On separate days, subjects cycled for 20 min at 30%, 60%, and 80% of their maximal exercise intensity. The last day consisted of 40 min of exercise at 60% of maximal exercise intensity. Venous blood was obtained before, during, and after exercise for analysis. Compared with rest, increases were observed during the 80% workload at 20 min postexercise -14,15-DHET (0.77 ± 0.21 vs. 0.93 ± 0.27 nM) - and at 2 min postexercise: 11,12-DHET (0.64 ± 0.22 vs. 0.71 ± 0.24 nM; p < .05). Also compared with rest, 40-min values during the 60% workload were 14,15-DHET 0.79 ± 0.22 vs. 0.91 ± 0.31 nM and at 2 min post 14,15 EET 0.12 ± 0.06 vs. 0.21 ± 0.16 nM (p < .05). Results suggest the CYP metabolites (i.e., DHETs) are released during short-term high-intensity and long-term moderate-intensity exercise.

Plasma and serum oxylipin, endocannabinoid, bile acid, steroid, fatty acid and nonsteroidal anti-inflammatory drug quantification in a 96-well plate format.

The goal of this research was to develop a high-throughput, cost-effective method for metabolic profiling of lipid mediators and hormones involved in the regulation of inflammation and energy metabolism, along with polyunsaturated fatty acids and common over-the-counter non-steroidal anti-inflammatory drugs (NSAIDs). We describe a 96-well plate protein precipitation and filtration procedure for 50 µL of plasma or serum in the presence of 37 deuterated analogs and 2 instrument internal standards. Data is acquired in two back-to-back UPLC-MS/MS analyses using electrospray ionization with positive/negative switching and scheduled multiple reaction monitoring for the determination of 145 compounds, including oxylipins, endocannabinoids and like compounds, bile acids, glucocorticoids, sex steroids, polyunsaturated fatty acids, and 3 NSAIDs. Intra- and inter-batch variability was <25% for >70% of metabolites above the LOQ in both matrices, but higher inter-batch variability was observed for serum oxylipins and some bile acids. Results for NIST Standard Reference Material 1950, compared favorably with the 20 certified metabolite values covered by this assay, and we provide new data for oxylipins, N-acylethanolamides, glucocorticoids, and 17-hydroxy-progesterone in this material. Application to two independent cohorts of elderly men and women showed the routine detection of 86 metabolites, identified fasting state influences on essential fatty acid-derived oxylipins, N-acylethanolamides and conjugated bile acids, identified rare presence of high and low testosterone levels and the presence of NSAIDs in ∼10% of these populations. The described method appears valuable for investigations in large cohort studies to provide insight into metabolic cross-talk between the array of mediators assessed here.