Molecular Pathways Linking Oxylipins to Nociception in Rats.

Oxylipins are lipid peroxidation products that participate in nociceptive, inflammatory, and vascular responses to injury. Effects of oxylipins depend on tissue-specific differences in accumulation of precursor polyunsaturated fatty acids and the expression of specific enzymes to transform the precursors. The study of oxylipins in nociception has presented technical challenges leading to critical knowledge gaps in the way these molecules operate in nociception. We applied a systems-based approach to characterize oxylipin precursor fatty acids, and expression of genes coding for proteins involved in biosynthesis, transport, signaling and inactivation of pro- and antinociceptive oxylipins in pain circuit tissues. We further linked these pathways to nociception by demonstrating intraplantar carrageenan injection induced gene expression changes in oxylipin biosynthetic pathways. We determined functional-biochemical relevance of the proposed pathways in rat hind paw and dorsal spinal cord by measuring basal and stimulated levels of oxylipins throughout the time-course of carrageenan-induced inflammation. Finally, when oxylipins were administered by intradermal injection we observed modulation of nociceptive thermal hypersensitivity, providing a functional-behavioral link between oxylipins, their molecular biosynthetic pathways, and involvement in pain and nociception. Together, these findings advance our understanding of molecular lipidomic systems linking oxylipins and their precursors to nociceptive and inflammatory signaling pathways in rats. PERSPECTIVE: We applied a systems approach to characterize molecular pathways linking precursor lipids and oxylipins to nociceptive signaling. This systematic, quantitative evaluation of the molecular pathways linking oxylipins to nociception provides a framework for future basic and clinical research investigating the role of oxylipins in pain.

Temperature and time-dependent effects of delayed blood processing on oxylipin concentrations in human plasma.

BACKGROUND: Oxidized derivatives of polyunsaturated fatty acids, collectively known as oxylipins, are labile bioactive mediators with diverse roles in human physiology and pathology. Oxylipins are increasingly being measured in plasma collected in clinical studies to investigate biological mechanisms and as pharmacodynamic biomarkers for nutrient-based and drug-based interventions. Whole blood is generally stored either on ice or at room temperature prior to processing. However, the potential impacts of delays in processing, and of temperature prior to processing, on oxylipin concentrations are incompletely understood. OBJECTIVE: To evaluate the effects of delayed processing of blood samples in a timeframe that is typical of a clinical laboratory setting, using typical storage temperatures, on concentrations of representative unesterified oxylipins measured by liquid chromatography-tandem mass spectrometry. DESIGN: Whole blood (drawn on three separate occasions from a single person) was collected into 5 mL purple-top potassium-EDTA tubes and stored for 0, 10, 20, 30, 60 or 120 min at room temperature or on wet ice, followed by centrifugation at 4 °C for 10 min with plasma collection. Each sample was run in duplicate, therefore there were six tubes and up to six data points at each time point for each oxylipin at each condition (ice/room temperature). Representative oxylipins derived from arachidonic acid, docosahexaenoic acid, and linoleic acid were quantified by liquid chromatography tandem mass spectrometry. Longitudinal models were used to estimate differences between temperature groups 2 h after blood draw. RESULTS: We found that most oxylipins measured in human plasma in traditional potassium-EDTA tubes are reasonably stable when stored on ice for up to 2 h prior to processing, with little evidence of auto-oxidation in either condition. By contrast, in whole blood stored at room temperature, substantial time-dependent increases in the 12-lipoxygenase-derived (12-HETE, 14-HDHA) and platelet-derived (thromboxane B2) oxylipins were observed. CONCLUSION: These findings suggest that certain plasma oxylipins can be measured with reasonable accuracy despite delayed processing for up to 2 h when blood is stored on ice prior to centrifugation. 12-Lipoxygenase- and platelet-derived oxylipins may be particularly sensitive to post-collection artifact with delayed processing at room temperature. Future studies are needed to determine impacts of duration and temperature of centrifugation on oxylipin concentrations.

Quantitation of Human Whole-Body Synthesis-Secretion Rates of Docosahexaenoic Acid and Eicosapentaenoate Acid from Circulating Unesterified α-Linolenic Acid at Steady State.

The rate at which dietary α-linolenic acid (ALA) is desaturated and elongated to its longer-chain n-3 polyunsaturated fatty acid (PUFA) in humans is not agreed upon. In this study, we applied a methodology developed using rodents to investigate the whole-body, presumably hepatic, synthesis-secretion rates of esterified n-3 PUFA from circulating unesterified ALA in 2 healthy overweight women after 10 weeks of low-linoleate diet exposure. During continuous iv infusion of d5-ALA, 17 arterial blood samples were collected from each subject at -10, 0, 10, 20, 40, 60, 80, 100, 120, 150, 180, and 210 min, and at 4, 5, 6, 7, and 8 h after beginning infusion. Plasma esterified d5-n-3 PUFA concentrations were plotted against the infusion time and fit to a sigmoidal curve using nonlinear regression. These curves were used to estimate kinetic parameters using a kinetic analysis developed using rodents. Calculated synthesis-secretion rates of esterified eicosapentaenoate, n-3 docosapentaenoate, docosahexaenoic acid, tetracosapentaenate, and tetracosahexaenoate from circulating unesterified ALA were 2.1 and 2.7; 1.7 and 5.3; 0.47 and 0.27; 0.30 and 0.30; and 0.32 and 0.27 mg/day for subjects S01 and S02, respectively. This study provides new estimates of whole-body synthesis-secretion rates of esterified longer-chain n-3 PUFA from circulating unesterified ALA in human subjects. This method now can be extended to study factors that regulate human whole-body PUFA synthesis-secretion in health and disease.

Dietary linoleic acid-induced alterations in pro- and anti-nociceptive lipid autacoids: Implications for idiopathic pain syndromes?

BACKGROUND: Chronic idiopathic pain syndromes are major causes of personal suffering, disability, and societal expense. Dietary n-6 linoleic acid has increased markedly in modern industrialized populations over the past century. These high amounts of linoleic acid could hypothetically predispose to physical pain by increasing the production of pro-nociceptive linoleic acid-derived lipid autacoids and by interfering with the production of anti-nociceptive lipid autacoids derived from n-3 fatty acids. Here, we used a rat model to determine the effect of increasing dietary linoleic acid as a controlled variable for 15 weeks on nociceptive lipid autacoids and their precursor n-6 and n-3 fatty acids in tissues associated with idiopathic pain syndromes. RESULTS: Increasing dietary linoleic acid markedly increased the abundance of linoleic acid and its pro-nociceptive derivatives and reduced the abundance of n-3 eicosapentaenoic acid and docosahexaenoic acid and their anti-nociceptive monoepoxide derivatives. Diet-induced changes occurred in a tissue-specific manner, with marked alterations of nociceptive lipid autacoids in both peripheral and central tissues, and the most pronounced changes in their fatty acid precursors in peripheral tissues. CONCLUSIONS: The present findings provide biochemical support for the hypothesis that the high linoleic acid content of modern industrialized diets may create a biochemical susceptibility to develop chronic pain. Dietary linoleic acid lowering should be further investigated as part of an integrative strategy for the prevention and management of idiopathic pain syndromes.

Diet-induced changes in n-3- and n-6-derived endocannabinoids and reductions in headache pain and psychological distress.

UNLABELLED: Omega-3 and omega-6 fatty acids are biosynthetic precursors of endocannabinoids with antinociceptive, anxiolytic, and neurogenic properties. We recently reported that targeted dietary manipulation-increasing omega-3 fatty acids while reducing omega-6 linoleic acid (the H3-L6 intervention)-reduced headache pain and psychological distress among chronic headache patients. It is not yet known whether these clinical improvements were due to changes in endocannabinoids and related mediators derived from omega-3 and omega-6 fatty acids. We therefore used data from this trial (N = 55) to investigate 1) whether the H3-L6 intervention altered omega-3- and omega-6-derived endocannabinoids in plasma and 2) whether diet-induced changes in these bioactive lipids were associated with clinical improvements. The H3-L6 intervention significantly increased the omega-3 docosahexaenoic acid derivatives 2-docosahexaenoylglycerol (+65%, P < .001) and docosahexaenoylethanolamine (+99%, P < .001) and reduced the omega-6 arachidonic acid derivative 2-arachidonoylglycerol (-25%, P = .001). Diet-induced changes in these endocannabinoid derivatives of omega-3 docosahexaenoic acid, but not omega-6 arachidonic acid, correlated with reductions in physical pain and psychological distress. These findings demonstrate that targeted dietary manipulation can alter endocannabinoids derived from omega-3 and omega-6 fatty acids in humans and suggest that 2-docosahexaenoylglycerol and docosahexaenoylethanolamine could have physical and/or psychological pain modulating properties. TRIAL REGISTRATION: ClinicalTrials.gov (NCT01157208) PERSPECTIVE: This article demonstrates that targeted dietary manipulation can alter endocannabinoids derived from omega-3 and omega-6 fatty acids and that these changes are related to reductions in headache pain and psychological distress. These findings suggest that dietary interventions could provide an effective, complementary approach for managing chronic pain and related conditions.

Dietary omega-6 fatty acid lowering increases bioavailability of omega-3 polyunsaturated fatty acids in human plasma lipid pools.

BACKGROUND: Dietary linoleic acid (LA, 18:2n-6) lowering in rats reduces n-6 polyunsaturated fatty acid (PUFA) plasma concentrations and increases n-3 PUFA (eicosapentaenoic (EPA) and docosahexaenoic acid (DHA)) concentrations. OBJECTIVE: To evaluate the extent to which 12 weeks of dietary n-6 PUFA lowering, with or without increased dietary n-3 PUFAs, alters unesterified and esterified plasma n-6 and n-3 PUFA concentrations in subjects with chronic headache. DESIGN: Secondary analysis of a randomized trial. Subjects with chronic headache were randomized for 12 weeks to (1) average n-3, low n-6 (L6) diet; or (2) high n-3, low n-6 LA (H3-L6) diet. Esterified and unesterified plasma fatty acids were quantified at baseline (0 weeks) and after 12 weeks on a diet. RESULTS: Compared to baseline, the L6 diet reduced esterified plasma LA and increased esterified n-3 PUFA concentrations (nmol/ml), but did not significantly change plasma arachidonic acid (AA, 20:4n-6) concentration. In addition, unesterified EPA concentration was increased significantly among unesterified fatty acids. The H3-L6 diet decreased esterified LA and AA concentrations, and produced more marked increases in esterified and unesterified n-3 PUFA concentrations. CONCLUSION: Dietary n-6 PUFA lowering for 12 weeks significantly reduces LA and increases n-3 PUFA concentrations in plasma, without altering plasma AA concentration. A concurrent increase in dietary n-3 PUFAs for 12 weeks further increases n-3 PUFA plasma concentrations and reduces AA.

Use of dietary linoleic acid for secondary prevention of coronary heart disease and death: evaluation of recovered data from the Sydney Diet Heart Study and updated meta-analysis.

OBJECTIVE: To evaluate the effectiveness of replacing dietary saturated fat with omega 6 linoleic acid, for the secondary prevention of coronary heart disease and death. DESIGN: Evaluation of recovered data from the Sydney Diet Heart Study, a single blinded, parallel group, randomized controlled trial conducted in 1966-73; and an updated meta-analysis including these previously missing data. SETTING: Ambulatory, coronary care clinic in Sydney, Australia. PARTICIPANTS: 458 men aged 30-59 years with a recent coronary event. INTERVENTIONS: Replacement of dietary saturated fats (from animal fats, common margarines, and shortenings) with omega 6 linoleic acid (from safflower oil and safflower oil polyunsaturated margarine). Controls received no specific dietary instruction or study foods. All non-dietary aspects were designed to be equivalent in both groups. OUTCOME MEASURES: All cause mortality (primary outcome), cardiovascular mortality, and mortality from coronary heart disease (secondary outcomes). We used an intention to treat, survival analysis approach to compare mortality outcomes by group. RESULTS: The intervention group (n=221) had higher rates of death than controls (n=237) (all cause 17.6% v 11.8%, hazard ratio 1.62 (95% confidence interval 1.00 to 2.64), P=0.05; cardiovascular disease 17.2% v 11.0%, 1.70 (1.03 to 2.80), P=0.04; coronary heart disease 16.3% v 10.1%, 1.74 (1.04 to 2.92), P=0.04). Inclusion of these recovered data in an updated meta-analysis of linoleic acid intervention trials showed non-significant trends toward increased risks of death from coronary heart disease (hazard ratio 1.33 (0.99 to 1.79); P=0.06) and cardiovascular disease (1.27 (0.98 to 1.65); P=0.07). CONCLUSIONS: Advice to substitute polyunsaturated fats for saturated fats is a key component of worldwide dietary guidelines for coronary heart disease risk reduction. However, clinical benefits of the most abundant polyunsaturated fatty acid, omega 6 linoleic acid, have not been established. In this cohort, substituting dietary linoleic acid in place of saturated fats increased the rates of death from all causes, coronary heart disease, and cardiovascular disease. An updated meta-analysis of linoleic acid intervention trials showed no evidence of cardiovascular benefit. These findings could have important implications for worldwide dietary advice to substitute omega 6 linoleic acid, or polyunsaturated fats in general, for saturated fats. TRIAL REGISTRATION: Clinical trials NCT01621087.

Lowering dietary linoleic acid reduces bioactive oxidized linoleic acid metabolites in humans.

Linoleic acid (LA) is the most abundant polyunsaturated fatty acid in human diets, a major component of human tissues, and the direct precursor to the bioactive oxidized LA metabolites (OXLAMs), 9- and 13 hydroxy-octadecadienoic acid (9- and 13-HODE) and 9- and 13-oxo-octadecadienoic acid (9- and 13-oxoODE). These four OXLAMs have been mechanistically linked to pathological conditions ranging from cardiovascular disease to chronic pain. Plasma OXLAMs, which are elevated in Alzheimer's dementia and non-alcoholic steatohepatitis, have been proposed as biomarkers useful for indicating the presence and severity of both conditions. Because mammals lack the enzymatic machinery needed for de novo LA synthesis, the abundance of LA and OXLAMs in mammalian tissues may be modifiable via diet. To examine this issue in humans, we measured circulating LA and OXLAMs before and after a 12-week LA lowering dietary intervention in chronic headache patients. Lowering dietary LA significantly reduced the abundance of plasma OXLAMs, and reduced the LA content of multiple circulating lipid fractions that may serve as precursor pools for endogenous OXLAM synthesis. These results show that lowering dietary LA can reduce the synthesis and/or accumulation of oxidized LA derivatives that have been implicated in a variety of pathological conditions. Future studies evaluating the clinical implications of diet-induced OXLAM reductions are warranted.