Lowering dietary linoleic acid reduces bioactive oxidized linoleic acid metabolites in humansā˜†

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Abstract

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.

Introduction

Oxidized linoleic acid metabolites (OXLAMs) are pleiotropic bioactive derivatives of linoleic acid (LA, 18:2n-6) that have been implicated in a variety of pathological conditions [1], [2], [3], [4], [5], [6], [7], [8], [9]. As a major component of oxidized low-density lipoprotein (LDL) [7], [10], [11] and atherosclerotic plaques [12], [13], OXLAMs are reported to play a central role in foam cell formation and the pathogenesis of atherosclerosis [4], [8], [14]. OXLAMs also can act as endogenous TRPV1 receptor channel activators (i.e. endovanilloids) [1], [2], facilitating peripheral and central pain sensitization. Circulating OXLAMs, which are elevated in Alzheimer's dementia [15] and non-alcoholic steatohepatitis (NASH) [3], have been proposed as mechanism-based biomarkers useful for indicating the presence and severity of both conditions.

LA is the most abundant polyunsaturated fatty acid in human diets [16], [17], [18], a major component of human tissues, and the direct precursor to the OXLAMs 9- and 13-hydroxy-octadecadienoic acid (9- and 13-HODE) and 9- and 13-oxo-octadecadienoic acid (9- and 13-oxoODE) [19] (Fig. 1). LA oxidation can proceed enzymatically via the actions of 12/15-lipoxygenase, cyclooxygenase or the cytochrome P450 enzyme family [19], [20], or non-enzymatically via free radical-mediated oxidation [21].

Because humans cannot synthesize LA de novo [17], [22], [23], dietary LA is the sole source of LA in blood and other tissues. These LA stores in turn serve as precursor pools for endogenous OXLAM synthesis. Hence, diet-induced reductions in LA content may subsequently decrease the abundance of OXLAMs in vivo, with potential implications for conditions characterized by TRPV1 hyperactivity (e.g. chronic pain), as well as cardiovascular, hepatic and neurodegenerative diseases. To our knowledge, however, the relationship between dietary LA and plasma OXLAMs has not been reported in humans.

We hypothesized that lowering dietary LA would reduce plasma OXLAMs as well as their precursor LA in circulating lipid pools, in humans with chronic headaches.

Section snippets

Patient characteristics

This study was approved by the Institutional Review Board of the University of North Carolinaā€“Chapel Hill. All patients provided written informed consent prior to participation. The cohort consisted of 67 subjects meeting the International Classification of Headache Disorders diagnostic criteria for Chronic Daily Headache [24]. Fifty-six of 67 (84%) randomized subjects completed the 12-week intervention phase. The mean age at randomization was 41.3 (range 20ā€“62; SD 11.5). Eighty-five percent of

Plasma fractions

Total lipids were extracted from 200Ā Ī¼l of plasma in 3Ā ml of 2:1 chloroform/methanol following the addition of unesterified heptadecaenoic acid (17:0) as an internal standard (0.14Ā nmol/Ī¼l) for unesterified fatty acids. KCl (0.5Ā M, 0.75Ā ml) was then added to separate the aqueous phase. The bottom chloroform layer was separated and re-extracted with 2Ā ml chloroform. The pooled extracts were dried down and separated into neutral lipid subclasses using thin layer chromatography on silica gel-60 plates

Results

Lowering dietary LA from 6.7 to 2.4% of calories for 12-weeks in 55 patients with chronic headaches significantly reduced the abundance of the four measured OXLAMs (9- and 13-HODEs, 9- and 13-oxoODEs), as well as their precursor LA in circulating lipid pools (Table 2). There were no significant differences in OXLAM reductions between the two low-LA intervention groups (Fig. 3), one with and one without added dietary n-3 fatty acids (Table 1), indicating that the observed reductions were

Discussion

We report for the first time that lowering dietary LA reduces OXLAMs in humans, here among subjects with chronic headaches. This link between dietary LA and OXLAMs may have important implications for pathological conditions linked to increased activity or abundance of OXLAMs (e.g. chronic pain, Alzheimer's dementia, cardiovascular disease and NASH) [1], [2], [3], [15]. To our knowledge, this is the first demonstration that changes in dietary LA can alter the abundance of plasma OXLAMs in

Acknowledgments

The authors would like to thank the following individuals for their research assistance: David Barrow for expertise with specimen collection, processing and management; Olafur Palsson, Beth Fowler, Carol Carr, Regina McCoy, and Tim McCaskill for design and functionality of the study website; Meg Mangan for 24-hour recall data collection and management; Jim Loewke for erythrocyte fatty acid analysis; Duk Hyun for nutrient composition analyses; and Chanee Lynch, Becky Coble and Angela Johnston

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    ā˜†

    The authors gratefully acknowledge funding support for this trial from the Mayday Fund (primary source); the National Institute on Alcohol Abuse and Alcoholism, NIH; the UNC Research Fellowship in Complementary and Alternative Medicine (Grant T32-AT003378, NCCAM, NIH); the North Carolina Clinical and Translational Sciences Institute (Grant UL1RR025747, NCRR, NIH); the UNC Nutrition Obesity Research Center, CHAI Core (Grant DK056350, NIDDK, NIH). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the Mayday Fund or the National Institutes of Health.

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