Polyunsaturated Fatty Acid Precursors Research Articles

Sex-dependent differences in tissue and blood n-3 PUFA levels following ALA or ALA + DHA feeding of liver-specific Elovl2-KO and control mice

Docosahexaenoic acid (DHA, 22:6n-3) must be consumed from the diet or synthesized from polyunsaturated fatty acid (PUFA) precursors, such as α-linolenic acid (ALA, 18:3n-3). Elongase 2 (encoded by Elovl2 gene) catalyzes two elongation reactions in the PUFA biosynthesis pathway and may be important in regulating the observed sex differences in n-3 PUFA levels. Our aim was to determine how targeted knockout of liver Elovl2 affects tissue and blood n-3 PUFA levels in male and female C57BL/6J mice. Twenty-eight-day old male and female liver Elovl2-KO and control mice were placed onto one of two dietary protocols for a total of 8 weeks (4–8 mice per genotype, per diet, per sex): 1) an 8-week 2 % ALA in total fat diet or 2) a 4-week 2 % ALA diet followed by a 4-week 2 % ALA + 2 % DHA diet. Following this 8-week feeding period, 12-week-old mice were sacrificed and serum, red blood cells (RBC), liver, heart and brain were collected and fatty acid levels measured. Significant interaction effects (p < 0.05, sex x genotype) for serum, RBC, liver and heart DHA levels were identified. In serum and liver, DHA levels were significantly different (p < 0.01) between all groups with male controls > female controls > female KO > male KO in serum and female controls > male controls > female KO > male KO in liver. In RBCs and the heart, female controls = male controls > female KO > male KO (p < 0.001). The addition of DHA to diet removed the interaction effects on DHA levels in the serum, liver and heart, yielding a significant sex effect in serum, liver (female > male, p < 0.01) and brain (male > female, p < 0.05) and genotype effect in serum and heart (control > KO, p < 0.05). Ablation of liver Elovl2 results in significantly lower blood and tissue DHA in a sex-dependent manner, suggesting a role for Elovl2 on sex differences in n-3 PUFA levels.

Predictors of upstream inflammation and oxidative stress pathways during early pregnancy

BackgroundInflammation and oxidative stress are critical to pregnancy, but most human study has focused on downstream, non-causal indicators. Oxylipins are lipid mediators of inflammation and oxidative stress that act through many biological pathways. Our aim was to characterize predictors of circulating oxylipin concentrations based on maternal characteristics. MethodsOur study was conducted among 901 singleton pregnancies in the LIFECODES Fetal Growth Study, a nested case-cohort with recruitment from 2007 to 2018. We measured a targeted panel of oxylipins in early pregnancy plasma and urine samples from several biosynthetic pathways, defined by the polyunsaturated fatty acid (PUFA) precursor and enzyme group. We evaluated levels across predictors, including characteristics of participants’ pregnancy, socioeconomic determinants, and obstetric and medical history. ResultsCurrent pregnancy and sociodemographic characteristics were the most important predictors of circulating oxylipins concentrations. Plasma oxylipins were lower and urinary oxylipins higher for participants with a later gestational age at sampling (13–23 weeks), higher prepregnancy BMI (obesity class I, II, or III), Black or Hispanic race and ethnicity, and lower socioeconomic status (younger age, lower education, and uninsured). For example, compared to those with normal or underweight prepregnancy BMI, participants with class III prepregnancy obesity had 45–46% lower plasma epoxy-eicosatrienoic acids, the anti-inflammatory oxylipins produced from arachidonic acid (AA) by cytochrome P450, and had 81% higher urinary 15-series F2-isoprostanes, an indicator of oxidative stress produced from non-enzymatic AA oxidation. Similarly, in urine, Black participants had 92% higher prostaglandin E2 metabolite, a pro-inflammatory oxylipin, and 41% higher 5-series F2-isoprostane, an oxidative stress indicator. ConclusionsIn this large pregnancy study, we found that circulating levels of oxylipins were different for participants of lower socioeconomic status or of a systematically marginalized racial and ethnic groups. Given associations differed along biosynthetic pathways, results provide insight into etiologic links between maternal predictors and inflammation and oxidative stress.

Protein concentrations and activities of fatty acid desaturase and elongase enzymes in liver, brain, testicle, and kidney from mice: Substrate dependency.

The synthesis rates of n-3 and n-6 polyunsaturated fatty acids (PUFAs) in rodents and humans are not agreed upon and depend on substrate availability independently of the capacity for synthesis. Therefore, we aimed to assess the activities of the enzymes for n-3 and n-6 PUFA synthesis pathways in liver, brain, testicle, kidney, heart, and lung, in relation to their protein concentration levels. Eight-week-old Balb/c mice (n = 8) were fed a standard chow diet (6.2% fat, 18.6% protein, and 44.2% carbohydrates) until 14 weeks of age, anesthetized with isoflurane and tissue samples were collected (previously perfused) and stored at -80°C. The protein concentration of the enzymes (Δ-6D, Δ-5D, Elovl2, and Elovl5) were assessed by ELISA kits; their activities were assayed using specific PUFA precursors and measuring the respective PUFA products as fatty acid methyl esters by gas chromatographic analysis. The liver had the highest capacity for PUFA biosynthesis, with limited activity in the brain, testicles, and kidney, while we failed to detect activity in the heart and lung. The protein concentration and activity of the enzymes were significantly correlated. Furthermore, Δ-6D, Δ-5D, and Elovl2 have a higher affinity for n-3 PUFA precursors compared to n-6 PUFA. The capacity for PUFA synthesis in mice mainly resides in the liver, with enzymes having preference for n-3 PUFAs.

Mice prenatally exposed to valproic acid do not show autism-related disorders when fed with polyunsaturated fatty acid-enriched diets

Dietary supplementations with n-3 polyunsaturated fatty acid (PUFA) have been explored in autism spectrum disorder (ASD) but their efficiency and potential in ameliorating cardinal symptoms of the disease remain elusive. Here, we compared a n-3 long-chain (LC) PUFA dietary supplementation (n-3 supp) obtained from fatty fish with a n-3 PUFA precursor diet (n-3 bal) obtained from plant oils in the valproic acid (VPA, 450 mg/kg at E12.5) ASD mouse model starting from embryonic life, throughout lactation and until adulthood. Maternal and offspring behaviors were investigated as well as several VPA-induced ASD biological features: cerebellar Purkinje cell (PC) number, inflammatory markers, gut microbiota, and peripheral and brain PUFA composition. Developmental milestones were delayed in the n-3 supp group compared to the n-3 bal group in both sexes. Whatever the diet, VPA-exposed offspring did not show ASD characteristic alterations in social behavior, stereotypies, PC number, or gut microbiota dysbiosis while global activity, gait, peripheral and brain PUFA levels as well as cerebellar TNF-alpha levels were differentially altered by diet and treatment according to sex. The current study provides evidence of beneficial effects of n-3 PUFA based diets, including one without LCPUFAs, on preventing several behavioral and cellular symptoms related to ASD.

FAAH Pro129Thr Variant Is Associated with Increased Cholesterol Levels in Normal-Weight Metabolically Unhealthy Subjects.

Introduction: The endocannabinoid system (ECS) plays an integral role in maintaining metabolic homeostasis, where an hyperactivation has been related with serum lipid alterations. The biological effects of ECS are limited by the activation of the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH) and by polyunsaturated fatty acid (PUFA) intake as precursors. The FAAH Pro129Thr variant has been associated with obesity in some populations. However, the association with metabolic phenotypes in the Mexican population has not been studied. This study aimed to analyze the association of the FAAH Pro129Thr variant with serum lipids and diet in Mexican adults with different metabolic phenotypes. Methods: This is a cross-sectional study with 306 subjects between 18 and 65 years of age. They were classified with normal weight (NW) or excess weight (EW) according to their body mass index (BMI). The EW group included individuals with overweight or obesity (BMI 25-39.9 kg/m2). The individuals were classified into two metabolic phenotypes, metabolically healthy and metabolically unhealthy (MUH), using the homeostatic model assessment of insulin resistance and the National Cholesterol Education Program-adenosine triphosphate III cutoff points for blood pressure, triglycerides, high-density lipoprotein cholesterol, and fasting glucose. Subjects with ≥2 of 5 altered parameters were classified as MUH. The FAAH Pro129Thr variant was determined by allelic discrimination with TaqMan® probes. Results: The total cholesterol and very low-density lipoprotein cholesterol levels were associated with the FAAH Pro129Thr variant in NW-MUH subjects. Moreover, a lower PUFA intake was found in EW-MUH subjects with the FAAH variant. Conclusions: FAAH Pro129Thr variant has an important role in lipid metabolism, especially in NW-MUH subjects. By contrast, a low dietary intake of endocannabinoid PUFA precursors may partly counteract the development of the altered lipid profile associated with overweight/obesity.

Linoleic and arachidonic fatty acids and their potential relationship with inflammation, pregnancy, and fetal development.

A healthy maternal diet must consider an appropriate supply of long-chain polyunsaturated fatty acids (LCPUFAs) precursors to ensure adequate growth and development of the fetus. In this regard, n-6 PUFAs, predominantly linoleic (C18:2 n-6, LA) and arachidonic acid (C20:4 n-6), have a central role in the development of the central nervous system because they are part of the membrane structure and participate in the metabolism and signal transduction of cells. Nevertheless, they can also be transformed into inflammatory metabolites promoting the pathogenesis of cardiovascular diseases, cancer, and autoimmune or inflammatory conditions. In modern westernized societies, there is a high dietary consumption of foods rich in n-6 PUFAs which could have detrimental consequences for the fetus and neonate due to excessive exposure to these fatty acids (FAs). To summarize the evidence of maternal, placental, and fetal alterations that an excessive intake of n-6 polyunsaturated FAs (PUFAs), LA, and AA), could produce during pregnancy. A thorough review of the literature regarding the effects of n-6 PUFAs during pregnancy and lactation including in vivo and in vitro models, was carried out using the PubMed database from the National Library of Medicine-National Institutes of Health. An elevated intake of n-6 PUFA, specifically LA, during pregnancy influences children's motor, cognitive, and verbal development during infancy and early childhood. Similarly, they could harm the placenta and the development of other fetal organs such as the fat tissue, liver, and cardiovascular system. Maternal diet, specifically LA intake, could have significant repercussions on fetal development and long-term consequences in the offspring, including the possibility of future metabolic and mental diseases. It would be necessary to focus on the prevention of these alterations through timely dietary interventions in the target population.

Differential effects of resolvin D1 and resolvin E1 on cementoblast function.

Resolvins are endogenous mediators of the resolution of inflammation. They are derived from omega-3 polyunsaturated fatty acid precursors. Resolvin D1 (RvD1) and Resolvin E1 (RvE1) are the best-characterized members for actively promoting periodontal regeneration in experimental animal models. Here, we evaluated the efficacy of RvD1 and RvE1 on cementoblasts, the key cells involved in dental cementum regeneration and the attachment of the tooth to the alveolar bone. Immortalized mouse cementoblasts (OCCM-30) were treated with different concentrations (0.1-1000ng/mL) of RvD1 and RvE1. Cell proliferation was measured using an electrical impedance-based real-time cell analyzer. Mineralization was evaluated with von Kossa staining. The mRNA expression of mineralized tissue-associated markers of bone sialoprotein (BSP), Type I collagen (COL I), osteocalcin (OCN), osteopontin (OPN), runt-related transcription factor 2 (RunX2), alkaline phosphatase (ALP), osteoprotegerin (OPG), receptor activator of nuclear factor kappa B (NF-κB) (RANK), receptor activator of NF-κB ligand (RANKL), and extracellular matrix-degrading enzymes [matrix metalloproteinase (MMP)-1, MMP-2, MMP-3, MMP-9, and their tissue inhibitors (TIMP-1, TIMP-2)], RvE1 receptor (ChemR23) and RvD1 receptor (ALX/PFR2), cytokines (tumor necrosis factor-alpha {TNF-α}, interleukin {IL}-1β, IL-6, IL-8, IL-10, IL-17), oxidative stress enzymes [superoxide dismutase (SOD), glutathione peroxidase (GPX), and cyclooxygenase-2 (Cox-2)] were analyzed using quantitative polymerase chain reaction (qPCR). Both RvD1 and RvE1 (10-100ng/mL) significantly increased the proliferation of cementoblasts and mineralized nodules at all concentrations (p<0.05). RvE1 increased BSP, RunX2, and ALP compared with the RvD1 dose and time-dependently, while RvD1 and RvE1 differentially regulated COL-I. RvE1 increased OPG mRNA expression, whereas RANK-RANKL mRNA expression decreased by RvE1. MMP-2, MMP-3, MMP-9, TIMP-1, and TIMP-2 expressions were reduced by RvE1 compared with RvD1. Treatment of cementoblasts with RvD1 and RvE1 differentially affected cytokine and oxidative stress enzymes while significantly increasing their receptor expressions (ChemR23 and ALX/PFR2). RvD1 and RvE1 regulate proliferation, mineralization, and gene expression in cementoblasts using similar pathways while differentially affecting tissue degradation, suggesting a targeted therapeutic approach for cementum turnover during periodontal regeneration.

Oxylipin Profiles during the First Day of Mechanical Ventilation in an Intensive Care Unit Cohort: Research Letter.

Lipid mediators derived from arachidonic acid and other precursor polyunsaturated fatty acids, collectively known as oxylipins, are metabolized along different distinct pathways and play important roles in the modulation of inflammation.1,2 Blood oxylipin profiles have been shown to correlate with the survival and development of acute respiratory distress syndrome in patients with sepsis or septic shock.3–5 We aimed to explore serum oxylipin profiles using liquid chromatography coupled to tandem mass spectrometry quantification as previously described6 during the first day of mechanical ventilation in the intensive care unit (ICU). Previously, we have noted elevated plasma concentrations of thromboxane B2, prostaglandin E2, 15-hydroxyeicosatetraenoate, and 11-hydroxyeicosatetraenoate in response to experimental hyperinflation lung injury7 in a large animal model. Based on this, we hypothesized that these metabolites would increase in response to mechanical ventilation in patients. Decreases in a subset of oxylipins have been observed, however, in relation to critical illness.8With ethical approval (Linköping, 2010/427-31 and 2018/16-32), all consecutive adult patients (n = 589) with indwelling arterial cannulas after admission to the ICU in Östersund Hospital, Sweden, between February 1, 2012, and January 31, 2013, were screened for inclusion in the study cohort unless transferred from another ICU.9 Blood samples were collected in EDTA tubes during 2012 and 2013. Within 30 min, the samples were spun for 10 min at 2,000g. The plasma was then frozen in aliquots to –70°C, then unfrozen, and analyzed in 2021. In the cohort,9 147 cases with complete data and samples were identified. Of these, 22 men and 3 women with mixed diagnoses were intubated and included in the study with case characteristics in Supplemental Table 1 (https://links.lww.com/ALN/D48). Samples were collected at the time of intubation and on the following morning.Of 67 oxylipins in the analysis panel, 57 were detected (fig. 1). Of these, 23 were above the limit of quantification in at least 80% of the samples, which was the predetermined limit for oxylipins to be included in formal analysis. The detected metabolites included all major metabolic pathways, cyclooxygenase, lipoxygenase, and cytochrome P450. Complete oxylipin analysis results are given in Supplemental Table 1 (https://links.lww.com/ALN/D48). None of the oxylipins was assumed to be normally distributed. After log transformation, 19 oxylipins were deemed to be normally distributed and were thus evaluated with paired Student’s t test and Hedges’ g. The remaining four oxylipins were compared using the Wilcoxon signed-rank test (table 1). Correction for multiple comparisons was performed with the false discovery rate set to 10% for α = 0.05, yielding a significance level at P < 0.001.For samples after up to 1 day of mechanical ventilation (after intubation), 12-hydroxyeicosatetraenoate showed significantly lower plasma concentrations, and no oxylipin increased (table 1).The current study supports decreased concentrations of 12-hydroxyeicosatetraenoate in response to mechanical ventilation in contrast to previous reports of 12-hydroxyeicosatetraenoate being considered a proinflammatory mediator.10 Thromboxane B2 and prostaglandin E2 were not among the oxylipins with sufficient detection frequencies for formal analysis. In contrast to the main hypothesis, 15-hydroxyeicosatetraenoate and 11-hydroxyeicosatetraenoate did not increase during the first day of mechanical ventilation.In conclusion, this study demonstrates that several oxylipins may be detected in intensive care patients and that plasma concentration of 12-hydroxyeicosatetraenoate decreases during the first day of mechanical ventilation in the ICU.This work was supported by the Swedish Research Council VR (2014–6354; Stockholm, Sweden), Marie Sklodowska Curie Actions, Cofund, Project INCA 600398 (European Union), Åke Wiberg’s Foundation (M17-0219; Stockholm, Sweden), research funding from Umeå University (Umeå, Sweden), and the ALF-LUA cooperative Region Västerbotten-Umeå University (Umeå, Sweden) intramural research funding system.Dr. Larsson has received speaker fees for lecturing in clinical symposia sponsored by Dräger Medical (Götesburg, Sweden), and not related to this specific topic. We do not consider this relevant to the report. The authors declare no other conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.Supplemental Digital Content, https://links.lww.com/ALN/D48

Application of Aroma Precursors in Food Plant Raw Materials: Biotechnological Aspect

The article is devoted to the analysis of the main factors accompanying the use of aroma precursors, in particular, of a lipid nature, in food raw materials. The prerequisites for the impact on the precursors of aroma with the help of plant enzymes are given. The purpose of the article is to analyze the biotechnological aspect, which is based on enzymatic reactions with aroma precursors and enzymes of plant origin. Features of the mechanism of action of lipid precursors are highlighted, their diversity causing various characteristic reactions is analyzed, and possible end products of reactions with certain odors are noted. The attention is paid to the issue of the status of the naturalness of flavor precursors in food products, which varies in different countries. A scheme of factors influencing the formation of aroma from lipid precursors has been developed. The influence of pigments of carotenoid nature on the aroma is considered, namely: examples of instantaneous change of watermelon aroma to pumpkin one due to isomerization of carotenoids are given. The main factors of enzymatic formation of aroma from precursors of polyunsaturated fatty acids for their effective use by creating micromicelles are summarized. A way to overcome the barrier of interaction between lipid precursors of a hydrophobic nature and hydrophilic enzymes has been substantiated. It is proposed to accelerate enzymatic reactions under in vitro conditions and use the vacuum effect to overcome the barrier between enzymes and precursors. To explain the effect of vacuum in a system with enzymes, ideas about disjoining pressure and the reasonable expediency of its use are considered. A schematic process flow diagram for the restoration of aroma lost during the technological processing of raw materials is given; it demonstrates the factors for ensuring interfacial activation conditions for enzymes and aroma precursors.

Plasma Oxylipins and Their Precursors Are Strongly Associated with COVID-19 Severity and with Immune Response Markers.

COVID-19 is characterised by a dysregulated immune response, that involves signalling lipids acting as mediators of the inflammatory process along the innate and adaptive phases. To promote understanding of the disease biochemistry and provide targets for intervention, we applied a range of LC-MS platforms to analyse over 100 plasma samples from patients with varying COVID-19 severity and with detailed clinical information on inflammatory responses (>30 immune markers). The second publication in a series reports the results of quantitative LC-MS/MS profiling of 63 small lipids including oxylipins, free fatty acids, and endocannabinoids. Compared to samples taken from ward patients, intensive care unit (ICU) patients had 2–4-fold lower levels of arachidonic acid (AA) and its cyclooxygenase-derived prostanoids, as well as lipoxygenase derivatives, exhibiting negative correlations with inflammation markers. The same derivatives showed 2–5-fold increases in recovering ward patients, in paired comparison to early hospitalisation. In contrast, ICU patients showed elevated levels of oxylipins derived from poly-unsaturated fatty acids (PUFA) by non-enzymatic peroxidation or activity of soluble epoxide hydrolase (sEH), and these oxylipins positively correlated with markers of macrophage activation. The deficiency in AA enzymatic products and the lack of elevated intermediates of pro-resolving mediating lipids may result from the preference of alternative metabolic conversions rather than diminished stores of PUFA precursors. Supporting this, ICU patients showed 2-to-11-fold higher levels of linoleic acid (LA) and the corresponding fatty acyl glycerols of AA and LA, all strongly correlated with multiple markers of excessive immune response. Our results suggest that the altered oxylipin metabolism disrupts the expected shift from innate immune response to resolution of inflammation.

Early biochemical observations point to nutritional strategies to manage non-alcoholic fatty liver disease

Abstract Non-alcoholic fatty liver disease (NAFLD) is the most prevalent liver disease globally. The first stage of NAFLD is steatosis, the accumulation of triacylglycerols within hepatocytes. Inflammation and oxidative stress both contribute to progression to more severe disease. In 2004 Clinical Science published two papers reporting on fatty acids and oxidative stress markers in the livers of patients with NAFLD; both these papers are highly cited. One paper reported an altered pattern of fatty acids within the livers of patients with NAFLD; there was a lower contribution of polyunsaturated fatty acids (PUFAs) including both n - 6 and n - 3 PUFAs and an altered balance between n - 6 and n - 3 PUFAs in favour of the former. Ratios of precursor PUFAs to their long chain more unsaturated derivatives were altered in NAFLD and were interpreted to indicate a reduced activity of the pathway of synthesis of long chain highly unsaturated PUFAs. The authors interpreted their findings to indicate that a low hepatic content of n - 3 PUFAs has a causal role in NAFLD. The second paper reported lower hepatic antioxidant defences and increased markers of oxidative stress in NAFLD, consistent with a role for oxidative stress in the disease. Many studies have now explored the effect of supplemental n - 3 PUFAs or antioxidants, including vitamin E, in patients with NAFLD with some benefits being reported. There remains much interest in n - 3 PUFAs and antioxidants as preventive and therapeutic strategies in NAFLD and therefore it seems likely that citation of the two papers from 2004 will be sustained.

Synechococcus elongatus PCC 7942 as a Platform for Bioproduction of Omega-3 Fatty Acids.

Alpha-linolenic acid and stearidonic acid are precursors of omega-3 polyunsaturated fatty acids, essential nutrients in the human diet. The ability of cyanobacteria to directly convert atmospheric carbon dioxide into bio-based compounds makes them promising microbial chassis to sustainably produce omega-3 fatty acids. However, their potential in this area remains unexploited, mainly due to important gaps in our knowledge of fatty acid synthesis pathways. To gain insight into the cyanobacterial fatty acid biosynthesis pathways, we analyzed two enzymes involved in the elongation cycle, FabG and FabZ, in Synechococcus elongatus PCC 7942. Overexpression of these two enzymes led to an increase in C18 fatty acids, key intermediates in omega-3 fatty acid production. Nevertheless, coexpression of these enzymes with desaturases DesA and DesB from Synechococcus sp. PCC 7002 did not improve alpha-linolenic acid production, possibly due to their limited role in fatty acid synthesis. In any case, efficient production of stearidonic acid was not achieved by cloning DesD from Synechocystis sp. PCC 6803 in combination with the aforementioned DesA and DesB, reaching maximum production at 48 h post induction. According to current knowledge, this is the first report demonstrating that S. elongatus PCC 7942 can be used as an autotrophic chassis to produce stearidonic acid.

Formation, Signaling and Occurrence of Specialized Pro-Resolving Lipid Mediators-What is the Evidence so far?

Formation of specialized pro-resolving lipid mediators (SPMs) such as lipoxins or resolvins usually involves arachidonic acid 5-lipoxygenase (5-LO, ALOX5) and different types of arachidonic acid 12- and 15-lipoxygenating paralogues (15-LO1, ALOX15; 15-LO2, ALOX15B; 12-LO, ALOX12). Typically, SPMs are thought to be formed via consecutive steps of oxidation of polyenoic fatty acids such as arachidonic acid, eicosapentaenoic acid or docosahexaenoic acid. One hallmark of SPM formation is that reported levels of these lipid mediators are much lower than typical pro-inflammatory mediators including the monohydroxylated fatty acid derivatives (e.g., 5-HETE), leukotrienes or certain cyclooxygenase-derived prostaglandins. Thus, reliable detection and quantification of these metabolites is challenging. This paper is aimed at critically evaluating i) the proposed biosynthetic pathways of SPM formation, ii) the current knowledge on SPM receptors and their signaling cascades and iii) the analytical methods used to quantify these pro-resolving mediators in the context of their instability and their low concentrations. Based on current literature it can be concluded that i) there is at most, a low biosynthetic capacity for SPMs in human leukocytes. ii) The identity and the signaling of the proposed G-protein-coupled SPM receptors have not been supported by studies in knock-out mice and remain to be validated. iii) In humans, SPM levels were neither related to dietary supplementation with their ω-3 polyunsaturated fatty acid precursors nor were they formed during the resolution phase of an evoked inflammatory response. iv) The reported low SPM levels cannot be reliably quantified by means of the most commonly reported methodology. Overall, these questions regarding formation, signaling and occurrence of SPMs challenge their role as endogenous mediators of the resolution of inflammation.

Altered oxylipin levels in human vitreous indicate imbalance in pro-/anti-inflammatory homeostasis in proliferative diabetic retinopathy

Proliferative diabetic retinopathy (PDR) is an advanced stage of diabetic retinopathy (DR), characterized by retinal neovascularization. It is a progressive fundus disease and a severe complication of diabetes that causes vision impairment. Hyperglycemia-induced persistent low-grade inflammation is a crucial factor underlying the pathogenesis of DR-associated damage and contributing to the progression of PDR. Highly enriched polyunsaturated fatty acids (PUFAs) in the retina are precursors to oxidized metabolites, namely, oxylipins, which exert pro-inflammatory or anti-inflammatory (resolving) effects under different pathological conditions and have been implicated in diabetes. To evaluate differences in oxylipin levels in the vitreous obtained from PDR and non-diabetic subjects, we performed a targeted assessment of oxylipins. A total of 41 patients with PDR and 22 non-diabetic control subjects were enrolled in this study. Vitreous humor obtained during routinely scheduled vitrectomy underwent a targeted but unbiased screening for oxylipins using mass spectrometry-based lipidomics. We found 21 oxylipins showing statistically significant differences in their levels between PDR and non-diabetic subjects (p < 0.05). Lipoxygenase (LOX)- and cytochrome P450 (CYP)- derived oxylipins were the most affected, while cyclooxygenase (COX) oxylipins were affected to a lesser extent. When categorized by their precursor PUFAs, ±19,20-EpDPE, a CYP product of docosahexaenoic acid (DHA) and 12S-HETE, a LOX product of arachidonic acid (ARA), were increased by the largest magnitude. Moreover, of these 21 oxylipins, 7 were considered as potential biomarkers for discriminating PDR patients from the non-diabetic controls. Our results indicate that altered oxylipin levels in the vitreous implicate an underlying imbalanced inflammation-resolution homeostasis in PDR.

Research Profile: Dr. Jeevan K. Prasain

Dr. Jeevan K. Prasain is an associate professor of pharmacology and toxicology at the University of Alabama at Birmingham. His current research focuses on delineation of unconventional cyclooxygenase (Cox)-independent prostaglandin (PG) synthesis mechanism, mass spectrometry-based metabolomics/lipidomics, biological activity, and uptake and metabolisms of dietary botanicals in vitro and in vivo. Dr. Prasain received his MSc in chemistry from Tribhuvan University located in Kathmandu, Nepal and went on to obtain his PhD in Japan from the Toyama Medical and Pharmaceutical University in pharmaceutical sciences. During his PhD tenure, he devoted much of his studies to natural product chemistry resulting in the discovery of several novel bioactive diarylheptanoids, subjects he went on to publish in several papers. After the completion of his PhD, he was awarded the Canadian Government Visiting Fellowship at the Biotechnology Research Institute (BRI) in Montreal, Canada to pursue postdoctoral research. During his time at BRI, Dr. Prasain was involved in drug discovery and high throughput screening of bioactive natural products from natural product libraries and their analysis by tandem mass spectrometry. Through this research, Dr. Prasain later went on to be a recipient of a Science and Technology Award at RIKEN in Tokyo Japan where he developed a mass spectrometry method for analysis of antibiotic cytochalasins. Based on his experiences in natural products research, he developed a great interest in dietary polyphenols in the promotion of health and prevention of chronic disease. Dr. Prasain joined the University of Alabama at Birmingham (UAB) as a postdoctoral fellow with Dr. Stephen Barnes in 2001, where he was later promoted to Assistant Professor in 2003. One of his research focuses was on the understanding of antidiabetic effects of puerarin, an isoflavone C-glucoside from Kudzu root and its metabolism and bioavailability. Kudzu root is well-known in traditional Chinese medicine, and various kudzu root based dietary supplements are available in the US market. Prasain's lab, in collaboration with Dr. Elias Meezan, also at UAB, demonstrated that puerarin treatment significantly improves glucose tolerance in C57 BL/6J ob/ob mice, an animal model of Type 2 diabetes mellitus. Specifically, puerarin blunts the rise in blood glucose levels after i.p. administration of glucose. Prasain et al. also found that in contrast to its O-glucoside isomer daidzin, puerarin is essentially absorbed without undergoing metabolism and is excreted in the urine mainly as an unchanged parent. In addition, in collaboration with Dr. Clinton Grubbs at UAB on the role of cranberry juice in bladder cancer prevention in animals, Prasain et al. demonstrated that commercially available cranberry juice concentrate can prevent urinary bladder cancers dose-dependently in N-butyl-N-(4-hydroxybutyl)-nitrosamine (OH-BBN) induced bladder carcinogenesis in rats when compared to the control group. This study also indicated that cranberry compounds once ingested reach the urinary bladder and concentrate in the urine in either the intact or in the metabolized forms and may prevent against the progression of carcinogenesis. This is because bladder cancers occur almost exclusively in the bladder epithelium, which is directly exposed to the urine stored in the bladder. In 2009, Dr. Prasain received an NIH R21 funding for investigating urinary metabolites of cranberry that protect against bladder cancer. The primary focus of this project was to understand what cranberry metabolites stored in the bladder are bioavailable and which metabolites are responsible against bladder carcinogenesis. The other focus of Dr. Prasain's lab is in the area of lipidomics, particularly profiling and characterizing PGs and other signaling lipids using tandem mass spectrometry. Using a combination of genetics, microinjection, and mass spectrometry, the late Dr. Michael Miller's and Dr. Prasain's labs discovered that C. elegans oocytes secrete sperm attractants F-series PGs derived from polyunsaturated fatty acid precursors. This NIH funded research led to an unanticipated discovery that C. elegans and mice generate specific F-series PGs without Cox enzymes. Over the past several years, much of the Dr. Prasain's lab effort has been to identify genes essential for Cox-independent PG synthesis and the signaling mechanisms that regulate PG functions in reproductive biology. The authors declare no conflict of interest.

The effect of feeding whole-crop barley or black oat silage in the fatty acid content of milk from cows in small-scale dairy systems in the highlands of Mexico.

Diets have an important effect on the fatty acid (FA) content in milk from dairy cows so that feeding strategies may modify the FA profile. There is little information on the fatty acid contents of whole-crop barley (BLY) and black oat (BKO) silages and on milk from cows fed these silages that are an alternative forage for small-scale dairy systems (SSDS). Given the growing interest in foods with favourable health attributes, the objective was to identify the FA content of milk from dairy cows fed whole-crop BLY and BKO silages in small-scale systems. Three inclusion levels of BLY and BKO silage were assessed in nine lactating cows at pasture for 8h/day and supplemented with 4.6kg/DM/cow/day commercial concentrate. Treatments were BLY: 10kg DM/cow/day BLY silage; BKO: 10kg DM/cow/d BKO silage; and BLY/BKO: 5kg BLY and 5kg DM/cow/d BKO silages (50:50). A 3 × 3 Latin square design repeated three times with nine cows and 14-day periods was utilised. Variables evaluated were the content of fatty acids present in feeds and milk. Contents of linoleic (C18:2n-6) and linolenic (C18:3n-3) acids, as precursors of polyunsaturated fatty acids, were 46 in BLY and 50 BKO g/100g, lower than for multispecies pasture (61g/100g). There were statistical differences in the content of short-chain fatty acids in milk (P < 0.05), being lower for BLY (18.9g/100g) compared with BKO (20.4g/100g) and BLY/BKO (20.6g/100g), the saturated fatty acids and linoleic acid (C18:2n-6c) were higher in BLY/BKO in relation to BLY and BKO. Content of health-promoting polyunsaturated fatty acids in milk was higher in BLY/BKO (P < 0.05). There were no differences (P < 0.05) in the atherogenic index, with values within reports. Small-grain cereal forages may produce milk with a favourable fatty acid content for human health.

Serum levels of n-3 and n-6 polyunsaturated fatty acids in patients with systemic lupus erythematosus and their association with disease activity: a pilot study

Objective Polyunsaturated fatty acids (PUFAs) may modulate the inflammatory process in systemic autoimmune diseases, including systemic lupus erythematosus (SLE). The aim of this study was to assess the serum concentrations of essential 18-carbon PUFAs and their long-chain derivatives in patients with SLE and healthy controls, and to analyse their associations with laboratory and clinical features of the disease. Method n-6 and n-3 PUFA composition was assessed in the sera of 30 SLE patients and 20 healthy controls using gas chromatography–mass spectrometry. We investigated the associations between PUFAs and disease activity measured with Systemic Lupus Erythematosus Activity Index (SLEDAI) scores, erythrocyte sedimentation rate, C-reactive protein, complement C3 and C4 concentrations, anti-nuclear antibody (ANA) titre, anti-double-stranded DNA (anti-dsDNA) antibody concentration, and medications. Results Serum linoleic acid (LA) and α-linolenic acid concentrations were significantly higher in SLE patients compared with healthy controls. LA concentration correlated positively with the ANA titre and corticosteroid doses; eicosapentaenoic acid (EPA) and docosahexaenoic acid correlated inversely with anti-dsDNA antibody concentration. Patients treated with immunosuppressants had significantly lower concentrations of LA, arachidonic acid, and EPA. Conclusion Both n-6 and n-3 PUFA precursors can participate in the inflammatory process in SLE patients. The mechanism of the PUFA metabolism disturbance needs further exploration.

Elovanoids downregulate SARS-CoV-2 cell-entry, canonical mediators and enhance protective signaling in human alveolar cells

The pro-homeostatic lipid mediators elovanoids (ELVs) attenuate cell binding and entrance of the SARS-CoV-2 receptor-binding domain (RBD) as well as of the SARS-CoV-2 virus in human primary alveoli cells in culture. We uncovered that very-long-chain polyunsaturated fatty acid precursors (VLC-PUFA, n-3) activate ELV biosynthesis in lung cells. Both ELVs and their precursors reduce the binding to RBD. ELVs downregulate angiotensin-converting enzyme 2 (ACE2) and enhance the expression of a set of protective proteins hindering cell surface virus binding and upregulating defensive proteins against lung damage. In addition, ELVs and their precursors decreased the signal of spike (S) protein found in SARS-CoV-2 infected cells, suggesting that the lipids curb viral infection. These findings open avenues for potential preventive and disease-modifiable therapeutic approaches for COVID-19.

The Influence of Phospholipase A 2 (PLA 2 ) Enzymes on Fatty Acid Release and Oxylipin Formation in Rat Hearts

Polyunsaturated fatty acids (PUFA) released by PLA2 are converted into diverse bioactive lipid mediators called oxylipins. However, little is known about the preference of PLA2 isoforms for PUFA release, and even less is known about how this further impacts the formation of oxylipins. Further, oxylipin levels in tissues do not always mimic the levels of their precursor PUFA, so conclusions about oxylipins cannot be drawn from PUFA content. Therefore, we aimed to determine the impact of PLA2 isoforms on PUFA release and oxylipin formation. We hypothesized that different PLA2 isoforms would impact PUFA release and oxylipin formation differently, and that oxylipin formation would not necessarily reflect changes seen in PUFA. Rat heart homogenates were incubated at 37C for 20 min with or without inhibitors, as the increase of both free PUFA and oxylipins was linear at 20 min incubation. Free PUFA and oxylipins were analyzed by HPLC-MS/MS. Two inhibitors were used: Varespladib (VAR) targeting secreted (sPLA2) isoforms IIA, V and X, and methyl arachidonyl fluorophosphonate (MAFP) targeting cytosolic (cPLA2) and calcium-independent (iPLA2) isoforms. Arachidonic Acid (ARA) release was similarly inhibited by both VAR and MAFP, however, while 15 out of 29 ARA oxylipins were inhibited with MAFP, only 3 out of 29 were inhibited with VAR. Docosahexaenoic acid (DHA) release was more strongly inhibited by VAR than by MAFP, which corresponded with 7 out of 12 DHA oxylipins being more strongly inhibited by VAR than by MAFP. Both alpha-linolenic acid (ALA) and eicosapentaenoic acid (EPA) were only inhibited by MAFP, which also inhibited 3 out of 5 ALA and 1 out of 3 EPA oxylipins. Further, 9 out of 10 LA oxylipins were inhibited by MAFP while none were affected by VAR. In conclusion, in the rat heart ARA is released by sPLA2 IIA, V, and/or X as well as by cPLA2 and/or iPLA2, DHA is released more by sPLA2 IIA, V, and/or X than by cPLA2 and/or iPLA2, and EPA and ALA are released primarily by cPLA2 and/or iPLA2. Effects on oxylipin formation largely reflected those on precursor PUFA release, with an exception, as inhibition of sPLA2 IIA, V and X by VAR mostly did not affect ARA oxylipin formation even though it inhibited ARA release. Therefore, careful evaluation of the effects on the oxylipin profile is needed when considering PLA2 inhibitors for potential treatments of diseases.

Mechanisms of TGFß in prostaglandin synthesis and sperm guidance in Caenorhabditis elegans.

The transparent epidermis of Caenorhabditis elegans makes it an attractive model to study sperm motility and migration within an intact reproductive tract. C elegans synthesize specific F-series prostaglandins (PGFs) that are important for guiding sperm toward the spermatheca. These PGFs are synthesized from polyunsaturated fatty acid (PUFA) precursors, such as arachidonic acid (AA), via a novel pathway, independent of the classical cyclooxygenases (Cox) responsible for most PG synthesis. While the enzyme(s) responsible for PG synthesis has yet to be identified, the DAF-7 TGFß pathway has been implicated in modulating PG levels and sperm guidance. We find that the reduced PGF levels in daf-1 type I receptor mutants are responsible for the sperm guidance defect. The lower level of PGs in daf-1 mutants is due in part to the inaccessibility of AA. Finally, lipid analysis and assessment of sperm guidance in daf-1;daf-3 double mutants suggest DAF-3 suppresses PG production and sperm accumulation at the spermatheca. Our data suggest that DAF-3 functions in the nervous system, and possibly the germline, to affect sperm guidance. The C elegans TGFß pathway regulates many pathways to modulate PG metabolism and sperm guidance. These pathways likely function in the nervous system and possibly the germline.