1. 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

2. 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 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

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

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

5. 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.

6. 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.

7. 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.

8. 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.

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

10. 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

11. 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

12. 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.

13. 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

14. 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

15. Omega-6 Consumption 1950 to 2000

16. 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

17. 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

18. 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

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

20. 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

21. 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.

22. 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.

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

24. 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 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

25. 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.

26. 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

27. ‘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

28. 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.

29. 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.

30. 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

31. 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

32. 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

33. 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

34. 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

35. 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

36. 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

37. 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

38. 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

39. 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

40. Was clinical improvement due to increased medication use?

41. 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

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

43. 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

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

45. 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

46. 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.

47. 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.

48. 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,

49. 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

50. 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

51. 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

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

53. 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

54. 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

55. 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

56. 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.

57. 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

58. 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

59. 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

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

61. 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)

62. 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

63. 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.

64. 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 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 Jan 18:1-10

65. Meal Plan Comparisons

66. 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.

67. 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

68. 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

69. 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: , 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:

70. References continued
Finkel AG, Yerry, JA, Mann JD. Dietary considerations in migraine management: Does a consistent diet improve migraine? Curr Pain Headache Rep 1: ,
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: , 2013.

71. 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

72. 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.