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

Summary

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].

Figure 1. Oxidative metabolism of linoleic acid.

LA can be enzymatically or non-enzymatically converted to 9- and 13-HpODE, with subsequent enzymatic conversion to hydroxy (9- and 13-HODE) and ketone (9- and 13-oxoODE) derivatives. The initial step (A) can be catalyzed by 12/15-lipoxygenase, cyclooxygenase, or cytochrome P450 enzymes. Abbreviations: LA: linoleic acid; LOX: lipoxygenase; COX: cyclooxygenase; HpODE; hydroperoxy-octadecadienoic acid; HODE: hydroxy-octadecadienoic acid; oxoODE: oxo-octadecadienoic acid.

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.

Materials and Methods

Patient characteristics

This study was approved by the Institutional Review Board of the University of North Carolina-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 20-62; SD 11.5). Eighty-five percent of randomized subjects were female and 88% were Caucasian.

Study Design

The main study was designed to evaluate the effects of lowering dietary LA, with or without concurrent increases in n-3 PUFAs, on plasma and erythrocyte fatty acids (primary outcomes) and clinical headache characteristics (secondary outcomes)(21). These outcomes will be reported in another publication. However, after our trial was already in progress, OXLAMs were implicated in the etiology of physical pain (1-2), and proposed as mechanism-based biomarkers in NASH (3). Recognizing that our LA lowering interventions provided a unique opportunity to evaluate whether diet-induced OXLAM reductions are possible in humans, we developed an interdisciplinary collaboration to specifically evaluate whether lowering dietary LA reduces plasma OXLAMs as well as their precursor LA in circulating lipid pools.

The dietary methods and methods employed to evaluate LA intake have been previously published [24]. In brief, after a 4-week baseline phase participants were randomized to one of two dietary interventions, to be maintained for 12 consecutive weeks. Both interventions were designed to markedly reduce LA intake. Group 1 consumed average US amounts of n-3 fatty acids; Group 2 also increased intake of n-3 fatty acids as shown in Table 2. Our diet method integrated the following key elements: 1) provision of foods accounting for two-thirds of caloric intake; 2) bi-monthly diet counseling; 3) self-monitoring; and 4) an intervention-specific website. All study participants were provided with, and instructed to exclusively use low-LA oils and fat sources, including vegetable oils and salad dressings. Because most US packaged food items contain substantial amounts of added LA, participants were also provided with a variety of low-LA substitute foods, including crackers, tortillas, breads and popcorn. Research foods were procured and prepared by the UNC Nutrition Research and Metabolism Core of the Clinical and Translational Research Center. Nutrient intakes were assessed using six unannounced telephone-administered 24-hour recalls; three administered prior to the intervention, and three administered in the final four weeks of the twelve-week intervention. Nutrient values were estimated using Nutritional Data System for Research software in version 2009-2010, developed by the Nutrition Coordinating Center, University of Minnesota, Minneapolis, MN [25].

Table 2.

Diet-induced reductions in circulating LA and its OXLAM products

Variable	Baseline (Median)	12-week (Median)	% Change (Median)	P-value
Diet (% of energy)
Dietary LA	6.74 (5.54, 8.11)	2.42 (2.09, 3.06)	−64.1	p<0.001
Plasma OXLAMs (nM)
9-HODE	266 (209, 365)	227 (182, 274)	−14.7	p = 0.001
13-HODE	268 (229, 376)	243 (205, 294)	−9.5	p < 0.001
Total HODEs	538 (442, 756)	474 (385, 564)	−12.0	p < 0.001
9-oxoODE	169 (140, 229)	146 (116, 203)	−13.8	p = 0.01
13-oxoODE	265 (213, 351)	204 (155, 273)	−23.2	p < 0.001
Total oxoODEs	431 (353, 555)	352 (265, 478)	−18.3	p < 0.001
Total OXLAMs	979 (823, 1310)	851 (693, 1007)	−13.1	p < 0.001
Concentration of LA in plasma fatty acid pools (nmol/ml)
Total plasma LA	3075 (2622, 3357)	2615 (2249, 3038)	−14.9	p < 0.001
Phospholipid-LA	813 (726, 941)	675 (593, 795)	−17.0	p < 0.001
Triglyceride-LA	567 (337, 749)	395 (293, 548)	−30.3	p < 0.001
Cholesteryl ester-LA	1592 (1429, 1816)	1440 (1191, 1665)	−9.6	p = 0.002
Free fatty acid-LA	58 (36, 81)	53 (42, 69)	−9.4	p = 0.35
Relative abundance of LA in circulating fatty acid pools (%Composition)
Phospholipid-LA	22.5 (20.9, 24.9)	20.0 (18.0, 21.7)	−11.2	p < 0.001
Triglyceride-LA	20.6 (17.1, 24.1)	17.3 (14.8, 19.6)	−16.0	p < 0.001
Cholesteryl ester-LA	55.5 (52.6, 58.5)	50.5 (46.7, 52.9)	−9.1	p < 0.001
Free fatty acid-LA	16.0 (14.0, 18.0)	15.2 (13.6, 16.4)	−5.2	p < 0.001
Erythrocyte-LA	12.0 (11.0, 12.7)	10.3 (9.3, 11.0)	−14.5	p < 0.001

Pre-to-post comparisons of dietary LA (n=55), plasma OXLAMs (n=55), LA fractions (n=55), and erythrocytes (n=52) were analyzed with the Wilcoxon Signed-Rank test for matched pairs. Blood collection was unsuccessful for one subject. Erythrocytes were not collected on four subjects. Abbreviations: LA: linoleic acid; OXLAMs: oxidized linoleic acid metabolites; HODE: hydroxy-octadecadienoic acid; oxoODE: oxo-octadecadenoic acid.

Sample collection

Sample preparation and analyses were performed by investigators who were blinded to study protocol and clinical data. Fasting blood was drawn at baseline and again after 12-weeks of exposure to the dietary interventions. Whole blood was collected into ethylenediaminetetraacetic acid (EDTA) tubes. Samples were immediately centrifuged at 2960 rpm for 15 min at room temperature. Plasma and erythrocyte aliquots were prepared and immediately transferred to a − 80°C freezer until analysis. Analyses of OXLAMs, plasma lipid fractions, and erythrocyte fatty acids were performed at the Cleveland Clinic Department of Cell Biology, National Institute on Aging Brain Physiology and Metabolism Section, and National Institute on Alcohol Abuse and Alcoholism Laboratory of Membrane Biochemistry and Biophysics, respectively.

OXLAM analysis

Plasma OXLAMs were analyzed as previously described [3]. Briefly, plasma (50 μl), internal standard [15(S)-HETE-d8, 10μL of 1000 ng/ml] and sodium hydroxide were added to the glass test tubes, overlaid with argon, and sealed. Lipids were hydrolyzed at 60°C under argon atmosphere for 2 h and then the released fatty acids were extracted into the hexane layer twice by liquid/liquid extraction using hexane and hexane/isopropanol with 4M acetic acid. With each extraction, tubes were capped under argon. The combined hexane layers were dried under nitrogen gas and then re-suspended in 200 μl 85% methanol/water (v/v). Fatty acid oxidation products (free plus esterified) in plasma were quantified using liquid chromatography online electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS) (Fig. 2) [3]. Briefly, 40 μl of lipid extract was injected onto an HPLC (Waters 2690 Separations Module, Franklin, MA) system, and the oxidized fatty acids and their precursors were separated through a C18 column (Phenomenex, ODS, 2 × 150 mm, 5 μm, Rancho Palos Verdes, CA). The oxidized fatty acids and their precursors were quantified on a triple quadrupole mass spectrometer (Quattro Ultima, Micromass, Manchester, UK) using ESI in negative ion mode and multiple reaction monitoring (MRM) using characteristic parent → daughter ion transitions for the specific molecular species monitored. The lipid peroxidation products analyzed included structurally specific species 9- and 13-HODE, 9- and 13-oxoODE, and their precursor LA. 15-HETE-d8 (Cayman Chemical, Ann Arbor, MI) was used as internal standard for calibration of oxidized fatty acids in plasma, as previously described [3].

Figure 2. Detection and quantification of OXLAM profile by ESI/LC/MS/MS.

Individual isomers of HODEs and oxoODEs formed by LA oxidation were quantified with a single injection. Lipid extracts were resolved by HPLC and monitored online by ESI/LC/MS/MS [3]. Abbreviations: HODE: hydroxy-octadecadenoic acid; oxoODE, oxo-octadecadenoic acid; 15-HETE-d8, internal standard.

Fatty acid analysis

Plasma Fractions

Total lipids were extracted from 200 μl of plasma in 3 ml of 2:1 chloroform/methanol following the addition of unesterified heptadecaenoic acid (17:0) as an internal standard (0.14 nmol/μl) for unesterified fatty acids. KCl (0.5M, 0.75 ml) was then added to separate the aqueous phase. The bottom chloroform layer was separated and re-extracted with 2 ml chloroform. The pooled extracts were dried down and separated into neutral lipid subclasses using thin layer chromatography on silica gel-60 plates (EM Separation Technologies, Gibbstown, NJ, USA). The lipid subclasses (total phospholipid, triacylglycerol, cholesteryl ester, unesterified fatty acid) were separated using the solvent system: heptane: diethylether: glacial acetic acid (60:40:3, v/v/v) [26]. Authentic standards of the lipid classes were run on separate lanes on the plates to identify lipid bands under ultraviolet light, after spraying with 0.03% 6-p-toluidine-2-naphthalene sulfonic acid in 50 mM Tris-HCl buffer (pH 7.4) (w/v). The bands were scraped into test tubes and methylated with 1% H₂SO₄-methanol for 3 h at 70°C [27]. Before methylation, di-17:0 PC was added to each tube as an internal standard for phospholipids, triglycerides and cholesteryl esters. The prepared fatty acid methyl esters (FAME) were analyzed using a gas-chromatography system (6890N, Agilent Technologies, Palo Alto, CA, USA) equipped with an SPTM-2330 fused silica capillary column (30 m × 0.25 mm i.d., 0.25 μm film thickness) (Supelco, Bellefonte, PA, USA) and a flame ionization detector. Fatty acid concentrations were calculated by proportional comparison of peak areas of samples to the area of the 17:0 internal standard.

Erythrocytes

Erythrocytes were obtained from EDTA blood samples after the plasma and buffy coat were removed. Following Bligh/Dyer extraction [28], erythrocyte aliquots were heated at 100°C for one hour with methanol containing 14% boron trifluoride to generate FAME. These were then extracted into hexane and analyzed with a GC/FID gas chromatograph (Agilent 6890) equipped with a 30-m DBFFAP capillary column. Fatty acids were identified through comparison with a standard fatty acid methyl ester mixture (GLC-462). Values are expressed as percentages of total red blood cell fatty acids.

Data Analysis

Non-parametric analyses were employed due to the presence of non-normal distributions. Pre-to-post intervention comparisons were tested with the Wilcoxon Signed-Rank test for matched pairs. A Mann Whitney U test was used for between group comparisons. P values less than 0.05 were considered significant. In addition, in an exploratory manner, Spearman’s coefficients were used to evaluate potential correlations between circulating LA pools and OXLAMs.

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 primarily due to the decrease in dietary LA. The combined results of the two LA-lowering intervention groups are presented in Table 2.

Figure 3. Comparable OXLAM reductions in the two LA lowering intervention groups.

There were no between-group differences in the median reductions in HODEs, oxo-ODEs or total OXLAMs, indicating that the observed changes were primarily due to the decrease in dietary LA rather than the n-3 fatty acids provided only to Group 2. Data for the combined groups are presented in subsequent tables and figures. The box-whisker plot is represented with the lower boundary of the box indicating the 25th percentile, the line within the box indicating the median value, and the upper boundary indicating the 75th percentile. The whiskers extend to the non-outlier range. P values represent between group comparisons using a Mann Whitney U test. Abbreviations: HODE: hydroxy-octadecadienoic acid; oxo-ODE: oxo-octadecadienoic acid; OXLAM: oxidized linoleic acid metabolite.

Table 1.

Median fatty acid contents of the LA lowering dietary interventions

Diet Group	Calories	Fat	SFA	PUFA	n-6 LA	n-3 ALA	n-3 EPA + DHA
Pre-intervention Phase
Combined (n=55)	1861	33.4	10.5	7.6	6.7	0.7	46 mg
Intervention Phase
Combined (n=55)	1742	30.7	13.2	4.4	2.4	1.1	305 mg
Group 1 (n=28)	1859	30.4	14.0	3.5	2.4	0.7	76 mg
Group 2a (n=27)	1596	30.7	12.9	6.0	2.5	1.6	1,482 mg

Fatty acid intake is expressed as a percentage of daily food energy (en%), except for n-3 EPA+DHA, which is expressed in mg. Based on six 24-hour dietary recalls administered on non-consecutive days. One of the 56 randomized subjects did not complete 24-hour recalls.

^aIndicates that dietary n-3 ALA, EPA and DHA were increased in Group 2 only. Abbreviations: Fat: total fat; SFA: total saturated fat; PUFA, total polyunsaturated fat; LA: linoleic acid; ALA: alpha-linolenic acid; EPA: eicosapentaenoic acid; DHA: docosahexaenoic acid.

Distinctions between OXLAM species and their precursor pools

Median reductions in the LA content of the four plasma fractions were significantly different using Friedman ANOVA (p=0.02), with the most pronounced changes in the phospholipid (PL) and triglyceride (TG) pools (Table 2). Unlike LA, the overall amount of total fatty acids did not change in any of these plasma lipid fractions (Table 3). Although reductions in oxoODEs tended to be more pronounced than HODEs (-18.3% vs. -12.0%), this difference was not statistically significant (p=0.45).

Table 3.

No changes in the total fatty acid content of plasma lipid classes

Plasma Lipid Classes (nmol/ml; n=55)	Baseline (Median)	12-week (Median)	% Change (Median)	P-value (Median)
Total fatty acids	9512 (7784, 11586)	9552 (7978, 11394)	+0.4	0.89
Total PL	3663 (3159, 4101)	3622 (3204, 3971)	−1.1	0.43
Total TG	2519 (1679, 3563)	2479 (1692, 3422)	−1.6	0.67
Total CE	2827 (2498, 3285)	2995 (2596, 3433)	+6.0	0.46
Total FFA	342 (235, 441)	362 (275, 446)	+6.0	0.63

Pre-to-post comparisons of total plasma fatty acids in plasma lipid classes were analyzed with the Wilcoxon Signed-Rank test for matched pairs. Abbreviations: PL: phospholipid; TG: triglyceride; CE: cholesteryl ester; FFA: free fatty acids.

Correlations between circulating LA pools and OXLAMs

Circulating OXLAMs correlated with the LA content of several OXLAM precursor pools at baseline, and after the 12-week dietary intervention. At baseline, total OXLAMs correlated with total plasma LA (Spearman rho(r)=0.48, p<0.01), as well as the LA content of erythrocytes (r=0.28, p=0.04). Of the plasma lipid fractions, the strongest OXLAM correlations were present in the phospholipid-LA (PL-LA) fraction (r=0.47, p<0.01), however baseline TG-LA (r=0.28, p=0.04) and CE-LA (r=0.33, p=0.01) were also significantly correlated with OXLAMs. After the 12-week intervention, total OXLAMs remained correlated with total plasma LA (r=0.40, p<0.01), PL-LA (r=0.50, p<0.01), TG-LA (r=0.37, p<0.01), and erythrocyte LA (r=0.42, p<0.01), but were no longer correlated with CE-LA (r=0.15, p=0.27). OXLAMs were not correlated with FFA-LA before (r=0.01, p=0.93) or after the dietary intervention (0.04, p=0.80).

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, 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 humans, or that lowering dietary LA reduces the abundance of OXLAMs in any tissue in a human or animal model. Our findings are consistent with a report [29] that a 4-fold increase in dietary LA (from corn oil) produced a 5-fold increase in the 9- and 13-HODE content of mammary tissue in female mice. Collectively, these observations indicate that dietary LA may hold proximal control over the production and/or accumulation of OXLAMs in certain tissues.

We also found that lowering dietary LA reduced the abundance of LA in several circulating lipid fractions that may serve as precursor pools for endogenous OXLAM synthesis. Importantly, the concentration of total fatty acids did not change in PL, TG, CE or FFA (Table 3), indicating that observed reductions in LA and OXLAMs were unlikely to be secondary to a general reduction in plasma lipoproteins. LA reductions were most pronounced in the PL and TG fractions, indicating that these esterified fractions may be particularly responsive to dietary modification. However, since the relative LA abundance was reduced in all measured pools, and the LA content of other potential OXLAM sources (e.g. liver, adipose) was not analyzed, it is not possible to attribute the observed OXLAM reductions to any specific plasma lipid pool. Future studies using LA-tracers [30] and/or tissue procurement for OXLAM analysis could help clarify the most relevant in vivo OXLAM precursor pool(s).

The robust correlations observed between circulating OXLAMs and LA in several LA pools both at baseline and after the 12-week intervention are consistent with the hypothesis that multiple LA pools contribute to in vivo OXLAM formation. The 12-week LA lowering intervention may not have been of sufficient duration to establish a new steady state of CE-LA in circulation. Consistent with this, there was a robust correlation between OXLAMs and CE-LA at baseline was no longer present following the 12-week intervention. Importantly, the CE pool contains about half of all circulating LA. Longer trials with serial analyses of LA and OXLAMs may therefore help characterize the temporal relations between diet-induced alterations in each circulating LA pool and OXLAMs.

Potential implications of dietary modulation of OXLAMs

OXLAMs are among the most abundant oxidized fatty acid derivatives in human plasma, with >50-fold higher concentrations than the more extensively studied prostanoid and isoprostane derivatives of arachidonic acid [15,31]. Unlike prostanoids however, OXLAMs are readily incorporated into, and released from, esterified lipid pools including PL [32,33], TG [34] and CE [11]. In rodents, plasma OXLAMs are largely esterified within lipoproteins [10,35]. As a major component of oxidized LDL [7] as well as the foam cells and migrating vascular smooth muscle cells found in atherosclerotic lesions [12,13], OXLAMs have been implicated in cardiovascular disease (CVD) pathogenesis [4,8,9]. Therefore, the diet-induced reductions in circulating OXLAMs demonstrated here may have clinical relevance for CVD risk reduction.

If comparable reductions in the abundance of OXLAMs are achievable in other tissues, dietary LA lowering may have diverse physiological and clinical consequences. Since LA is the parent compound for OXLAM synthesis, diet-induced changes in tissue LA may induce corresponding alterations in local OXLAM concentrations. Human and animal trials have established that modifications in dietary LA alter the LA content of numerous tissue types [36,37,38]. Sensitivities to alterations in dietary LA are likely to be tissue-specific [39,40,41], with marked diet-induced alterations in the LA content of peripheral nerves [39], small intestine [41], liver [39], testes [39], adipose tissue [37,39], and skeletal muscle [39],and more modest changes in myelin and brain [39,40,42]. Remarkably, the abundance of LA in rat sciatic nerve increased linearly from 3 to 15% of total fatty acids when dietary LA was increased from 2 to 12% of energy [39]. In rodents, circulating LA readily crosses the blood brain barrier [42], where the majority is quickly oxidized to unknown products. It is presently unknown what proportion of these oxidized LA products are 9- and 13-HODEs and oxoODEs.

Diet-induced modifications in the OXLAM content of neuronal tissues such as sciatic nerve and brain may have important implications for conditions characterized by TRPV1 hyperactivity. OXLAM-mediated TRPV1 activation in rodents induced hyperalgesia and allodynia [1,2], which was reversed with OXLAM specific immunological neutralization [2], indicating that OXLAM-mediated signaling contributes to physical pain. Notably, pathologies characterized by TRPV1 hyperactivity are common in tissues that readily accumulate LA (e.g. fibromyalgia [43], irritable bowel syndrome [44,45], sciatica [46]).While it is not yet known whether lowering dietary LA reduces neuronal and glial OXLAM synthesis and accumulation, such a reduction would be expected to attenuate TRPV1 activation[47], and ameliorate conditions characterized by TRPV1 hyperactivity including physical pain. As TRPV1 is widely distributed in peripheral and central nervous system tissues[48], the ability to modulate endovanilloid tone through diet could have broad implications.

In the United States, per capita dietary LA tripled from about 2% of energy (en%) to 7 en% during the 20^th century [49], which coincided with a marked increase in adipose tissue LA [50,51,52]. Because human adipose tissue LA has a half-life of 1-2 years [36,53,54], the gradual mobilization of LA from adipose stores into circulation is expected to lengthen the time to steady state when lowering dietary LA. Hence, more robust OXLAM reductions may be achievable by extending the duration of LA lowering dietary interventions beyond 12-weeks. However, the chronic pain population studied here may have been especially compliant due to the potential for pain relief. Therefore, the magnitude of OXLAM reductions observed in this 12-week trial may not be readily achievable in other populations.

In conclusion, dietary LA lowering reduces circulating oxidized LA derivatives that have been implicated in a variety of pathological conditions. Future studies evaluating the metabolic and clinical implications of diet-induced OXLAM reductions are warranted.