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. (1). DIETARY OMEGA-6 FATTY ACID LOWERING INCREASES THE BIOAVAILABILITY OF PLASMA OMEGA-3 FATTY ACIDS IN HUMAN PLASMA LIPID POOLS. 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, thus bioavailability for maintaining brain and heart integrity. Objective: To evaluate whether dietary n-6 PUFA lowering, with or without increases in dietary n-3 PUFAs, reduces plasma n-6 PUFA and increases n-3 PUFA concentrations, and to compare magnitude of changes between groups in human subjects. Design: Subjects were randomized to: (1) average n-3, low n-6 (L6) diet;or (2) high n-3, low n-6 LA (H3-L6) diet for 12 weeks. Esterified and unesterified plasma fatty acids were quantified at baseline and after 12 weeks on diet. Results: Compared to baseline, the L6 diet reduced esterified LA and increased esterified n-3 PUFA concentrations, but did not significantly change arachidonic acid (AA, 20:4n-6) concentration. Only the unesterified EPA concentration was increased significantly. The H3-L6 diet decreased esterified LA and AA concentrations, and produced more marked increases in the n-3 PUFA esterified and unesterified concentrations. Conclusions: Dietary n-6 PUFA lowering for 12 weeks reduced LA and increased n-3 PUFA concentrations in plasma, without altering the plasma AA concentration. A concurrent increase in dietary n-3 PUFA for 12 weeks further increased n-3 PUFA plasma concentrations, and also reduced AA. Dietary n-6 lowering can increase circulating n-3 PUFA bioavailability particularly when combined with dietary n-3 PUFAs, for maintaining brain and heart integrity. Research is being prepared for submission. IDENTIFICATION OF OXIDIZED LINOLEIC ACID METABOLITES (OXLAMS) IN PLASMA BY QUADRUPOLE TIME-OF-FLIGHT MASS SPECTROMETRY. Linoleic acid (LA) and LA-esters are the precursors of LA hydroperoxides, which are readily converted to 9- and 13-hydroxy-octadecadienoic acid (HODE) and 9- and 13-oxo-octadecadienoic acid (oxo ODE) metabolites in vivo. These oxidized LA metabolites (OXLAMs) have been implicated in a variety of pathological conditions. Therefore, their accurate measurement may provide mechanistic insights into disease pathogenesis. We published a novel quadrupole time-of-flight mass spectrometry (Q-TOFMS) method for quantitation and identification of target OXLAMs in plasma, using rat plasma. In this method, the esterified OXLAMs were base-hydrolyzed and followed by liquid-liquid extraction. Quantitative analyses were based on one-point standard addition with isotope dilution. The Q-TOFMS data of target metabolites were acquired and multiple reaction monitoring extracted-ion chromatograms were generated post-acquisition with a 10 ppm extraction window. The limit of quantitation was 9.7-35.9 nmol/L depending on the metabolite. The method was reproducible with a coefficient of variation of <18.5%. Mean concentrations of target metabolites in rat plasma were 57.8, 123.2, 218.1 and 57.8 nmol/L for 9-HODE, 13-HODE, 9-oxoODE and 13-oxoODE, respectively. Plasma levels of total OXLAMs were 456.9 nmol/L, which correlated well with published concentrations obtained by gas chromatography/mass spectrometry (GC/MS). The concentrations were also obtained utilizing a standard addition curve approach. The calibration curves were linear with correlation coefficients of >0.991. Concentrations of 9-HODE, 13-HODE, 9-oxoODE and 13-oxoODE were 84.0, 138.6, 263.0 and 69.5 nmol/L, respectively, which were consistent with the results obtained from one-point standard addition. Target metabolites were simultaneously characterized based on the accurate Q-TOFMS data. This is the first study of secondary LA metabolites using Q-TOFMS. (2). QUANTIFYING HEPATIC SECRETION OF DOCOSAHEXAENOIC ACID (DHA). DHA is a polyunsaturated a fatty acid enriched in brain tissue that has important structural and signaling implications for addictive disorders and neurological function. Rodent data and human trials suggest that dietary omega-6 linoleic acid interferes with liver synthesis-secretion of omega-3 DHA. Therefore high omega-6 linoleic acid diets may contribute to deficits in omega-3 DHA, leading to suboptimal neurological function. A reduction in dietary omega-6 linoleic acid may increase liver synthesis-secretion and tissue accumulation of omega-3 DHA from its dietary precursor omega-3 alpha-linolenic acid (LNA). To test this, we plan to use the BPMS established infusion method and model to quantify liver synthesis-secretion rates of long-chain omega-3 polyunsaturated fatty acids, (omega-3 EPA and DHA) from circulating unesterified omega-3 LNA in humans consuming an average US diet for 12 weeks. We plan to initiate an IRB approved protocol that requires a 22-hour hospital admission to the NIAAA inpatient unit, including an 8-hour infusion of d5-LNA with serial blood draws and LC/GC analysis. The primary outcome will be the liver synthesis-secretion rate of docosahexaenoic acid (DHA) from infused d5-LNA. PUFAS IN BIPOLAR DISORDER. Several publication indicate a distortion of plasma concentrations of n-3 and n-6 polyunsaturated fatty acids and their metabolites in bipolar disorder, possibly related to general inflammatory risk. We are initiating a collaboration with Dr. Erika Saunders at Penn State School of Medicine to measure concentrations of these compounds in patient with bipolar disorder in relation to diet and drug history.

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Saunders, Erika Fh; Reider, Aubrey; Singh, Gagan et al. (2015) Low unesterified:esterified eicosapentaenoic acid (EPA) plasma concentration ratio is associated with bipolar disorder episodes, and omega-3 plasma concentrations are altered by treatment. Bipolar Disord 17:729-42
Ramsden, Christopher E; Faurot, Keturah R; Zamora, Daisy et al. (2015) Targeted alterations in dietary n-3 and n-6 fatty acids improve life functioning and reduce psychological distress among patients with chronic headache: a secondary analysis of a randomized trial. Pain 156:587-96
Liu, Joanne J; Green, Pnina; John Mann, J et al. (2015) Pathways of polyunsaturated fatty acid utilization: implications for brain function in neuropsychiatric health and disease. Brain Res 1597:220-46
Taha, Ameer Y; Cheon, Yewon; Faurot, Keturah F et al. (2014) Dietary omega-6 fatty acid lowering increases bioavailability of omega-3 polyunsaturated fatty acids in human plasma lipid pools. Prostaglandins Leukot Essent Fatty Acids 90:151-7
Yuan, Zhi-Xin; Rapoport, Stanley I; Soldin, Steven J et al. (2013) Identification and profiling of targeted oxidized linoleic acid metabolites in rat plasma by quadrupole time-of-flight mass spectrometry. Biomed Chromatogr 27:422-32
Ramsden, Christopher E; Ringel, Amit; Feldstein, Ariel E et al. (2012) Lowering dietary linoleic acid reduces bioactive oxidized linoleic acid metabolites in humans. Prostaglandins Leukot Essent Fatty Acids 87:135-41