Bioavailability of Specialized Pro-resolving Mediators in Obese Humans
Phase: NA
Conditions: Obesity Adult Onset
Sponsor: University of North Carolina, Chapel Hill

Bioavailability of SPMs in Obese Humans 
NCT number NCT04701138 
Document Date 06/05/2020 

 
1 
Complete Title: Bioavailability of Specialized Pro-resolving Mediators in Obese Humans 
 
 
Short Title: Immunometabolic Response to SPMs  
 
Drug or Device Name(s): SPM Active  
 
FDA IND/IDE (if applicable): N/A 
 
Sponsor: NC TracS Grant # 550KR242033  
 
Protocol Date: 2/10/20  
 
Amendment 1 Date: 6/05/20 
Amendment 2 Date:  
Amendment 3 Date: 
Amendment 4 Date: 
 
 
NC Translational and Clinical Sciences Institute (TraCS) 
Brinkhous-Bullitt Building, Dental Cir 
Chapel Hill, NC 27599 
USA 
Saame Raza Shaikh, PhD 
2203 McGavran-Greenberg 
Chapel Hill, NC, 27514 
Phone (919) 843-4348  
email: shaikhsa@email.unc.edu 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
2 
 
PROTOCOL TITLE: Bioavailability of Specialized Pro-Resolving Mediators in Obese Humans 
 
 
 
 
Short Title:  Immunometabolic Response to SPMs 
 
  
 
 
 
 
 
   Lead Investigator: 
Saame Raza Shaikh, Ph.D. 
 University of North Carolina at Chapel Hill 
Protocol Version: 2 
Version Date: June 5th 2020 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
6/5/20 

 
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TABLE OF CONTENTS 
Table of Contents ....................................................................................................... 3 
Abbreviations and Definitions of Terms ....................................................................... 4 
Protocol Synopsis ....................................................................................................... 5 
1 BACKGROUND  AND RATIONALE ............................................................................. 8 
2 STUDY OBJECTIVES ................................................................................................. 9 
3 INVESTIGATIONAL PLAN ........................................................................................ 10 
4 STUDY PROCEDURES............................................................................................. 12 
5 STUDY EVALUATIONS AND MEASUREMENTS .......................................................... 13 
6 STATISTICAL CONSIDERATIONS .............................................................................. 14 
7 STUDY INTERVENTION (DEVICE OR OTHER INTERVENTION) ..................................... 17 
8 STUDY INTERVENTION ADMINISTRATION ............................................................... 17 
9 SAFETY MANAGEMENT ......................................................................................... 17 
10  DATA COLLECTION AND MANAGEMENT ................................................................ 17 
11 RECRUITMENT STRATEGY ...................................................................................... 18 
12 CONSENT PROCESS ............................................................................................... 18 
13 PUBLICATION ....................................................................................................... 19 
14 REFERENCES ........................................................................................................ 19 
Appendix .................................................................................................................. 20 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
4 
 
ABBREVIATIONS AND DEFINITIONS OF TERMS 
 
 
 
Abbreviation 
Definition 
14-HDHA 
14-hydroxy docosahexaenoic acid 
17-HDHA 
17-hydroxy docosahexaenoic acid 
18-HEPE 
18-hydroxy eicosapentaenoic acid 
BMI 
Body mass index 
CyTOF 
Cytometry at time of flight 
DHA 
Docosahexaenoic acid 
EPA 
Eicosapentaenoic acid 
HDL 
high density lipoprotein 
LDL 
Low density lipoprotein 
NSAIDs 
non-steroidal anti-inflammatory drugs 
PBMC 
Peripheral blood mononuclear cells 
PCA 
Principal component analysis 
PHI 
Personal health information 
PUFA 
Polyunsaturated fatty acid 
SPMs 
Specialized Pro-resolving Mediators 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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PROTOCOL SYNOPSIS 
Study Title 
Immunometabolic Response to Specialized Pro-Resolving 
Mediators in Obese Humans  
Funder 
Departmental funds 
Clinical Phase 
Pilot study 
Study Rationale 
Obesity is associated with chronic, low-grade inflammation and 
impaired immunity, which contributes to the development of 
metabolic complications such as diabetes, cardiovascular disease, 
and increased susceptibility to infection due to impaired humoral 
immunity. The metabolism of immune cells has emerged as a 
major therapeutic target for improving chronic inflammation. 
Specialized pro-resolving mediators (SPMs) in particular are lipid-
derived metabolites critical in orchestrating the resolution phase 
of inflammation. However, SPMs are lowered with obesity. The 
innovation lies in the study of SPMs as therapeutic agents for 
immune resolution of chronic inflammation that can lead to an 
improved humoral immune response in the obese. Our first 
objective is to establish the concentration of SPMs in plasma, 
serum and peripheral blood mononuclear cells (PBMCs) of obese 
subjects in response to dietary supplementation of SPMs (aim 1a). 
We will further determine the immunological fitness of the 
leukocyte pool, specifically examining B cell antibody production 
in response to ex-vivo stimulation (aim 1b). Finally, we will 
correlate metabolic profiles with SPM concentrations (aim 2). 
Overall, this will help us determine whether SPM 
supplementation mitigates the impaired humoral immune 
response of obese subjects and determine the use of SPMs as 
potential therapeutic agents for the obese. 
Study Objective(s) 
Primary Aim 1: To compare immunological fitness pre- and post- 
oral SPM administration in the obese.  
• Aim 1a: To quantify SPM concentrations in plasma 
(pg/mL), serum (pg/mL) and PBMCs before and after SPM 
supplementation. 
• Aim 1b: To measure antibody responses of B cells in PBMC 
pool with in vitro stimulation and cytokine production 
before and after SPM supplementation.  
Secondary Aim 2: To compare metabolic profiles pre- and post- 
supplementation in response to an oral dose of SPMs in the 
obese.  
• Aim 2a: To measure fasting insulin (ng/dL) and fasting 
glucose (mg/dL) before and after intervention. 
• Aim 2b: To correlate pre- and post- metabolic 
measurements with measured pre- and post- SPM 
concentrations. 

 
6 
Test Article(s) 
(If Applicable) 
The ‘SPM Active’ dietary supplement will be supplied by 
Metagenics for intervention.  
Study Design 
 
A non-randomized uncontrolled clinical trial with convenience 
sampling. At total of 24 individuals (12 males & 12 females) will 
be selected against inclusion/exclusion criteria. Pre- and post- i-
ntervention measurements will be collected and analyzed.  
Subject Population 
key criteria for Inclusion 
and Exclusion: 
Inclusion Criteria 
1. Male and (post-menopausal) female subjects age 50-65 
years with BMI 30-40 (obese) who are euglycemic and 
pre-diabetic  
Exclusion Criteria 
1. Subjects with active autoimmune disease, liver disease, 
coagulopathy, hypothyroidism, known allergy to fish or 
shellfish, those taking active asthma medications, 
anticoagulants, estrogen, testosterone, daily aspirin or 
NSAIDs, anyone with inability to give informed consent 
2. Subjects receiving immunomodulatory or 
immunosuppressant therapy, with known active 
malignancy or undergoing treatment for malignancy, and 
those consuming n-3 PUFA supplements or high 
consumption of fatty fish (>2 servings per week) 
Number Of Subjects  
24 
Study Duration 
The entire study is expected to last 6-9 months. This includes an 
enrollment period of 3-4 months and data analysis. 
Study Phases 
Screening 
Study Treatment 
Follow-Up   
(1) Screening: Screening for eligibility will be performed by Dr. 
Butler and his designated study coordinator at UNC Family 
Medicine Center during standard pre-operative checkups in a 
private, closed-door room. Subjects will be asked questions orally 
during the primary visit to determine if eligibility criteria are met, 
at which time informed consent and HIPPA authorization will be 
obtained.  
(2) Intervention: The intervention is the dietary supplement ‘SPM 
Active’ supplied by Metagenics. All subjects will be administered 4 
capsules per day for 4 weeks +/- 2-4 days. 12-hour fasting blood 
and serum will be drawn pre- and post-intervention for each 
subject by a licensed phlebotomist at the UNC Family Medicine 
Lab and under the direction of Dr. Butler.  
(3) Follow-Up: The study coordinator will follow up twice by 
phone at the end of week 1 and week 3 of supplementation and 
relay any health concerns to Dr. Butler who may determine if an 
in-person visit is necessary. Following 4 weeks intervention, 
patients will discontinue supplementation follow up in a routine 
manner as designated by their UNC Family Medicine physician. 

 
7 
Efficacy Evaluations 
If SPM administration does not lead to increased uptake of SPMs 
in PBMCs or serum with the first 9 subjects, then we will not 
conduct CyTOF. Instead, we will spend the funds in conducting a 
pilot metabololipidomic study of 9 subjects of one sex to 
determine if 8 weeks of intervention leads to an elevation in the 
three metabolites of interest. We envision no technical limitations 
to the study as all the proposed protocols are optimized. 
Safety Evaluations 
While adverse events are unlikely, if 50% or more of total study 
participants report intolerable adverse reactions from taking the 
supplement, we will stop the study. 
Sample Size Rationale 
The primary aim of the study is designed and powered to 
measure an increase in 18-HEPE, 17-HDHA, and 14-HDHA in Aim 
1. Additional measures in Aim 2 are exploratory, but necessary 
for future grant applications to demonstrate the feasibility of the 
approach. At a family-wise error rate of 5% controlling for testing 
of three metabolites mentioned above, n=9 will provide more 
than 80% power to detect a 50%-fold change increase in 
metabolite levels within each subject. We are assuming a similar 
effect size for both males and females and are doubling the 
sample size (n=18, 9 per sex) to allow us to subset the data in 
case we find divergent results between sexes. To account for 
possible loss-to-follow up, we increased the sample size to 24 
(n=12 male + 12 female). Power analysis was conducted 
assuming a paired t-test on pre- and post-treatment metabolite 
levels for three pre-specified metabolites. Expected variability of 
within subject differences in metabolite levels was assessed from 
a previous dataset and the median standard deviation was 
calculated across all metabolites.1 Power analyses were calculated 
by Dr. Michael Love in the UNC Department of Biostatistics. 
Statistical & Analytic 
Plan 
Generally, most datasets will be assessed for normality and 
homogeneity of variance using the Shapiro-Wilks test and Bartlett 
test respectively. However, normality will also be assessed via a 
qqplot of the standardized residuals and we will visualize the 
distribution of our data with a histogram. In addition, to 
corroborate the Bartlett test we will check the homogeneity of 
variance of the residuals (homoscedasticity) using a scale-location 
plot where the square root of the standardized residuals is plotted. 
Data that passes both assumptions will be analyzed via a paired 
two sample Student T-test. Data that fails the assumption of 
heteroscedasticity will be analyzed using a paired Welch T-test. 
Another alternative would be to apply a log or square root 
transformation to achieve heteroscedasticity or approach a 
Gaussian distribution. However, if the data still fails the 
assumptions of normality and heteroscedasticity it will be analyzed 
with a paired Wilcoxon rank sum test. All p-values from these 

 
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analyses will undergo either a Bonferroni or Benjimani-Hochberg p-
value adjustment at a 5% false discovery rate. 
DATA AND SAFETY 
MONITORING PLAN 
Data will be kept on an encrypted desktop computer protected by 
multiple passwords in a lab, located on a hall protected by card 
access in Michael Hooker Research Center, Chapel Hill, NC. De-
identified data will be stored on REDCap for use by research team 
personnel only. Data management will be performed by the PI 
and the study coordinators. All data acquisition will meet HIPPA 
and confidentiality standards. The PIs will be responsible for 
monitoring and reviewing subject data, lab results, study 
progress, subject safety and confidentiality, and the accuracy and 
security of the emerging data. They will also be responsible for 
running the assays and entering all data into redcap for statistical 
analysis. The co-I, Dr. Butler, will follow-up with patients as 
necessary; his contact information and that of the study 
coordinator will also be given to the study subjects.  
 
 
1 
BACKGROUND AND RATIONALE 
1.1 
Introduction 
Chronic inflammation, a central component of obesity, contributes toward the development of numerous 
metabolic complications such as type 2 diabetes, cardiovascular diseases, and increased susceptibility to 
infection. The metabolism of immune cells has emerged as a major therapeutic target for improving chronic 
inflammation, and thereby, whole-body metabolism. Specialized pro-resolving mediators (SPMs) in particular 
are lipid-derived metabolites that have been shown to orchestrate the resolution of inflammation, 
regeneration of damaged tissues, and even improve outcomes to infections.2,3 However, SPMs are lowered 
with obesity. It is therefore hypothesized that exogenous administration of SPMs to obese humans may 
have therapeutic value as insulin and glucose sensitizing agents by targeting chronic inflammatory pathways 
in obesity. Until recently, a major roadblock in the field of obesity and SPMs is that translation of findings 
from the preclinical level to humans has remained limited. However, this obstacle can now be overcome as 
the first oral formulation of SPMs has emerged on the market as a dietary supplement. We will lead the 
field by taking the first steps toward precision administration of SPMs as therapeutic agents for immune 
resolution of chronic inflammation in the obese. Specifically, we will establish the concentration of SPMs in 
plasma, serum and peripheral blood mononuclear cells (PBMCs) of obese subjects in response to dietary 
supplementation with ‘SPM Active’. We will further determine the immunological fitness of the leukocyte 
pool, specifically examining B cell antibody production in response to ex-vivo stimulation. This will 
determine whether SPM supplementation mitigates the impaired humoral immune response of obese 
subjects. Overall, this work will help determine the use of SPMs as potential therapeutic agents for the 
obese. 
 
1.2 
Name and Description of Investigational Product or Intervention  
The study will provide the dietary supplement ‘SPM Active,’ manufactured by Metagenics, as intervention. 
Each capsule contains 145 mg of the specialized pro-resolving mediators (SPMs) 18-HEPE (25 mg), 17-HDHA 
(40 mg) and 14-HDHA (80 mg) from fractionated marine lipid concentrate. SPMs are synthesized 
enzymatically from n-3 polyunsaturated fatty acids (PUFA) known as eicosapentaenoic (EPA) and 

 
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docosahexaenoic acids (DHA) and are known to play a critical role in resolving chronic, low-grade 
inflammation.  
 
1.3 
Non-Clinical and Clinical Study Findings 
The same ‘SPM Active’ dietary supplement to be administered to obese subjects in this clinical trial was 
previously used as intervention in a human clinical trial by Dalli et al, and found to be safe at doses up to 
4.5g in healthy individuals.4 The investigators concluded that SPM supplementation leads to an increase in 
peripheral blood SPM concentrations and reprograms peripheral blood cells, thus indicating a role for SPMs 
in mediating immunological activity.4  
 
1.4 
Relevant Literature and Data 
 
Obesity is a growing national and global epidemic. In the United States; over 35% of Americans are 
currently obese (Body mass index (BMI) >30 kg/m2), and another one-third overweight (BMI: 25-29.9 
kg/m2).5 Obesity is associated with numerous chronic health conditions, including insulin resistance, type-2 
diabetes, hyperlipidemia and hypertension, leading to what is termed as the metabolic syndrome.6 Chronic 
inflammation, a central component of obesity, significantly contributes to the development of these 
metabolic complications and has been associated with accumulation of visceral adipose tissue and immune 
cell infiltration.7-9 Recently, the metabolism of immune cells has emerged as a major therapeutic target for 
improving chronic inflammation and whole-body metabolism, as immune cells have fundamentally different 
metabolic programming between pro- and anti-inflammatory subsets.10-13  
 
Specialized pro-resolving mediators (SPMs) have recently emerged as a target for improving chronic 
inflammation. SPMs known as protectins, resolvins, lipoxins and maresins, are potent metabolites 
enzymatically synthesized from n-3 polyunsaturated fatty acids (PUFA) eicosapentaenoic (EPA) and 
docosahexaenoic acids (DHA).14 Our research, in agreement with other labs, have discovered that 
metabolites of the SPM family are decreased across a range of tissues in mouse models of obesity and in 
circulation of obese individuals.15-18 To exemplify, we have reported that the DHA-derived SPMs 14-HDHA 
and 17-HDHA are lowered in the spleens of obese C57BL/6J mice.15 We are also finding a strong reduction 
in the EPA-derived SPM known as 18-HEPE in white adipose tissue and liver of obese mice relative to lean 
controls (Fig. 1). Overall, our results are highly consistent with data from a recent study to show that 
14HDHA, 17-HDHA, and 18-HEPE are lowered in circulation of obese subjects.17 
 
SPMs have insulin/glucose-normalizing properties. Rigorous literature reports demonstrate that EPA- and 
DHA-derived SPMs improve varying aspects of murine metabolism including insulin resistance and hepatic 
steatosis.20-22 We have investigated the effects of resolvin E1 (the downstream product of 18-HEPE) on 
hyperglycemia and hyperinsulinemia of obese male C57BL/6J mice. Administration of resolvin E1 to obese 
male mice at 300ng per mouse per day for 4 days prior to sacrifice has no effect on body weight (Fig. 2A) 
or food intake (data not shown). Resolvin E1 completely restores high fat diet-induced hyperglycemia (Fig. 
2B) and strongly improves hyperinsulinemia (Fig. 2C). 
 
2 
STUDY OBJECTIVE  
The purpose of the study is to determine the efficacy of SPM supplementation on chronic inflammation in 
obese human subjects. The premise is based on the notion that SPMs are lowered in obese individual. 
Therefore, it is hypothesized that exogenous administration of SPMs to obese individuals may have 
therapeutic value as insulin/glucose sensitizing agents by targeting inflammatory pathways.19 This application 
breaks new ground by taking the first steps toward translating SPMs from the pre-clinical level to obese 

 
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humans. We propose a bioavailability study in which we will establish the concentration of SPMs in plasma 
and PBMCs of obese subjects (primary aim) in response to dietary supplementation with SPMs. Additional 
exploratory measurements that correlate metabolic parameters with SPM concentration are proposed in 
our secondary aim. 
 
2.1 
Primary Objective 
Our primary objective is to compare immunological fitness pre- and post- oral SPM administration in the 
obese:  
- 
Aim 1a: To quantify SPM concentrations in plasma, serum and PBMCs before and after oral SPM 
supplementation. We will measure SPMs in 100µl of plasma and 2 x 106 PBMCs. PBMCs will be isolated 
with established protocols in our lab.15, 26 The rationale for PBMCs vs. plasma is to compare whether 
SPM levels in PBMCs are higher than plasma or vice versa, indicating SPM uptake into the immune cells 
or lack thereof. We will also measure other lipid metabolites involved in the inflammatory resolution 
process including thromboxanes, leukotrienes, prostaglandins, epoxy fatty acids, eoxins, mono-, di-, and 
tri-hydroxy fatty acids, and hepoxilins. Blinded samples will be shipped to our collaborator Dr. Reisdorph 
for two-dimensional reverse phase HPLC tandem mass spectrometry. 
 
- 
Aim 1b: To measure antibody responses of B cells in PBMC pool with in vitro stimulation and cytokine 
production before and after SPM supplementation. B cells will be directly stimulated in the PBMC isolate 
to mimic their natural environment. Then, antibody concentrations will be measured from the 
supernatants of the cultured cells via ELISA for pre- and post-intervention samples. 
 
2.2 
Secondary Objective 
Our secondary objective is to compare metabolic profiles in response to an oral dose of SPMs in the obese:  
- 
Aim 2a: To measure fasting insulin and fasting glucose before and after intervention. Blood samples 
from baseline and post-intervention will be used to measure fasting glucose (mg/dL) and fasting insulin 
(ng/mL) through McClendon labs. Exploratory studies will test if obese adult participants taking a daily 
dose of SPMs have lower mean fasting levels of serum insulin and glucose after 4 weeks of SPM 
administration, relative to baseline. Participants will be asked by the study coordinator about the extent 
to which they complied with dosing at the end of week 2 and week 3 following the start of 
supplementation 
 
- 
Aim 2b: To correlate pre- and post- metabolic measurements with measured pre- and post- SPM 
concentrations. Anthropomorphic and blood pressure measurements will be taken. Each subject will 
have his/her insulin (in ng/dL) and glucose (in mg/dL) measured pre/post supplementation through 
Family Medicine and the Reisdorph lab will determine SPM concentrations in the plasma pre/post 
supplementation. Insulin and glucose measurements will then be correlated with measured SPM levels. 
- 
 
3 
INVESTIGATIONAL PLAN (brief overview) 
3.1 
Study Design 
We will conduct a non-randomized uncontrolled clinical trial to measure the efficacy of SPM administration 
in the obese. We will selectively recruit (n=24) obese subjects (BMI 30-40 kg/m2) and administer an SPM 
formulation (“SPM Active”) provided by Metagenics for 4 weeks. The study design will rely on blood 
samples collected at baseline and at the end of the 4-week intervention. All analyses will rely on 

 
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comparisons between pre- and post-intervention. The concentration of SPMs, in addition to other PUFA-
derived metabolites that share the same enzymatic pathways as SPMs, will be established at baseline and 
post-intervention using mass spectrometry-based metabololipidomics. The concentration of all metabolites 
will be measured for plasma and peripheral blood mononuclear cells (PBMCs). 
 
Overview of Study Phases 
 
Screening/Baseline: Subjects will be recruited in person at UNC Family Medicine Center at 540 Manning 
Drive, Chapel Hill, NC 27599 in a private, closed-door room during a regularly scheduled office visit with Dr. 
Butler. We are using a convenience sample method, therefore everyone age 50-65 will be screened 
according to the criteria set forth for the study. During their visit, Dr. Butler will ask subjects if they wish to 
learn more about the study. If yes, the subjects will be directed to meet with the study coordinator. If the 
study coordinator is unavailable, Dr. Butler can complete the consent process during the office visit. 
Recruitment and informed consent will then follow the support documents provided, with either 
preoperative or post-operative consent and scripts being read by the study coordinator to the potential 
study subject. Upon full understanding and answering of any questions, the study coordinator will obtain 
written and verbal informed consent along with providing a copy of the consent form for the subject to 
keep themselves. We will recruit a total of 24 (n=12 men + 12 women) obese (BMI 30-40 kg/m2) 
euglycemic and pre-diabetic subjects who will have similar distributions in their age (50-65 years), race, and 
smoking status. There will be no randomization for this study.  
 
Intervention/Treatment: All 24 subjects will be administered 4 capsules of ‘SPM Active’ per day for 4 weeks 
+/- 2-4 days. There are 3 metabolites of the SPM family in the capsule, which are 17-HDHA (40mg) + 14-
HDHA (80mg) + 18-HEPE (25mg). All three metabolites feed into pathways shown to improve insulin 
resistance and all three metabolites are decreased in obese subjects.15, 17, 19, 24 The rationale for the dosing 
and time of intervention is based on the recommendations by the manufacture.  The supplement will be 
provided by Metagenics. Note the manufacturer has not conducted bioavailability studies focused on the 
obese. Subjects will be instructed to consume 2 capsules with breakfast and 2 with dinner (4 total per day). 
Fasting blood will be drawn pre- and post-intervention using phlebotomy available under the direction of Dr. 
Butler. 
 
Baseline measurements will be taken prior to intervention so that each subject may serve as his/her own 
control. 12-hour fasting blood and serum will be drawn pre- and post-intervention for each subject by a 
licensed phlebotomist at the UNC Family Medicine Lab and under the direction of Dr. Butler, the co-I. Each 
blood sample will be the size of 10 10mL plasma and serum tubes (9 plasma tubes + 1 serum tube) to 
ensure that enough B cells are collected from PBMCs for activations (see section 4.1 below). Therefore, a 
total of 10 tubes or ~100 ml of blood will be drawn twice during the study, 4 weeks apart. Finally, each 
subject will have his/her height (ft), body weight (lbs), blood pressure (mmHg), and pulse (bpm) taken at 
baseline and 4 weeks of intervention. 
 
Follow up: Subjects will have two face-to-face visits and two phone contacts during the study. The first visit 
will be at baseline (pre-intervention) and the second visit will conclude the study (post-intervention). We 
will continuously monitor subject health status throughout the study via phone contact at the end of week 1 
and week 3 following the start of supplementation. At the end of the study, all patients will follow up in a 
routine manner as designated by their physician at UNC Family Medicine. 
 
Unscheduled Visits: If the study coordinator relays health concerns to Dr. Butler that will necessitate in-
person visit.  

 
12
 
3.2 
Allocation to Treatment Groups and Blinding (if applicable) 
There will be no randomization or blinding for this study. All 24 subjects will receive the “SPM Active” 
intervention. Baseline measurements will be taken prior to intervention so that each subject may serve as 
his/her own control. 
 
3.3 
Study Duration, Enrollment and Number of Subjects 
Each subject’s participation will last up to 4 weeks +/- 2-4 days, with no additional time involved in this 
study outside normal pre- and post-operative care. The entire study is expected to last 6-9 months. This 
includes an enrollment period of 3-4 months and data analysis. 
 
3.4 
Target Population 
Inclusion Criteria:  
- 
Male and female subjects with age 50-65 years and obese (i.e. BMI 30-40) who are 
euglycemic and pre-diabetic (i.e. fasting glucose 70-125 mg/DL or HbA1c of 5.7-6.4%).  
- 
Only post-menopausal females will be recruited in the female cohort to reduce the confounding 
inflammatory effects of estrogen with supplementation.  
Exclusion Criteria: 
- 
Those with fasting glucose values > 126 mg/dL or known type 2 diabetes  
- 
Females who are pre-menopausal, pregnant, planning to become pregnant, breastfeeding or 
lactating  
- 
Subjects consuming n-3 PUFA supplements in the last 3 months prior to enrollment, high 
consumption of fatty fish (>2 servings per week), and subjects with active autoimmune disease, 
liver disease, coagulopathy, hypothyroidism, known allergy to fish or shellfish, inability to give 
informed consent, or taking anticoagulants (e.g. warfarin and direct acting anticoagulants), those 
taking estrogen or testosterone, and anyone taking daily aspirin, NSAIDs, or active asthma 
medications.  
- 
Subjects receiving immunomodulatory or immunosuppressant therapy (corticosteroids or 
monoclonal antibodies) in the 4 weeks prior to study enrollment, and subjects with known active 
malignancy or undergoing treatment for malignancy will be excluded. 
 
Demographics: No exclusions will be made based on race, gender, or ethnicity. Since all female subjects will 
be post-menopausal, this study will not include female subjects who are pregnant or become pregnant. This 
is not because we expect risk to mother or fetus in pregnancy but rather because of the hormonal effects 
of high estrogen in pre-menopausal women on the lipidomic profile that we are studying. Moreover, the 
inflammatory effects of estrogen will confound the lipidomic profile measured from the subjects. For this 
reason, the female cohort will include only post-menopausal women.  
 
4 
STUDY PROCEDURES  
4.1 
Screening/Baseline Visit procedures 
Insulin (ng/dL), glucose (mg/dL), BMI (kg/m2), weight (lbs), height (ft), disease status, and medications will be 
abstracted from medical charts. These metabolic measurements will be compared pre- and post- 

 
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supplementation for aim2a, and used to investigate their association with SPM concentrations for 
aim2b.  
 
12-hour fasting blood and serum will be drawn for each subject by a licensed phlebotomist at the UNC 
Family Medicine Lab and under the direction of Dr. Butler at baseline. Each blood sample will be the size of 
10 10mL plasma and serum tubes (9 plasma tubes + 1 serum tube). PBMCs will be isolated from each 
blood sample. SPM concentration will be compared in the plasma, serum and PBMCs obtained for 
each patient, in accordance with aim 1a. For aim 1b, we will plate 15 wells of B cells (isolated from the 
PBMCs) for activations with 200,000 B cells per well (3 x 10^6 total). We are assuming 1 mL blood yields 
approximately 1 x 10^6 PBMCs. We also assume 0.5 x 10^6 PBMCs per well with 5% B cells. Since, on 
average, 7-8 mL of blood is collected per 10mL tube, we will collect 10 tubes to ensure we have at least 60 
mL blood for the 60 x 10^6 PBMCs needed to collect 3 x 10^6 B cells for activations. Therefore, a total of 
10 tubes or ~100 ml of blood will be drawn.  
 
In addition to blood collection, each subject will have his/her height (ft), body weight (lbs), blood pressure 
(mmHg), and pulse taken (bpm).  
 
4.2 
Intervention/Treatment procedures 
All subjects will receive the dietary supplement “SPM Active”, provided free of cost by Metagenics. Subjects 
will be responsible for taking 4 capsules per day (2 at breakfast and 2 at dinner) for a total of 4 weeks.  
 
4.3 
Follow- up procedures 
There is no scheduled follow up procedure apart from that detailed in the study design. The patients will 
follow up in a routine manner as designated by their physician at UNC Family Medicine. 
 
4.4 
Unscheduled visits 
The study coordinator may relay health concerns to Dr. Butler that will necessitate an in-person visit.  
 
4.5 
Concomitant Medication documentation 
All prescription or OTC drugs taken by study subjects will be examined by Dr. Butler and his team prior to 
enrollment in the study. No previous adverse events have been observed in previous studies (such as the 
human clinical trial done by Dalli et al.) with “SPM Active,” though we will continuously monitor subject 
health status via phone contact at the end of week 1 and week 3.  
 
4.6 
Subject Completion/ Withdrawal procedures 
Subjects will be compensated $250 cash for their participation and completion of the study ($100 at 
baseline visit + $150 at week 4 visit upon completion). No prorated compensation will be applied for this 
pilot study. This amount of compensation is not deemed coercive; the incentive is meant to compensate 
subjects for their time in the study which include two study visits, two phone visits, and two blood draws.   
 
This is a voluntary research study. Refusal to participate or withdrawal of participation and/or consent can 
occur at any time, for any reason. Subjects may be withdrawn if: (1) they experience intolerable adverse 
reactions or allergic reaction to study supplement or (2) are not at least 90% compliant with taking study 
supplement during the 4 weeks active phase. In the event of withdrawal, all personal health information 
(PHI), electronic or hard copy, will be immediately purged and permanently destroyed. Withdrawal or 

 
14
refusal will not influence ability, timing, or quality of clinical care, type of clinical care administered, standing 
with UNCHC system, or ability to participate in future research studies. The investigators also have the right 
to stop participation at any time. This could be because participants have chosen to stop supplementation, 
or have failed to follow instructions or because the entire study has been stopped.  
 
4.7 
Screen failure procedures 
Study participants will be checked to determine if they meet the inclusion and exclusion criteria. Those that 
are not eligible will be excluded from the study. If the study coordinators are not able to contact study 
subjects in the follow-up phone calls they will be considered as “loss to follow-up” and no longer enrolled in 
the study.  
 
5 
STUDY EVALUATIONS AND MEASUREMENTS  
Samples taken from pre- and post-intervention visits will be immediately transferred to Michael Hooker 
Research Center Room 2204 (Shaikh Lab) for immediate processing. Tubes will be labelled with a de-
identified research code and will not include any PHI. We will measure SPMs in 100µl of plasma and serum 
and 2x106 PBMCs. PBMCs will be isolated with established protocols in our lab 1, 2. The rationale for PBMCs 
vs. plasma and serum is to compare biomarkers for SPM uptake. We will also measure other lipid 
metabolites including thromboxanes, leukotrienes, prostaglandins, epoxy fatty acids, eoxins, mono-, di-, and 
tri-hydroxy fatty acids, and hepoxilins. Blinded samples will be shipped to our collaborator Dr. Reisdorph 
(pay-for-service, see support letter) for two-dimensional reverse phase HPLC tandem mass spectrometry. 
Blood samples from baseline and post-intervention will be used to measure fasting glucose (mg/dL), fasting 
lipid panel (total cholesterol in mg/dL, LDL in mg/dL, HDL in mg/dL, triglyceride levels in mg/dL) and fasting 
insulin (ng/mL) through McClendon labs. 
 
5.1 
Efficacy Evaluation 
There will be no efficacy evaluation due to the pre-/post- study design, which requires that data be obtained 
through the end of the clinical trial. However, the study may be stopped due to safety concerns (see safety 
evaluation section 5.2).  
 
5.2 
Safety Evaluations 
Any health conditions that arise over the course of the study that were not present in the subjects prior to 
their enrollment will be monitored by Dr. Butler who will evaluate whether to withdraw the subject from 
the study.   
 
6 
STATISTICAL CONSIDERATION    
6.1 
Primary Endpoint 
The primary aim of the study is designed and powered to measure an increase in 18-HEPE, 17-HDHA, and 
14-HDHA in Aim 1. At a family-wise error rate of 5% controlling for testing of three metabolites mentioned 
above, n=9 will provide more than 80% power to detect a 50%-fold change increase in metabolite levels 
within each subject. We are assuming a similar effect size for both males and females and are doubling the 
sample size (n=18, 9 per sex) to allow us to subset the data in case we find divergent results between 
sexes. To account for possible loss-to-follow up, we increased the sample size from 18 to 24 (n=12 male + 
12 female). Power analysis was conducted assuming a paired t-test on pre- and post-treatment metabolite 
levels for three pre-specified metabolites. Expected variability of within subject differences in metabolite 
levels was assessed from a previous dataset and the median standard deviation was calculated across all 

 
15
metabolites.1 Power analyses were calculated by Dr. Michael Love in the UNC Department of Biostatistics. 
Note that all analyses will be conducted in the R statistical software. 
 
- 
Aim 1a: SPM lipidomic measurements will be analyzed pre/post supplementation. Each SPM metabolite’s 
distribution will be assessed for normality and homogeneity of variance using the Shapiro-Wilks test and 
Bartlett test respectively. However, normality will also be assessed via a qqplot of the standardized 
residuals and we will visualize the distribution of our data with a histogram. In addition, to 
corroborate the Bartlett test we will check the homogeneity of variance of the residuals 
(homoscedasticity) using a scale-location plot where the square roots of the absolute values of the 
standardized residuals are plotted. If the metabolite’s distribution satisfies the assumption of 
normality & homogeneity of variance then a paired two-tailed Student’s T-test will be used to generate 
p-values corresponding to differences before and after supplementation. If the assumption of 
heteroscedasticity is not met, the data will be analyzed using a paired Welch T-test. Another alternative 
would be to apply a log or square root transformation. However, if the data still fails the assumptions 
of normality and heteroscedasticity it will be analyzed with a paired Wilcoxon rank sum test. All p-values 
will undergo a Benjimani-Hocherberg adjustment (optimal for large-scale multiple hypothesis testing) at 
a false discovery rate of 5% to account for multiple hypothesis testing between metabolites. 
  
 
- 
Aim 1b: Antibody concentrations measured via ELISA will be tested for the assumptions of normality and 
homogeneity of variance using the Shapiro-Wilks test and Bartlett test respectively. However, normality 
will also be assessed via a qqplot of the standardized residuals and we will visualize the 
distribution of our data with a histogram. In addition, to corroborate the Bartlett test we will check 
the homogeneity of variance of the residuals (homoscedasticity) using a scale-location plot where 
the square root of the standardized residuals is plotted. Data that passes both assumptions will be 
analyzed via a paired two sample Student T-test. Data that fails the assumption of heteroscedasticity will 
be analyzed using a paired Welch T-test. Another alternative would be to apply a log or square root 
transformation. However, if the data still fails the assumptions of normality and heteroscedasticity it 
will be analyzed with a paired Wilcoxon rank sum test. All p-values from these analyses will undergo a 
Bonferroni p-value adjustment (optimal for smaller-scale multiple hypothesis testing). 
  
 
6.2 
Secondary Endpoint 
 
- 
Aim 2a: The data will be assessed for normality and homogeneity of variance using the Shapiro-Wilks test and 
Bartlett test respectively. However, normality will also be assessed via a qqplot of the standardized residuals 
and we will visualize the distribution of our data with a histogram. In addition, to corroborate the Bartlett test 
we will check the homogeneity of variance of the residuals (homoscedasticity) using a scale-location plot 
where the square root of the standardized residuals is plotted. Data that passes both assumptions will be 
analyzed via a paired two sample Student T-test. Data that fails the assumption of heteroscedasticity will be 
analyzed using a paired Welch T-test. Another alternative would be to apply a log or square root 
transformation. However, if the data still fails the assumptions of normality and heteroscedasticity it will be 
analyzed with a paired Wilcoxon rank sum test. All p-values from these analyses will undergo a Bonferroni p-value 
adjustment. 
 
- 
Aim 2b: The data will also be assessed for normality using the Shapiro-Wilks test, qqplots, and we will 
visualize the distribution of the data with a histogram. If the data are normal or it cannot be 
transformed to approximate a Guassian distribution, we will utilize a Pearson correlation to 

 
16
determine associations between SPM concentration and metabolic outcomes (insulin/glucose measures). 
If the data is not normal we will utilize a Spearman correlation. All p-values from these analyses will undergo 
a Bonferroni p-value adjustment. 
 
6.3 
Statistical Methods 
 
Data will be assessed for normality and homogeneity of variance using the Shapiro-Wilks test and Bartlett test 
respectively. However, normality will also be assessed via a qqplot of the standardized residuals and we will 
visualize the distribution of our data with a histogram. In addition, to corroborate the Bartlett test we will 
check the homogeneity of variance of the residuals (homoscedasticity) using a scale-location plot where the 
square root of the standardized residuals is plotted. Data that passes both assumptions will be analyzed via a 
paired two sample Student T-test. Data that fails the assumption of heteroscedasticity will be analyzed using a 
paired Welch T-test. Another alternative would be to apply a log or square root transformation. However, if 
the data still fails the assumptions of normality and heteroscedasticity it will be analyzed with a paired 
Wilcoxon rank sum test. All p-values will undergo either a Bonferroni or Benjimani-Hocherberg p-value 
adjustment. 
 
Further analyses include determining patterns of metabololipidomic signatures and immune cell phenotypes 
from both aims. To do this we will conduct a principal component analysis (PCA) on the metabololipidomics 
and ex-vivo B cell antibody production data. PCA will aid in reducing the biological noise in the dataset and 
identify trends between metabolites and B cell function to determine correlated metabololipidomic 
signatures. To identify clusters of signatures shared among individuals, k-means clustering on the PCA 
analysis will be conducted to visualize the partitioned metabolite-cellular landscapes pre- and post-
intervention. Moreover, statistical estimates of population parameters will be tabulated along with 
corresponding confidence intervals (CIs) and/or standard errors (SEs) to convey levels of precision / 
imprecision. 
 
6.4 
Sample Size and Power 
Experimental rigor is ensured through the analyses of blinded samples. The study is designed and powered 
to measure an increase in 18-HEPE, 17-HDHA, and 14-HDHA in Aim 1. Additional measures in Aim 2 are 
exploratory, but necessary for future grant applications to demonstrate the feasibility of the approach. At a 
family-wise error rate of 5% controlling for testing of three metabolites mentioned above, n=9 will provide 
more than 80% power to detect a 50%-fold change increase in metabolite levels within each subject. We 
are assuming a similar effect size for both males and females and are doubling the sample size (n=18, 9 per 
sex) to allow us to subset the data in case we find divergent results between sexes. To account for possible 
loss to follow-up, we will increase sample size from 18 to 24 (n=12 male + 12 female). Power analysis was 
conducted assuming a paired t-test on pre- and post-treatment metabolite levels for three pre-specified 
metabolites. Expected variability of within subject differences in metabolite levels was assessed from a 
previous dataset and the median standard deviation was calculated across all metabolites.1 Power analyses 
were calculated by Dr. Michael Love in the UNC Department of Biostatistics.  
 
6.5 
Interim Analysis 
Given the pre-/post- design of the study, doing an interim analysis in the middle of the study is not feasible, 
for we will have to obtain and compare pre- and post- data. Additionally, the metabolipidomics analyses that 

 
17
we are performing will take several months to complete. Finally, with the small number of subjects, this study 
is not expected to span a long period of time.  
 
7 
STUDY INTERVENTION  
The “SPM Active” supplement will be shipped all at once from Metagenics at room temperature to the UNC 
Family Medicine clinic with expiration that covers the full duration of the intervention. Study participants 
will be instructed to keep the supplement at room temperature. Subjects who have consented to the study 
will receive the exact number of tables required for the study. They will be instructed to consume 4 
capsules per day (2 capsules with breakfast and 2 with dinner) of “SPM Active” for 4 weeks. Each capsule 
contains 145 mg of SPMs for a total daily dose of 580 mg. There are 3 metabolites of the SPM family in the 
capsule, which are 17-HDHA (40mg) + 14-HDHA (80mg) + 18-HEPE (25mg). All three metabolites feed into 
pathways shown to improve insulin resistance and all three metabolites are decreased in obese subjects.15, 
17, 19, 24 The rationale for the dosing and time of intervention is based on the recommendations by the 
manufacturer. Appropriate dosing will be monitored with the follow-up phone calls at weeks 2 and 3. 
Participants who have chosen to stop supplementation, or have failed to follow supplement instructions will 
be withdrawn from the trial.  
 
8 
STUDY INTERVENTION ADMINISTRATION  
No randomization or blinding/unblinding procedures will be used for study.  
 
9 
SAFETY MANAGEMENT 
Safety standards will be overseen by Co-Investigator, Dr. Erik Butler, DO and his designated study 
coordinator. Subjects will be screened for adverse events during two follow-up phone calls at Weeks 1 
and 3 of intervention, as well during last study visit, Week 4. Subjects will be given the study 
coordinator’s phone number to report any Adverse Events or Serious Adverse Events.   All reported 
Adverse Events (AEs) will be given to the co-investigator, Dr. Butler, each week of the study.   
 
Should a Serious Adverse Event (SAE) occur, Dr. Butler, co-investigator, will be immediately notified.  
The subject will be instructed to stop study drug, and seek immediate medical attention.  SAEs 
include but are not limited to bleeding, chest pain/pressure, shortness of breath, weakness, altered 
mental status, vision changes or extreme pain. 
 
A log of all AEs will be maintained throughout the study and will be assigned to each patient. Note 
that this study will not require a Data Safety Monitoring Board (DSMB) due to its small sample size 
and the low risk of study drug. However, Dr. Erik Butler, co-Investigator, will review weekly the 
adverse event reporting log for all subjects. Any clinically significant AEs will be follow-up by Dr. 
Butler and the study coordinator to ensure resolution.  Patients will be further evaluated by Dr. Butler 
or other health care professional for ongoing AEs.   
 
Though highly unlikely, if 50% or more of total study participants report intolerable adverse reactions from 
taking the supplement, we will stop the study.  
 
10 
DATA COLLECTION AND MANAGMENT  
 
Personal identifiers and medical history will be collected following patient recruitment and informed 
consent.  
 

 
18
Data from this study will be kept in electronic and hard copy forms with the respective security measures. 
Electronic data will be kept on an encrypted desktop computer protected by multiple passwords in a lab, 
located on a hall protected by card access in Michael Hooker Research Center, Chapel Hill, NC. De-identified 
data will be stored on REDCap for use by research team personnel only. Data management will be 
performed by the PI and the study coordinators. All data acquisition will meet HIPPA and confidentiality 
standards. Electronic data will be stored for a minimum of five years in the case that this data needs to be 
verified. When deemed appropriate, all files will be purged and permanently deleted from research 
computers. Hard copy data will be shredded. Human biological samples (serum/plasma) will be stored for a 
minimum of five years in the case that these samples are needed for duplication of or verification of study 
results. Please see Part C for further details. These samples will be stored in locked freezers in locked 
research laboratories in secure facilities that require card access. When all analysis of human biological 
samples is completed, all samples will be disposed of according to university and environmental health 
guidelines. Data that links dummy identifiers to any personal identifying information will be shredded and 
disposed of, or, in the case of electronic data, will be permanently deleted. Limited clinical data (glucose, 
insulin, lipid panel) may be part of subjects’ secure electronic medical record in Epic. 
Note that the biological samples and data collected from subjects who are lost to follow-up will be 
handled in this way as well.  
The PIs will be responsible for monitoring and reviewing subject data, lab results, study progress, subject 
safety and confidentiality, and the accuracy and security of the emerging data. They will also be responsible 
for running the assays and entering all data into redcap for statistical analysis. 
 
11 
RECRUITMENT STRATEGY 
Subjects will be recruited at UNC Family Medicine Center at 540 Manning Drive, Chapel Hill, NC 27599. Dr. 
Butler and the study coordinator will be responsible for recruiting subjects. We are using a convenience 
sample method, therefore everyone age 50-65 years will be screened according to the criteria set forth for 
the study. Subjects will be asked questions orally during the primary visit to determine if eligibility criteria 
are met, at which time informed consent and HIPPA authorization will be obtained. Recruitment will take 
place in a private room during a normally scheduled clinical visit. Subjects will be briefed on the scientific 
premise of the study along with details on participation involvement, risks, benefits to the individual, 
benefits to scientific knowledge, ability to refuse participation and/or ability to withdraw participation at any 
time. 
 
12 
CONSENT PROCESS 
Subjects will be asked questions orally during the primary visit to determine if eligibility criteria are met, at 
which time informed consent and HIPPA authorization will be obtained. Subjects will be recruited and 
consented in a private, closed-door room with preoperative or post-operative consent and scripts being 
read by the study coordinator to the potential study subject. Upon full understanding and answering of any 
questions, the study coordinator will obtain written and verbal informed consent along with providing a 
copy of the consent form for the subject to keep themselves. Dr. Butler or the study coordinator will 
consent subjects in a professional and non-coercive manner. 
 
 
 

 
19
13 
PLANS FOR PUBLICATION 
If our hypothesis is correct, data from aims 1 and 2 will provide a solid foundation for an NIH R21 grant. 
The lead PI, after consultation with the others on the investigative team, will communicate with program 
officials to tailor an R21 grant to NIDDK PA-18-104 that would focus on the mechanistic links between 
adipose tissue pro-resolving lipid mediators and lymphocyte metabolism. 
 
14 
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oduction. 
 
15      APPENDIX 
 
Fig. 1: A metabolite of the EPA-derived SPM family is lowered in obese mice. C57BL/6J male mice 
were administered a lean control or high fat diet for 15 weeks. Visceral white adipose tissue and 
liver were subjected to mass spectrometry based metabololipidomics for PUFA-derived mediators. 
For simplicity, we present 18-HEPE. Data are average ± SEM from 5 mice per diet. *p<0.05. 
Experiments with females are underway. Analyses were conducted blinded to ensure strong rigor. 

 
21
 
Fig. 2: Short-term administration of EPA-derived SPM improves fasting glucose/insulin levels of 
obese mice. Male C57BL/6J mice were fed as described in Fig.1. Prior to sacrifice, a cohort of mice 
was i.p. injected daily for 4 days with 300ng resolvin E1 or vehicle control. (A) Body weights, (B) 
fasting glucose, and (C) fasting insulin levels. Data are average + SEM from 11-18 mice per diet. 
**p<0.01, ***p<0.001, ****p<0.0001.