Clinical and metabolic effects of altering omega-3 and omega-6 fatty acids in migraine February 21, 2014 Doug Mann, MD, Professor of Neurology University of North Carolina, Chapel Hill

Outline Specific Aims Rationale for testing dietary modification for chronic pain Preliminary studies: Basic hypotheses, Chronic Daily Headache study, results from animal studies Molecular mechanisms linking endogenously-produced lipid mediators to physical pain Current R01 : study design, questions. Three studies: 1.) Completed – CDH funded by Mayday and forms the bases of for other studies. 2 arms 2.) RO1 funded Sept 2013 and about to launch: Episodic Migraine and diet --- 3 arms. 3.) Post traumatic headache in soldiers, pending funding: 3 arms. Quick overview some of the relevant fatty acid biochemistry useful for understanding the presentation Randomized trial – hypothesis, design, findings using 45Ca2+ TRPV1 assays, transcriptomics and tissue fatty acid analyses Briefly discuss Lipidomics assay - foster collaboration among NIH pain and neuroscience investigators

Studies of Diet and Chronic Pain at UNC, Program on Integrative Medicine 1.) Closed to enrollment – Chronic Daily Headache (CDH) funded by Mayday Fund 2011- 2013. Ongoing data analysis outcomes. 2.) Starting - RO1 funded Sept 2013. Episodic Migraine (EM) and diet --- 3 arms. 153 subjects. 3.) Pending Review - Post traumatic headache in soldiers --- 3 arms.

Specific Aims: R01 Migraine study Clinical and metabolic effects of altering omega-3 and omega-6 fatty acids in migraine

Study Purpose To assess whether: targeted PUFA modifications designed to increase dietary n-3 EPA and DHA, with or without concurrent reduction in n-6 LA, can increase analgesic derivatives of n-3 EPA and DHA, and improve headache-related clinical outcomes.

Specific Aim 1 To assess the efficacy of the dietary interventions in inducing the predicted changes in circulating PUFA endovanilloid derivatives. (1a) Compared to Diet C, Diet A will produce significant increases in analgesic derivatives of n-3 EPA and DHA and reductions in pro-nociceptive n-6 AA-derived and LA-derived endovanilloids. (1b) Diet B (High n-3, High n-6 LA) will result in values for analgesic derivatives of n-3 EPA and DHA intermediate between Diet A and Diet C.

Specific Aim 2 To compare the clinical efficacy of the dietary interventions in adults with migraine. Compared to Diet C, Diet A will produce significant improvement in: (2a) the Headache Impact Test—a headache-specific quality of life measure, and (2b) a significantly steeper rate of decrease in headache hours per day as compared with Diet C. (2c) Diet B will result in changes in clinical outcomes intermediate between Diet A and Diet C.

Specific Aim 3 To test our model of the proposed causal chain linking changes in n-3 and n-6 PUFAs, endovanilloid derivatives, and HA endpoints.

rationale Clinical and metabolic effects of altering omega-3 and omega-6 fatty acids in migraine

Rationale Migraine common (16% women, 7% men) Debilitating, painful, costly, life-impacting Good abortive therapies (cost & side effects) Preventive therapies overall somewhat disappointing Timing ideal for a new strategy

Rationale: food and headache Traditional consensus with limited evidence beyond patient reports. Neurotransmitter precursors in red wine, aged cheese, pickled foods, processed meats, other. Allergies – gluten, dairy. Allergy testing for specific foods. Patient reports of specific food triggers: citrus, aspartame, caffeine, eggs, breads, other. Elimination diet (chicken and rice) with add-backs

Rationale: other potential dietary influences Magnesium intake as a daily supplement is effective in some with EM. No predictive elements except menses. No dietary studies. Riboflavin as a daily supplement is effective – replicated outcomes in EM. No dietary studies. Alpha-lipoic acid – weak clinical evidence of effectiveness in EM. No dietary studies.

Migraine Co-Morbidities Obesity --- headache (inflammation) Crohn’s disease --- migraine Ulcerative colitis --- migraine Irritable bowel syndrome (Aydinlar, 2013) Omega-6 FA consumption Gastrointestinal symptoms Nausea, diarrhea, cramping, bloating, pain

Headache and Fatty Acids One prior study of Omega-3 FA supplements in migraine (Pradlier, 2001) Negative clinical, migraine-related outcomes. Minimally affected group (average < 3 migraines/month) No confirmation of compliance, no biomarkers No biochemical outcomes No consideration of diet and ratios of omega-3 to omega-6 FA intake. Active intervention for 16 weeks 196 subjects (96 intervention, 87 placebo) Strong placebo effect

Omega-6 Consumption 1950 to 2000

Polyunsaturated Fats in U.S. Diets 2008 n-6 AA n-3 EPA+DHA Additive in all kinds of food – mostly carbohydrates, starting around 1960. n-6 LA 7 % of energy Estimated from Day 1 of 24-hour dietary recall interviews conducted in What We Eat In America, NHANES 2007-8

US per capita consumption of vegetable oils in the 20th century kg/p/y 2 4 6 8 10 12 14 Canola 0.9 Year Palm Peanut Safflower Sesame Sunflower 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 1909 1919 1929 1939 1949 1959 1969 1979 1989 1999 Coconut Corn Cottonseed Olive Soybean Linoleic Acid Corn oil, cottonseed, peanut soybean, safflower, Ollive oil, macadamian nut, coconut, Blasbalg et al AJCN 2011

US per capita apparent consumption of n-6 LA and n-3 ALA in the 20th century n-6 LA LA 2.2 n-3 ALA ALA 0.35 Blasbalg et al AJCN 2011

Overview: biochemistry of n-3 and n-6 fatty acids Quick overview some of the relevant fatty acid biochemistry useful for understanding the presentation

Essential Dietary Fats and their Bioactive Metabolites Omega-6 Omega-3 Linoleic Acid α-Linolenic Acid 9-HOTrE EndoVanilloids Eicosanoids Eicosapentaenoic Acid 9-HODE 13-HODE 18-HEPE EndoVanilloids Arachidonic Acid Docosahexaenoic Acid Resolvin E1 Eicosanoids EndoVanilloids Known as essential fats because humans lack the enzymatic machinery to synthesize de novo, therefore must get from diet. Major components of nervous system tissues and glia, immune tissues. Amounts consume influence amounts in tissues Precursor POOLS FOR ENDOGENOUS SYNTHESIS OF VARIETY OF LIPID MEDIATIORS (e.g. classical eicosanoids, also endocannabinoids, endovanilloids)Extremely Take away here – oversimplified (inflammation) – presented as if we know what is going on – extremely complex, tip of the iceberg in understanding In general and with a few notable exceptions, lipid mediators derived from n-6 LA and AA pro-nociceptive, and those derived from n-3 EPA and DHA anti-nociceptive Docosanoids/ Protectins EndoCannabinoids Prostaglandin E2 15-HETE EndoCannabinoids Protectin D1 Docosahexaenoyl Ethanolamide Synaptamide Arachidonoyl Ethanolamide

Diet and Physical Pain: Hypotheses Targeted changes in dietary fatty acids alter tissue fatty acids. Alters the endogenous production of bioactive lipid mediators (e.g. eicosanoids, endovanilloids, endocannabinoids, resolvins). Alters the neurochemical milieu in a manner that may attenuate physical pain.

Rationale for targeted dietary intervention General model Mechanisms linking n-3 & n-6 fatty acids to physical pain General model I want to provide an overview of our proposed model linking dietary n-3 and n-6 fatty acids to pain to Help understand the rationale used to design our targeted dietary intervention As major components of vascular, immune, myelin, glial and neuronal cell membranes, n-3 and n-6 fatty acids are biosynthetic precursors to lipid mediators with putative anti-nociceptive and pro-nociceptive properties (e.g. eicosanoids, endovanilloids, resolvins) The H3-L6 intervention increases the abundance of n-3 EPA and DHA (blue) and decreases the abundance of n-6 LA and AA (red) in fatty acid precursor pools, including the membranes of neurons, glia, endothelial cells, platelets, and immune cells. These changes in precursor abundance alter the concentrations of n-3 and n-6 derived analgesic and pro-nociceptive mediators, including E-series resolvins, D-series resolvins, maresins, prostaglandins, endovanilloids, and endocannabinoids. C. Changes in the milieu of lipid mediators alter the activities of receptors involved in pain signaling, including the vanilloid receptor (TRPV1) and several G-protein coupled receptors (e.g. E-prostanoid receptors, resolvin receptors, cannabinoid receptors). D. Increases in analgesic mediators and decreases in pro-nociceptive mediators reduce pain signaling in the trigemino-vascular system and central pain signaling pathways.

Preliminary studies Clinical and metabolic effects of altering omega-3 and omega-6 fatty acids in migraine

of dietary n-3 and n-6 fatty acids for treatment of chronic headaches: Targeted alteration of dietary n-3 and n-6 fatty acids for treatment of chronic headaches: a randomized trial Published detailed study methods in the journal Trials 2011. Mayday funded trial, 2 years completed in week of Thanksgiving (2011) Discuss design and methods employed briefly here today. Present some preliminary metabolic and clinical results Ramsden CE, Faurot K, Mann JD et al., Trials 2011, BJN 2012, PAIN 2013

Chronic Daily Headache Up to 5% of adults. Migraine - 16 and 6 % 15 or more headache days per month. HA for 4 hours or more per headache day. Six months or more duration. With or without migraine features. Excluded “organic causes”. Medication responses considered in the dx. Population of interest.

Dietary Interventions H3-L6 intervention Increase n-3 EPA and DHA Reduce n-6 LA L6 intervention Maintain low n-3 EPA and DHA intakes (typical of US) Reduce n-6 LA and n-6 AA Two active interventions that were both hypothesized to have anti-nociceptive effects – one greater than other Integrated method –due to ubiquity LA in food supply – most challenging aspect is to reduce LA. No previous trial reduced to less than about 4 en%, goal here is about 2 en% Laboratory analyzed foods, provided all oils and fat sources, foods for 2 meals and 2 snacks per day, Web-based intervention materials Importantly, designed to be equally intensive and credible in terms of food provision, interactions with study dietitian and other investigators, the intensity and breadth of the dietary advice and intervention materials, You’ll see later both groups reported comparable, moderate expectation benefit MacIntosh BA, Ramsden CE et al. BJN 2012

‘Chronic Daily Headache’ Patient population ‘Chronic Daily Headache’ Headache characteristics  15 headache days per month  4 headache hours per day Chronic migraine bumpybrains.com Chronic tension-type headache Ramsden CE, Faurot K, Mann JD et al., Trials 2011

Methods Adults meeting Inclusions/Exclusions Provided 2 meals and two snacks per day We provided all oils and fats All provided food - biochemical analyses at NIH Intensive dietary counseling by study dietitian Guidelines for cooking, shopping, dining out Baseline phase - 4 weeks; intervention 12 weeks Visits every 2 weeks during the intervention for dietary counseling and food pick up, diary review.

Methods No supplements: requested they not start on them during the study. Not given other dietary suggestions relating to traditional consensus advice re: diet. If they were on fish oil or PUFAs for headache, they were excluded. Web based headache diary Continue care with neurologist or other MD.

Methods Arm 1. Low omega-6 (L6) Arm 2. Low omega-6, high omega-3 (L6 H3) Both possibly anti-nociceptive Clinical and biochemical primary endpoints Multiple secondary outcome measures

Overview of Trial Design H3-L6 intervention L6 intervention USUAL CARE THROUGHOUT Randomized, parallel-group clinical trial Dietitian counseling and food provision every 2 weeks Patients continued usual headache care throughout trial H3-L6 intervention

Clinical Outcomes Headache-related quality-of-life (HIT-6) Headache days per month Headache hours per day Headache medication use Psychological distress (BSI-18) Physical and mental function (SF-12) Dual clinical and biochemical outcomes. Clinical outcome – HIT-6 (common)HA related QOL and disability HA diary validated UNC HA clinic PIM previous trials Biochemical – carefully collected processed samples – first trial to lower n-6 LA

Biochemical Outcomes Circulating fatty acid biosynthetic precursor pools **Erythrocytes (n-6 LA, AA; n-3 EPA, DHA) Anti- and pro-nociceptive n-3 and n-6 metabolites Eicosapentaenoic acid (e.g. 18-HEPE) Docosahexaenoic acid (e.g. 17-HDHA) Linoleic acid (e.g. 9- and 13-HODE and -oxoODEs) Arachidonic acid (e.g. 5-, 8-, 9-, 11-, 12-, 15-HETE) Dual clinical and biochemical outcomes. Clinical outcome – HIT-6 (common)HA related QOL and disability HA diary validated UNC HA clinic PIM previous trials Biochemical – carefully collected processed samples – first trial to lower n-6 LA Ramsden CE, Mann JD et al., Trials 2011, PAIN 2013

Trial Profile Assessed for eligibility Total Screened: n=211 Ineligible (n=144) Enrollment Randomized (n=67) Allocated to H3-L6 intervention (n=33) Allocated to L6 intervention (n=34) Allocation What I want to show you is that (1) subjects met initial eligibility critera entered 4-6 week baseline phase – headache diary, those with >15 d, 4 hours eligible. Baseline testing battery, baseline dietary assessment, blood collection 70 subjects Randomized to either (1) Low n-6 diet (average US n-3 PUFAs and all other nnutrients), Half randomized to combined Low n-6, high n-3 intervention. Discontinued intervention (n=5) Discontinued intervention (n=6) Follow-Up Intention-to-treat Analysis HIT-6 and Headache Days (n=33) Intention-to-treat Analysis HIT-6 and Headache Days (n=34) Analysis

Chronic Daily Headache Patient Characteristics Also show you in a minute that average 23 headache days per month, 10 hours per day. All subjects headache physician More than 70% neurology headache specialist – clearly some degree of resistance to conventional pharmacologic care

LA, EPA and DHA Consumption in Chronic Daily Headache Trial (g) No supplements, all through the diet. *LA intake is expressed as a percentage of daily food energy (%E). Median intakes assessed via six 24-hour dietary recalls administered on non-consecutive days. MacIntosh BA, Ramsden CE, Mann JD et al. BJN 2012

Diets altered erythrocyte fatty acid content in a manner predicted to reduce physical pain Erythrocyte fatty acid serve as a representative precursor pool here. We also have very novel data 4 plasma lipid fractions but beyond scope of presentation WE HYPOTHESIZEd THAT BOTH INTERVENTIONS WILL SHOW ANTI_NOCICEPTIVE EFFECTS, AND THAT THE H3-L6 MUCH MORE PRONOUNCED CLINICAL BENFIT Ramsden CE, Mann JD et al., Trials 2011, PAIN 2013

Between-group comparisons H3-L6 intervention produced greater pain reduction HIT-6 Headache days/ month -50 -40 -30 -20 -10 Headache hours/ day Severe Headache days L6 H3-L6 % change (12 weeks) Between-group comparisons p<.001 p<.02 p<.02 p<.01

Headache hours per day by diet group P-diff = 0.01 Great deal of metabolic results from numerous collaborations, In the Remaining time focus (zero in on) one MECHANISM of interest believe has great potential for chronic pain – TRPV1 Ramsden CE, Mann JD et al., Trials 2011, PAIN 2013

Was clinical improvement due to increased medication use?

Change in medication use by diet group Great deal of metabolic results from numerous collaborations, In the Remaining time focus (zero in on) one MECHANISM of interest believe has great potential for chronic pain – TRPV1

Clinical effects of the H3-L6 and L6 interventions on SF 12 SF-12 Health related Ramsden, Faurot, Mann et al, unpublished

Daily ratings of general health self-reported health* 20 40 60 80 2 . 4 6 8 3 days since randomization H3L6 Group difference p-value = 0.03** L6 * 1= poor; 4= excellent ** Proportional odds longitudinal model, group x time interaction

Clinical effects of the H3-L6 and L6 interventions on psychological distress (BSI-18) Ramsden, Faurot, Mann et al, unpublished

Diet-induced changes in anti- and pro-nociceptive lipid mediators Great deal of biochemical/mechanistic results from numerous collaborations, In the Remaining time focus (zero in on) one MECHANISM of interest believe has great potential for chronic pain – TRPV1

Since the H3-L6 intervention targets some of the same pain-related biochemical pathways as common headache medications (e.g. aspirin, valproate), shared mechanisms may help explain the reductions in medication use in the H3-L6 group.

Results Summary The H3-L6 intervention: Produced statistically significant, clinically relevant improvements in: Headache hours per day Severe headache days Quality of life Physical function Emotional distress Produced marked alterations in circulating n-3 and n-6 derived: Endovanilloids Eicosanoids Resolvin pathway precursors – to be analyzed Endocannabinoids** to be analyzed Targeted dietary manipulation of n-3 and n-6 fatty acids reduced headache pain and improved quality-of-life in this population with chronic headaches, and could represent a novel strategy for treating chronic pain in general. Given the promising findings of this trial, future trials evaluating clinical efficacy and elucidating biochemical mechanisms in populations with chronic pain are warranted.

Limitations Small trial Changes in fatty acids not independent No true control group Mediators still unclear Used only blood tissues—unable to determine if other pertinent tissues changed Although there was sufficient power to detect between-group differences in the targeted biochemical and clinical outcomes,

Potential mechanisms responsible for pain reduction Great deal of biochemical/mechanistic results from numerous collaborations, In the Remaining time focus (zero in on) one MECHANISM of interest believe has great potential for chronic pain – TRPV1

Attenuation of TRPV1 hyper-activation Anti-nociception Attenuation of TRPV1 hyper-activation It looks like we might have had substantial clinical benefit However, difficult to put pieces together b/c we have almost no data on the fatty acid composition of the eVN precursor pool in relevant tissues

TRPV1-mediated neurogenic inflammatory cascade Endogenous vanilloid agonists Substance P CGRP TRPV1 Physical Pain Anxiety Alcohol Craving NK1 Receptor Vasodilation Tolerance Dependence Withdrawal CGRP Receptor pH Heat + - Endogenous vanilloid inhibitors

Oxidized Linoleic Acid Metabolites are TRPV1 Agonists 9-HODE 13-HODE 9-oxo-ODE 13-oxo-ODE Name enzymes:

D-series resolvins and NPD1 inhibit TRPV1 activation Docosahexaenoic Acid 14-hydroxy-DHA 17-hydroxy-DHA Name enzymes: Protectin D1 7 (S) Maresin 1 Resolvin D2

Endovanilloids and the neurochemistry of pain Bear with me here….only a couple of take home points from this slide…highlight at end. Identifed by David Julius, 1997, proximal or upstream – drug target. Whether you know it or not, you are familiar with this receptor if you’ve ever eaten a hot pepper. Take home points – 1) the fatty acid composition of neuron, actually other local tissues matters. 2) Improved understanding of this pathway presented opportunity for highly targeted (specific) interventions, targeted toward TRPV1 – act upstream, potential to be more selective for pain than current treatments, hopeful less side effects

Can diet alter endovanilloids in key ‘pain tissues’? It looks like we might have had substantial clinical benefit However, difficult to put pieces together b/c we have almost no data on the fatty acid composition of the eVN precursor pool in relevant tissues

Diet-induced changes in anti- and pro-nociceptive lipid mediators in circulation Since the H3-L6 intervention targets some of the same pain-related biochemical pathways as common headache medications (e.g. aspirin, valproate), shared mechanisms may help explain the reductions in medication use in the H3-L6 group.

Summary & Conclusions Targeted dietary manipulation of n-3 and n-6 fatty acids: Reduced pain and improved quality of life in chronic headache Could represent a novel strategy for treating chronic pain in general Future trials evaluating clinical efficacy and elucidating biochemical mechanisms in chronic pain are warranted

Future Directions It looks like we might have had substantial clinical benefit However, difficult to put pieces together b/c we have almost no data on the fatty acid composition of the eVN precursor pool in relevant tissues

Ramsden, Rapoport, Yuan, Remaley NIH Clinical Center LC/MS/MS assay for lipid mediators of nociception 9-HODE 13-HODE COX/LOX 15-LOX-1 MaR1 14-HDoHE 12-LOX 15-LOX NPD1 17-HDoHE RvD1 RvD2 RvD3 RvD4 LA PGE2 COX1/2 5-LOX 5-HETE LTB4 15-LOX-2 15-HETE LXA4 & LXB4 TXB2 AA DHA EPA RvE1 18-HEPE COX2/CYP RvE2 12-HETE EH 9-OxoODE 13-OxoODE PGD2 PGF2α 20-OH-LTB4 20-COOH-LTB4 5,15-DiHETE 5-HEPE 12-HEPE 15-HEPE RvD5 RvD6 4-HDoHE 7-HDoHE PGE3 TXB3 PGD3 PGF3α CYP LXA5 & LXB5 5,15-DiHEPE RvE3 12/15-LOX 8-HETE 9-HETE 11-HETE Ramsden, Rapoport, Yuan, Remaley Indicates mediators assayed for Chronic Daily Headache trial

Design of R01 trial: Diet and Migraine Clinical and metabolic effects of altering omega-3 and omega-6 fatty acids in migraine

Three arm trial ARM 1: Diet A—High n-3 EPA+DHA, Low n-6 LA ARM 2: Diet B—High n-3 EPA+DHA, High n-6 LA ARM 3: Diet C—Low n-3 EPA+DHA, High n-6 LA (average U.S. intake; control group)

Overview of Trial Design Randomization, Blood Collection Study End, 6 wk f/u, USUAL CARE THROUGHOUT Randomized, parallel-group clinical trial Dietitian counseling and food provision every 2-3 weeks Patients continue usual headache care throughout trial H3-L6 intervention

Fatty Acid Targets Main diet changes to achieve nutrient targets: Low n-6, high n-3 Ave n-6, high n-3 Ave n-6, Ave n-3 (Control) Total Fat (%en) 30% Monounsaturated (%en) 15% 12% Polyunsaturated (%en) 4% 7.5% Saturated Fat (%en) 11% Linoleic acid (n-6) (%en) 2% 7.2% EPA + DHA (mg) 1500 150 Main diet changes to achieve nutrient targets: Replace all oils, spreads and salad dressings with those provided by the study Avoid processed foods that contain oils. Replace with foods provided by the study Consume study provided fish daily (or consume low fat meat, fish and poultry on Control) NHANES data 2009 – 2010: total fat 33%, MUFA 12%, PUFA 7.2%, SF 11%, LA 6.4%, EPA+DHA 90mg The research kitchen will prepare special oil mixes for each for each of the diets to achieve the nutrient targets. The low n-6, high n-3 diet will consume oil that is 25% EVOO and 75% Macadamia Nut Oil and butter. The Ave n-6, high n-3 and Control diet will consume 25% EVOO and 75% corn oil as will as a butter mix that is 50% butter and 50% corn oil. The high n-3 diets will receive very high n-3 canned tuna and salmon as well as from high n-3 fish. The Control group will receive low fat fish and low fat poultry products.

16-Wk Diet Intervention, 6-Wk f/u Personalized nutrition counseling–9 sessions Food provision -prepared and unprepared foods Diet education materials Diet website Seven day meal plan Self-monitoring 24-hr dietary recalls baseline, intervention, post-intervention Nutrition counseling – targeted and tailored counseling. Participants meet with the registered dietitian, who is experienced in research, every 2 -3 weeks. Food provision – prepared and unprepared foods that were designed or selected to meet the fatty acid targets. 2-3 week supply is packed out in rolling coolers during nutrition counseling visits. Foods provided approximately 2/3rd of calorie needs. Diet education materials include research diet guidelines, food lists (allowed, limit, not allowed), how to read food labels, grocery shopping guides and dining out guide. Website includes all diet education materials and access to more than 75 recipes that fit the diet guidelines. 7-day meal plan helps participants plan their meals. The meal plan is available in 3 calorie levels (2,000, 2,500 and 3,000). Participants are assigned the meal plan for weight maintenance based on 35kcals/kg. Self monitoring – a daily checklist is provided to help participants stay on track. The checklist can be completed quickly and is reviewed with the dietitian during counseling. 24-hr, telephone administered, dietary recalls are conducted prior to starting the diet intervention phase and in the last 4 weeks of the diet intervention. Diet Methods Paper: MacIntosh BA, Ramsden CE, Raurot KR, Zamora D, Mangan M, Hibbeln JR, Mann JD. Low n-6 and low n-6 plus high n-3 diets for use in clinical research. Br J Nutr. 2013 Jan 18:1-10

Meal Plan Comparisons

Measures-Clinical (in addition to daily diary) Base line Wk 4 Wk 10 Wk 16 Wk 22 Demographics X Headache History/Physical Exam x HIT-6 PROMIS-29 Profile Comorbid pain assessment MIDAS Satisfaction with care/diet Periodic Assessment of Care for Headaches and Pregnancy Status Expectation of Benefit Food Preference / Dispensing Blood draw ALSO SEEN AT WEEKS 2,7,13 FOR FOOD PICK-UPS, REVIEW PREFERENCES AND ADVERSE EVENTS.

Measures-Biochemical Analyte Formula Method n-6 fatty acids LA 18:2 n-6 GC AA 20:4 n-6 DTA 22:4 n-6 DPA n-6 22:5 n-6 n-3 fatty acids ALA 18:3 n-3 EPA 20:5 n-3 DPA (n-3) 22:5 n-3 DHA 22:6 n-3 Omega-3 Index EPA + DHA Other fatty acids Palmitic acid 16:0 Oleic acid 18:1 n-9   Precursor Endovanilloids (LA and AA-derived TRPV1 agonists) 9-HODE LC/MS/MS 13-HODE 9-oxoODE 13-oxoODE 12-HETE 15-HETE

Measures-Biochemical Endovanilloids (EPA and DHA-derived TRPV1 antagonists) 17-hydroxy-DHA (Primary Outcome) -precursor to D-series resolvins DHA LC/MS/MS 18-HEPE -precursor to E-series resolvins EPA Endocannabinoids 2-AG AA AEA 2-DHG DHEA OEA Oleic acid PEA Palmitic acid Eicosanoids   PGE2 LTB4 TXB2 Lipoxin A4 Other analytes Substance P N/A ELISA Calcitonin gene-related peptide Cytokines (IL-1, Il-6, IL-10, TNF, MCP-1) Multiplex-ELISA

References Ramsden CE, Mann JD, Faurot KR, Lynch C, Imam ST, MacIntosh BA, Hibbeln JR, Loewke J, Smith S, Coble R, Suchindran C and Gaylord SA. Low omega-6 vs. low omega-6 plus high omega-3 dietary intervention for Chronic Daily Headache:  Protocol for a randomized clinical trial.  Trials Journal 12: 97-105, 2011. MacIntosh BA, Ramsden CE, Faurot KR, Zamora D, Mangan M, Hibbeln JR, Mann JD. Low n-6 and low n-6 plus high n-3 diets for use in clinical research. Br J Nutr. 18: 1-10, 2013. Ramsden CE, Ringel A, Feldstein AE,  Taha AY, Macintosh BA, Hibbeln JR, Majchrzak- Hong SF, Faurot KR, Rapoport SI, Cheon Y, Chung Y, Berk M, Mann, JD. Lowering dietary linoleic acid reduces bioactive oxidized linoleic acid metabolites in humans. Prostaglandins Leukot Essential Fatty Acids 87: 135-141. 2012.

References continued Finkel AG, Yerry, JA, Mann JD. Dietary considerations in migraine management: Does a consistent diet improve migraine? Curr Pain Headache Rep 1: 373-376, 2013. Ramsden CE, Faurot KR, Zamora D, Suchindran CM, Macintosh BA, Gaylord S, Ringel A, Hibbeln JR, Feldstein AE, Mori TA, Barden A, Lynch C, Coble R, Mas E, Palsson O, Barrow DA, Mann JD. Targeted alteration of dietary n-3 and n-6 fatty acids for the treatment of chronic headaches: a randomized trial. Pain, 154: 2441-51, 2013.

Acknowledgements UNC Program on Integrative Medicine UNC Dept of Physical Medicine and Rehabilitation UNC Department of Neurology. UNC NCCAM T-32 Integrative Medicine Fellowship Mayday Fund UNC TraCS Intramural Program of NIAAA UNC-Chapel Hill CTSA UNC NORC UNC CHAI Core John M. Davis

Acknowledgements Chris Ramsden Douglas Mann Kim Faurot Beth MacIntosh C. Suchindran Susan Gaylord Daisy Zamora Chanee Lynch Angela Johnston Becky Coble Amit Ringel David Barrow Marjorie Busby Olafur Palsson Beth Fowler Carol Carr Tim McCaskill Merit McMannis Regina McCoy Gus Swenson Meg Mangan Joseph R Hibbeln Sharon Majchrzak-Hong Jim Loewke Ariel Feldstein Alexandros Makriyannis Jodi Wood Trevor Mori Anne Barden Departments of Physical Medicine & Rehabilitation and Neurology.