Adrenic acid induces oxidative stress in hepatocytes.

Adrenic acid (ADA), which is an endogenously synthesized polyunsaturated free fatty acid, was significantly increased in nonalcoholic fatty liver disease (NAFLD) patients and NAFLD-model mice compared with the corresponding controls in our previous study. To elucidate the involvement of ADA in NAFLD and nonalcoholic steatohepatitis (NASH), we examined ADA-induced lipotoxicity in human hepatocarcinoma HepG2 cells. The ROS production in HepG2 cells was increased by exposure to ADA. It was also shown that the treatment with ADA decreased cell viability in a dose-dependent manner. The N-Acetyl-L-Cysteine pretreatment counteracted this ADA-induced ROS production and cell death. Furthermore, ADA modulated the expressions of SOD2, HO-1 and Gpx1 as antioxidant enzymes. These findings suggest that ADA could induce oxidative stress accompanied by cell death, providing new insights into lipotoxicity that is involved in the pathogenesis of NAFLD and NASH.

The C18:3n6/C22:4n6 ratio is a good lipid marker of chronic kidney disease (CKD) progression.

BACKGROUND: Chronic kidney disease (CKD) is a major challenge for public health due to increased risk of cardiovascular diseases (CVD) and premature death. The aim of this study was to determine the clinical picture of FA and the course of the pathophysiological mechanisms of CKD. METHODS: The study involved 149 patients with CKD and a control group including 43 people. Fatty acid profiles were investigated using gas chromatography. A total of 30 fatty acids and their derivatives were identified and quantified. The omega3, omega6, SFA, MUFA, and PUFA fatty acid contents were calculated. The correlation matrix was obtained for parameters relating to patients with CKD vs. FA, taking patients' sex into consideration. The index C18:3n6/C22:4n6 was calculated according to the length of the treatment. Statistica 12.0 software (Tulsa, Oklahoma, USA) was used for the statistical analyses. RESULTS: The results showed decreased levels of total PUFA and increased concentrations of MUFA, including the activation of the palmitic and oleic acid pathway. An increase in the levels of n-6 9C22: 4n6 family fatty acids in all the patients and a reduction in the n-3 family (EPA, DHA) were observed. C18:3n6 was negatively correlated and C22:4n6 was positively correlated with the duration of the treatment. The index C18:3n6/C22:4n6 was defined as a new marker in the progression of the disease. Moreover, the index C18:3n6/ C22:4n6 was drastically decreased in later period. Nervonic acid was higher in the CKD group. In the group of men with CKD, there was a negative correlation between the excretion of K+, anthropometric measurements, and the levels of EPA and DHA. CONCLUSIONS: The course of inflammation in CKD occurs through the decrease in PUFA and the synthesis of MUFA. The dominating cascade of changes is the elongation of GLA-C18:3n6 into DGLA-C20:3n6 and AA-C20:4n6. As CKD progresses, along with worsening anthropometrical parameters and increased secretion of potassium, the activity of Ʌ6-desaturase decreases, reducing the synthesis of EPA and DHA. The synthesis of AdA-C22:4n6 increases and the ratio C18:3n6/C22:4n6 drastically decreases after 5 years. This parameter can be used to diagnose disease progression.

Analysis on the Substrate Specificity of Recombinant Human Acyl-CoA Synthetase ACSL4 Variants.

Acyl-CoA synthetase long-chain family members (ACSLs) are a family of enzymes that convert long-chain free fatty acids into their acyl-CoAs. ACSL4 is an ACSL isozyme with a strong preference for arachidonic acid (AA) and has been hypothesized to modulate the metabolic fates of AA. There are two ACSL4 splice variants: ACSL4V1, which is the more abundant transcript, and ACSL4V2, which is believed to be restricted to the brain. In the present study, we expressed recombinant human ACSL4V1 and V2 in Spodoptera frugiperda 9 (Sf9) cells using the baculovirus expression system and then partially purified both variants by cobalt affinity column chromatography. We then established a novel ACSL assay system with LC-MS/MS, which is highly sensitive and applicable to various kinds of fatty acids, and used it to investigate the substrate specificity of recombinant human ACSL4V1 and V2. The results showed that both ACSL4 variants preferred various kinds of highly unsaturated fatty acids (HUFAs), including docosahexaenoic acid (DHA), adrenic acid (docosatetraenoic acid) and eicosapentaenoic acid (EPA), as well as AA as a substrate. Moreover, our kinetic studies revealed that the two variants had similar relative affinities for AA, EPA and DHA but different reaction rates for each HUFA. These results confirmed the importance of both of ACSL4 variants in the maintenance of membrane phospholipids bearing HUFAs. Structural analysis of these variants might reveal the molecular mechanism by which they maintain membrane phospholipids bearing HUFAs.

Untargeted Profiling of Concordant/Discordant Phenotypes of High Insulin Resistance and Obesity To Predict the Risk of Developing Diabetes.

This study explores the metabolic profiles of concordant/discordant phenotypes of high insulin resistance (IR) and obesity. Through untargeted metabolomics (LC-ESI-QTOF-MS), we analyzed the fasting serum of subjects with high IR and/or obesity ( n = 64). An partial least-squares discriminant analysis with orthogonal signal correction followed by univariate statistics and enrichment analysis allowed exploration of these metabolic profiles. A multivariate regression method (LASSO) was used for variable selection and a predictive biomarker model to identify subjects with high IR regardless of obesity was built. Adrenic acid and a dyglyceride (DG) were shared by high IR and obesity. Uric and margaric acids, 14 DGs, ketocholesterol, and hydroxycorticosterone were unique to high IR, while arachidonic, hydroxyeicosatetraenoic (HETE), palmitoleic, triHETE, and glycocholic acids, HETE lactone, leukotriene B4, and two glutamyl-peptides to obesity. DGs and adrenic acid differed in concordant/discordant phenotypes, thereby revealing protective mechanisms against high IR also in obesity. A biomarker model formed by DGs, uric and adrenic acids presented a high predictive power to identify subjects with high IR [AUC 80.1% (68.9-91.4)]. These findings could become relevant for diabetes risk detection and unveil new potential targets in therapeutic treatments of IR, diabetes, and obesity. An independent validated cohort is needed to confirm these results.

Adrenic acid as an inflammation enhancer in non-alcoholic fatty liver disease.

BACKGROUND: This study was designed to identify novel links between lipid species and disease progression in non-alcoholic fatty liver disease (NAFLD). METHODS: We analyzed lipid species in the liver and plasma of db/db mice fed a choline-deficient l-amino acid-defined, high-fat diet (CDAHFD) using liquid chromatography/mass spectrometry (LC/MS). An in vitro experiment was performed using HepG2 cells stimulated with recombinant human TNFα or IL1ß. The expression of steatosis-, inflammation-, and fibrosis-related genes were analyzed. Plasma samples from NAFLD patients were also analyzed by LC/MS. RESULTS: The CDAHFD-fed db/db mice with hepatic steatosis, inflammation, mild fibrosis, obesity, and hypercholesterolemia displayed significantly higher hepatic and plasma levels of free adrenic acid (p < 0.05). The accumulated adrenic acid in the CDAHFD-fed db/db mice was associated with increased expression of ELOVL2 and 5, and the suppression of the acyl-CoA oxidase 1 gene during peroxisomal ß-oxidation. The pretreatment of HepG2 cells with adrenic acid enhanced their cytokine-induced cytokines and chemokines mRNA expression. In NAFLD patients, the group with the highest ALT levels exhibited higher plasma adrenic acid concentrations than the other ALT groups (p-value for trend <0.001). CONCLUSION: Data obtained demonstrated that adrenic acid accumulation contributes to disease progression in NAFLD.

7,10,13,16-Docosatetraenoic acid impairs neurobehavioral development by increasing reactive oxidative species production in Caenorhabditis elegans.

AIMS: To investigate human breast milk (HBM) lipids that may adversely affect infant neurodevelopment. MAIN METHODS: We performed multivariate analyses that combined lipidomics and psychologic Bayley-III scales to identify which HBM lipids are involved in regulating infant neurodevelopment. We observed a significant moderate negative correlation between 7,10,13,16-docosatetraenoic acid (omega-6, C22H36O2, the common name adrenic acid, AdA) and adaptive behavioral development. We further studied the effects of AdA on neurodevelopment by using Caenorhabditis elegans (C. elegans) as a model. Worms from larval stages L1 to L4 were supplemented with AdA at 5 nominal concentrations (0 µM [control], 0.1 µM, 1 µM, 10 µM, and 100 µM) and subjected to behavioral and mechanistic analyses. KEY FINDINGS: Supplementation with AdA from larval stages L1 to L4 impaired neurobehavioral development, such as locomotive behaviors, foraging ability, chemotaxis behavior, and aggregation behavior. Furthermore, AdA upregulated the production of intracellular reactive oxygen species. AdA-induced oxidative stress blocked serotonin synthesis and serotoninergic neuron activity and inhibited expression of daf-16 and the daf-16-regulated genes mtl-1, mtl-2, sod-1, and sod-3, resulting in attenuation of the lifespan in C. elegans. SIGNIFICANCE: Our study reveals that AdA is a harmful HBM lipid that may have adverse effects on infant adaptive behavioral development. We believe this information may be critical for AdA administration guidance in children's health care.

The imperative of arachidonic acid in early human development.

This review is about the role of arachidonic acid (ArA) in foetal and early growth and development. In 1975 and '76, we reported the preferential incorporation of ArA into the developing brain of rat pups, its conservation as a principal component in the brains of 32 mammalian species and the high proportion delivered by the human placenta for foetal nutrition, compared to its parent linoleic acid (LA). ArA is quantitatively the principal acyl component of membrane lipids from foetal red cells, mononuclear cells, astrocytes, endothelium, and placenta. Functionally, we present evidence that ArA, but not DHA, relaxes the foetal mesenteric arteries. The placenta biomagnifies ArA, doubling the proportion of the maternal level in cord blood. The proportions of ArA and its allies (di-homo-gamma-linolenic acid (DGLA), adrenic acid and ω6 docosapentaenoic acid) are similar or higher than the total of ω3 fatty acids in human milk, maintaining the abundant supply to the developing infant. Despite the evidence of the importance of ArA, the European Food Standard Agency, in 2014 rejected the joint FAO and WHO recommendation on the inclusion of ArA in infant formula, although they recommended DHA. The almost universal dominance of ArA in the membrane phosphoglycerides during human organogenesis and prenatal growth suggests that the importance of ArA and its allies in reproductive biology needs to be re-evaluated urgently.

Changes in Phospholipid Composition of the Human Cerebellum and Motor Cortex during Normal Ageing.

(1) Background: Changes in phospholipid (phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine, i.e., PC, PE and PS) composition with age in the mitochondrial and microsomal membranes of the human cerebellum and motor cortex were examined and compared to previous analyses of the prefrontal cortex, hippocampus and entorhinal cortex. (2) Methods: Nano-electrospray ionization on a hybrid triple quadrupole−linear ion trap mass spectrometer was used to analyse the brain regions of subjects aged 18−104 years. (3) Results: With age, the cerebellum showed many changes in the major phospholipids (>10% of the phospholipid class). In both membrane types, these included increases in PE 18:0_22:6 and PS 18:0_22:6, decreases in PE 18:0_20:4 and PS 18:0_18:1 and an increase in PC 16:0_16:0 (microsomal membrane only). In addition, twenty-one minor phospholipids also changed. In the motor cortex, only ten minor phospholipids changed with age. With age, the acyl composition of the membranes in the cerebellum increased in docosahexaenoic acid (22:6) and decreased in the arachidonic (20:4) and adrenic (22:4) acids. A comparison of phospholipid changes in the cerebellum, motor cortex and other brain areas is provided. (4) Conclusions: The cerebellum is exceptional in the large number of major phospholipids that undergo changes (with consequential changes in acyl composition) with age, whereas the motor cortex is highly resistant to change.

Extended lipid profile in Romanian ischemic stroke patients in relation to stroke severity and outcome: a path analysis model.

INTRODUCTION: Our aim was to evaluate the extended lipid profile in ischemic stroke patients and the relationship with stroke type, severity and outcome. MATERIAL AND METHODS: We prospectively enrolled 124 ischemic stroke patients and 40 healthy controls; baseline plasma and erythrocyte membrane fatty acids concentrations and common lipid profile were analysed. Stroke severity was evaluated by NIHSS on admission, while the functional outcome was defined by mRS at discharge and after 3 months. RESULTS: Total cholesterol, triglycerides, HDL-cholesterol, DHA, adrenic, stearic and lauric acid were all lower in patients, taking into account that 87.7% of patients did not receive statins before admission. There was a different pattern in plasma and erythrocyte membrane of fatty acids between patients and controls, also omega-3 index was significantly lower in patients. Patients with poor outcome without statins had significantly lower triglyceride (p = 0.028), while the total cholesterol levels were significantly lower in patients with poor outcome (p = 0.03) but with treatment initiated after admission. Bivariate analysis revealed that patients with poor outcome had significantly lower triglyceride levels regardless the statins use, while the total cholesterol and HDL-cholesterol levels were significantly lower in patients with poor outcome under statin treatment. The long-term outcome were positively influenced by age (ß̂ = 0.22, p = 0.001), and NIHSS score at admission (ß̂ = 0.55, p < 0.001), and negatively by cholesterol levels (ß̂ = -0.17, p = 0.031). CONCLUSIONS: DHA, adrenic, stearic and lauric acid were lower in stroke patients; plasma adrenic acid was consumed during the acute phase. The most important predictors for long-term outcome was NIHSS at admission followed by age and total cholesterol.

The Contribution of Cytosolic Group IVA and Calcium-Independent Group VIA Phospholipase A2s to Adrenic Acid Mobilization in Murine Macrophages.

Adrenic acid (AA), the 2-carbon elongation product of arachidonic acid, is present at significant levels in membrane phospholipids of mouse peritoneal macrophages. Despite its abundance and structural similarity to arachidonic acid, very little is known about the molecular mechanisms governing adrenic acid mobilization in cells of the innate immune system. This contrasts with the wide availability of data on arachidonic acid mobilization. In this work, we used mass-spectrometry-based lipidomic procedures to define the profiles of macrophage phospholipids that contain adrenic acid and their behavior during receptor activation. We identified the phospholipid sources from which adrenic acid is mobilized, and compared the data with arachidonic acid mobilization. Taking advantage of the use of selective inhibitors, we also showed that cytosolic group IVA phospholipase A2 is involved in the release of both adrenic and arachidonic acids. Importantly, calcium independent group VIA phospholipase A2 spared arachidonate-containing phospholipids and hydrolyzed only those that contain adrenic acid. These results identify separate mechanisms for regulating the utilization of adrenic and arachidonic acids, and suggest that the two fatty acids may serve non-redundant functions in cells.

Plasma eicosanoids as noninvasive biomarkers of liver fibrosis in patients with nonalcoholic steatohepatitis.

BACKGROUND: Eicosanoid and related docosanoid polyunsaturated fatty acids (PUFAs) and their oxygenated derivatives have been proposed as noninvasive lipidomic biomarkers of nonalcoholic steatohepatitis (NASH). Therefore, we investigated associations between plasma eicosanoids and liver fibrosis to evaluate their utility in diagnosing and monitoring NASH-related fibrosis. METHODS: Our analysis used baseline eicosanoid data from 427 patients with biopsy-confirmed nonalcoholic fatty liver disease (NAFLD), and longitudinal measurements along with liver fibrosis staging from 63 patients with NASH and stage 2/3 fibrosis followed for 24 weeks in a phase II trial. RESULTS: At baseline, four eicosanoids were significantly associated with liver fibrosis stage: 11,12-DIHETE, tetranor 12-HETE, adrenic acid, and 14, 15-DIHETE. Over 24 weeks of follow up, a combination of changes in seven eicosanoids [5-HETE, 7,17-DHDPA, adrenic acid, arachidonic acid (AA), eicosapentaenoic acid (EPA), 16-HDOHE, and 9-HODE) had good diagnostic accuracy for the prediction of ⩾1 stage improvement in fibrosis (AUROC: 0.74; 95% CI: 0.62-0.87), and a combination of four eicosanoids (7,17-DHDPA, 14,15-DIHETRE, 9-HOTRE, and free adrenic acid) accurately predicted improvement in hepatic collagen content (AUROC: 0.72; 95% CI: 0.50-0.77). CONCLUSION: This study provides preliminary evidence that plasma eicosanoids may serve as noninvasive biomarkers of liver fibrosis and may predict liver fibrosis improvement in NASH.

Exposure to a Farm Environment During Pregnancy Increases the Proportion of Arachidonic Acid in the Cord Sera of Offspring.

Growing up in a farm environment is protective against allergy development. Various explanations have been put forward to explain this association. Fatty acids are regulators of immune function and the composition of fatty acids in the circulation system may affect immune development. Here, we investigate whether the fatty acid composition of cord serum differs for infants born to Farm (n = 26) or non-Farm mothers (n =29) in the FARMFLORA birth-cohort. For comparison, the levels of fatty acids in the maternal diet, serum and breast milk around 1 month post-partum were recorded. The fatty acids in the cord sera from infants born to Farm mothers had higher proportions of arachidonic acid (20:4 n-6) and adrenic acid (22:4 n-6) than those from infants born to non-Farm mothers. No differences were found for either arachidonic acid or adrenic acid in the diet, samples of the serum, or breast milk from Farm and non-Farm mothers obtained around 1 month post-partum. The arachidonic and adrenic acid levels in the cord blood were unrelated to allergy outcome for the infants. The results suggest that a farm environment may be associated with the fatty acid composition to which the fetus is exposed during pregnancy.

Increased isoprostanoid levels in brain from murine model of Krabbe disease - Relevance of isoprostanes, dihomo-isoprostanes and neuroprostanes to disease severity.

Krabbe disease (KD) is a rare and devastating pediatric leukodystrophy caused by mutations in the galactocerebrosidase (GALC) gene. The disease leads to impaired myelin formation and extensive myelin damage in the brain. Oxidative stress is implicated in the pathogenesis of KD but insofar few information is available. The gray and white matter of the brain are rich in docosahexaenoic acid and adrenic acid respectively and under non-enzymatic oxidative stress, release isoprostanoids, i.e. F4-neuroprostanes (F4-NeuroPs) and F2-dihomo-isoprostanes (F2-dihomo-IsoPs). In this study, the formation of isoprostanoids in brain tissue was investigated in a well-established KD mouse model (twitcher) that recapitulates the human pathology. According to the genotype determinations, three groups of mice were selected: wild-type control mice (n = 13), heterozygotes mice (carriers of GALC mutations, n = 14) and homozygous twitcher mice (n = 13). Measurement of F2-dihomo-IsoP and F4-NeuroP levels were performed on whole brain tissue obtained at day 15 and day 35 of the life cycle. Brain isoprostanoid levels were significantly higher in the twitcher mice compared to the heterozygous and wild-type control mice. However, F2-dihomo-IsoP and F4-NeuroP levels did not differ in brain of day 15 compared to day 35 of the heterozygote mice. Interestingly, isoprostanoid levels were proportionally enhanced with disease severity (F2-dihomo-IsoPs, rho = 0.54; F4-NeuroPs, rho = 0.581; P values ≤ 0.05; n = 13). Our findings are the first to show the key role of polyunsaturated fatty acid oxidative damage to brain grey and white matter in the pathogenesis and progression of KD. This shed new insights on the biochemical indexes of KD progression, and potentially provide information for novel therapeutic targets.

Identification of lipidomic markers of chronic 3,3',4,4',5-pentachlorobiphenyl (PCB 126) exposure in the male rat liver.

Exposure to PCB 126, an environmentally relevant aryl hydrocarbon receptor agonist, is an environmental factor causing hepatic steatosis in rodent models; however, the lipidome of PCB 126-exposed rats has not been investigated in-depth. The objective of the present study was therefore to characterize dose-dependent changes in the lipid profile in the liver of male Sprague-Dawley rats exposed to PCB 126. Rats were exposed for three month to intraperitoneal injections of 0.01, 0.05 and 0.2µmol/kg bw PCB 126 in corn oil. Control animals were exposed in parallel and received corn oil alone. Lipids were extracted from whole liver homogenate and levels of polar lipids and fatty acids incorporated into triglycerides (FATAGs) were determined with tandem mass spectrometry using electrospray ionization. PCB 126 exposure increased the hepatic content of polar lipids and FATAGs. Protein adjusted levels of several polar lipid classes, in particular phosphatidylserine levels, decreased, whereas FATAGs levels typically increased with increasing PCB 126 dose. Sensitive, dose-dependent endpoints of PCB 126 exposure included an increase in levels of adrenic acid incorporated into triglycerides and changes in levels of certain ether-linked phospholipid and 1-alkyl/1-alkenyldiacylglycerol species, as determined using partial least square discriminant analysis (PLS-DA) and ANOVA. These changes in the composition of polar lipids and fatty acid in the liver of PCB 126 exposed rats identified several novel markers of PCB 126-mediated fatty liver disease that need to be validated in further studies.

Current development in non-enzymatic lipid peroxidation products, isoprostanoids and isofuranoids, in novel biological samples.

Isoprostanoids and isofuranoids are lipid mediators that can be formed from omega-3 and omega-6 polyunsaturated fatty acids (PUFAs). F2-isoprostanes formed from arachidonic acid, especially 15-F2t-isoprostane, are commonly measured in biological tissues for decades as the biomarker for oxidative stress and diseases. Recently, other forms of isoprostanoids derived from adrenic, eicosapentaenoic, and docosahexaenoic acids namely F2-dihomo-isoprostanes, F3-isoprostanes, and F4-neuroprostanes respectively, and isofuranoids including isofurans, dihomo-isofurans, and neurofurans are reported as oxidative damage markers for different metabolisms. The most widely used samples in measuring lipid peroxidation products include but not limited to the blood and urine; other biological fluids, specialized tissues, and cells can also be determined. In this review, measurement of isoprostanoids and isofuranoids in novel biological samples by gas chromatography (GC)-mass spectrometry (MS), GC-MS/MS, liquid chromatography (LC)-MS, and LC-MS/MS will be discussed.