Artigo Lipocina No Leite Humano Associada À Adiposidade Infantil

Brown fat-activating lipokine 12,13-diHOME in human milk is associated with infant adiposity
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Danielle Wolfs, M.P.H.1; Matthew D. Lynes, Ph.D.1; Yu-Hua Tseng, Ph.D.1; Stephanie Pierce, M.D.2;
Valerie Bussberg, B.S.c.3, Abena Darkwah, B.S.c.3, Vladimir Tolstikov, Ph.D.3, Niven R. Narain, Ph.D.3,
Michael C. Rudolph, Ph.D.5, Michael A. Kiebish, Ph.D.3, Ellen W. Demerath, Ph.D.4; David A. Fields,
Ph.D.5; Elvira Isganaitis, M.D., M.P.H.1,6
1. Department of Integrative Physiology and Metabolism, Joslin Diabetes Center, Boston, MA
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2. Department of Obstetrics and Gynecology, University of Oklahoma Health Sciences Center,
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Oklahoma City, OK
3. BERG, Framingham, MA
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4. Division of Epidemiology and Community Health, University of Minnesota School of Public Health,
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Minneapolis, MN
5. Department of Pediatrics, University of Oklahoma Health Sciences Center, Oklahoma City, OK

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6. Department of Pediatrics, Harvard Medical School, Boston, MA
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Corresponding author:
Elvira Isganaitis, M.D., M.P.H.

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Assistant Investigator and Pediatric Endocrinologist

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Joslin Diabetes Center

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One Joslin Place, room 655A

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Boston, MA 02215
Tel: 617-732-2603
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Fax: 617-309-2593
Elvira.Isganaitis@joslin.harvard.edu
Clinical trial registry #: www.clinicaltrials.gov, NCT02535637
© The Author(s) 2020. Published by Oxford University Press on behalf of the Endocrine
Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
Disclosure summary: We would like to thank those who made this study possible. DAF and EWD
are supported by the Eunice Kennedy Shriver National Institute of Child Health & Human
Development of the National Institutes of Health under award number R01HD080444. YT receives

funding from United States Army Medical Research grant W81XWH-17-1-0428 and National
Institutes of Health (NIH) grants: R01DK077097 and R01DK102898. SP receives funding from the
University of Oklahoma College of Medicine Alumni Association. DAF, DW, SP, and EI receive funding
from the Harold Hamm Diabetes Center Team Science Grant. MDL was supported by NIH grant
K01DK111714. DW, MDL, EI, and YT by the DRC award (P30DK036836 –PI: George King). The authors
report no conflicts of interest related to the study. All authors read and approved the final
manuscript.
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ABSTRACT
Context: Little is known about the specific breastmilk components responsible for protective effects
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on infant obesity. Whether 12,13-diHOME, an oxidized linoleic acid metabolite and activator of
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brown fat metabolism, is present in human milk, or linked to infant adiposity, is unknown.
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Objective: To examine associations between concentrations of 12,13-diHOME in human milk and
infant adiposity.
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Design: Prospective cohort study between 2015-2019, following participants from birth to 6-months.
Setting: Academic medical centers.

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Participants: Volunteer sample of 58 exclusively breastfeeding mother-infant pairs; exclusion criteria

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included smoking, gestational diabetes, and health conditions with the potential to influence
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maternal or infant weight gain.

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Main outcome measures: Infant anthropometric measures including weight, length, BMI, and body
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composition at birth and at 1, 3 and 6 months postpartum.

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Results: We report for the first time that 12,13-diHOME is present in human milk. Higher milk 12,13-
diHOME was associated with increased weight-for-length (WFL) Z-score at birth (=0.5742,
p=0.0008), lower infant fat mass at 1-month (p=0.021), and reduced gain in body mass index (BMI)
Z-score from 0 to 6-months (=-0.3997, p=0.025). We observed similar associations between infant
adiposity and milk abundance of related oxidized linoleic acid metabolites 12,13-epOME and 9,10-
diHOME, and metabolites linked to thermogenesis including succinate and lyso-phosphatidylglycerol

18:0. Milk abundance of 12,13-diHOME was not associated with maternal BMI, but was positively
associated with maternal height, milk glucose concentration, and was significantly increased after a

bout of moderate exercise.
Conclusions: We report novel associations between milk abundance of 12,13-diHOME and adiposity
during infancy.
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Keywords: breastmilk, thermogenic metabolites, infant adiposity, brown fat activators, 12,13-
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diHOME, infant metabolism
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INTRODUCTION
Despite efforts to promote breastfeeding as a way to combat childhood obesity, relatively

little is still known about specific components in breastmilk responsible for its protective effects. A
recent analysis uncovered a role for alkylglycerol lipids in human milk as regulators of mitochondrial
metabolism and adipocyte “beige-ing” (1), suggesting that breastfeeding may activate adipocyte
thermogenesis in the neonate. Whether this effect is limited to alkylglycerols, and whether
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additional signaling lipids and metabolites in human milk might also modulate adipocyte metabolism
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or activate brown fat, remains unknown.
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Human milk is rich in lipids, and several signaling lipids, or “lipokines”, have been reported to
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act like hormones to regulate inflammation, insulin sensitivity, and systemic metabolism (2,3). One
recently identified lipokine, 12,13-dihydroxy-9Z-octadecenoic acid (12,13-diHOME), an oxidized

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derivative of linoleic acid, was shown to regulate brown adipose tissue fuel uptake and
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thermogenesis in mice and humans (4). The term “thermogenic metabolite” has been used to
describe the brown-fat activating potential of 12,13-diHOME and other metabolites (e.g. succinate,
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BAIBA, 12-HETE, 12-HEPE, uridine, etc.). Plasma levels of 12,13-diHOME are increased in response to
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cold exposure and acute exercise (4-7) and inversely related to body mass index (BMI), plasma
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triglycerides, insulin, and markers of liver function (4,8). Moreover, 12,13-diHOME in newborn feces
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has been linked to higher risk of asthma and atopic diseases (9). However, this lipid has not
previously been identified in human milk.

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With this analysis, we tested whether 12,13-diHOME is present in human milk, and
examined its associations with infant weight gain, BMI, and body composition during the early
postnatal period. We additionally examined associations between milk abundance of other
thermogenic metabolites and infant adiposity. We report that human milk concentration of 12,13-
diHOME, related oxylipids including 12,13-epOME and 9,10-epOME, the Krebs cycle metabolite
succinate, and lyso-phosphatidylglycerol 18:0 (Lyso-PG 18:0), are inversely associated with infant
adiposity. Our data add to the emerging evidence that human milk contains lipids and metabolites
that can modulate adipocyte metabolism (1) and provide further support for the possibility that

differences in milk composition could be functionally related to early life obesity risk.
METHODS
Mother-infant participants and study procedures.
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This prospective cohort study enrolled 58 mother-infant pairs at Oklahoma University Health
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Sciences Center and University of Minnesota (CTI: NCT02535637). Eligibility requirements included
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intention to breastfeed exclusively from birth to 6-months and a singleton, term pregnancy resulting
in a healthy infant. Exclusion criteria included maternal smoking, gestational or pre-gestational

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diabetes, and any health conditions with the potential to influence maternal weight gain during
pregnancy or infant growth, including but not limited to: hypertension, pre-eclampsia, use of oral
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glucocorticoids, fetal congenital anomalies, etc. Medical records and patient questionnaires provided
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demographic and clinical information. The mother-infant pairs presented to the study site for weight
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measurements and to provide milk samples at 1, 3, and 6-months (± 5 days) post-delivery. Milk
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samples were collected between 08:00 and 10:00 hours, at least 1.5 hours after the last infant
feeding, and with the mother fasted for at least 1 hour. The infant’s weight was measured by a high
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sensitivity scale (Seca 728) and the mothers then breastfed from both breasts (for a full and complete
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milk expression). Upon completion of all testing protocols, the mothers provided a single breast
expression sample by breast pump from whichever side they felt could provide the fullest expression.
The breast milk was collected in 150 mL BPA-free polypropylene vessels, aliquoted, and kept at -80°C
until the time of analysis. The University of Oklahoma Health Sciences Institutional Review Board
approved all the procedures and protocols detailed in this study.

Acute exercise bout.
To test whether milk abundance of 12,13-diHOME is influenced by maternal exercise, we

performed a pilot study of acute exercise in a distinct volunteer sample of sixteen breastfeeding
mother-infant pairs recruited separately at Oklahoma University Health Sciences Center between
2018-2020. The exercise bout was done at 1 month postpartum, and consisted of the mother
walking for three bouts of 10 minutes (with 2-5 minutes rest between each bout) on a treadmill at
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65-75% of heart rate reserve per the Karvonen Formula: [Target heart rate = (max heart rate –
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resting heart rate) x intensity + resting heart rate]. Speed/grade was adjusted to maintain the target
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intensity. Milk samples were collected at baseline and 90 minutes after exercise.
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Infant body composition analysis.
A digital scale (Seca 728) and infantometer (Seca 416) were used to measure the infant’s
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weight and length (crown-to-heel) at 1, 3, and 6 months. Air-displacement plethysmography (ADP,

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COSMED) was performed on infants at their 1-month appointment. At 6-months, a dual-energy x-ray
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absorptiometry (DXA, Lunar scanner, GE Healthcare) device was used to measure infant body
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composition (% fat, total fat and fat-free mass). Skin fold thicknesses were measured at the
abdomen, subscapular, thigh and triceps in duplicate using Harpenden skinfold calipers by the same
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technician (DAF). Further, arm and waist circumferences were taken in duplicate with a tension tape
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measure. The same investigator performed the DXA scans and positioned the infants to minimize
variability.
Assay Methodology
Lipidomic Analysis and 12,13-diHOME Quantification

Concentrations of 12,13-diHOME and other lipokines were measured through liquid
chromatography-tandem mass spectrometry (LC-MS/MS). Breast milk samples were thawed at room
temperature and immediately placed on ice. Aliquots of 200 µL were taken and added to 600 µL of
methanol for a protein crash. Ten microliters of a mixture of 5 deuterated internal standards were
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spiked into the samples, vortexed and stored at -20⁰C overnight. The deuterated standard mixture
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contains d4-9-HODE, d4-9,10-diHOME, d8-5(S)-HETE, d4-LTB4, and d4-PGE2 at a concentration of 100
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ng/mL. Breast milk samples were then subjected to a solid phase extraction. C18 cartridges at
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500mg/6mL (Biotage, Uppsala, Sweden) were conditioned with 10 mL of methanol followed by 10
mL of water. The samples were centrifuged at 14000 g, and the pH of the supernatant of the
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samples was adjusted by adding pH 3.5 water before loading the samples onto the C18 cartridges.
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The cartridges were washed with 5 mL water followed by 5 mL hexanes. Fractions were dried down
under a stream of N2 gas, and reconstituted in 50 µL methanol:water (1:1, by vol). Samples were
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centrifuged at 20000 g and the supernatant was transferred to LC-MS vials for analysis.
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Electrospray ionization (ESI) LC-MS/MS was performed on a TripleTOF 6600 (Sciex,

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Framingham, MA, USA) coupled to an Eksigent Ekspert MicroLC 200 (Sciex, Framingham, MA, USA)
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system with a Synergi Fusion-RP capillary C18 column (150 x 0.5mm, 4 µm; Phenomenex) with a
column oven heated to 40⁰C. Eleven µL of sample was injected at a flow rate of 20 µL/min and were
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separated by reverse phase chromatography with mobile phases A (100% H2O, 0.1% acetic acid) and
B (100% MeOH, 0.1% acetic acid). The gradient started at 60% B for 0.5 minutes, increased to 80% B
by 5 minutes, increased to 95% B by 9 minutes, held at 95% B for 1 minute, and decreased to 60% B
by 12 minutes to equilibrate. Samples were only acquired in negative mode due to the chemical
structure of the targeted lipids. The ESI DuoSpray Ion Source parameter settings were set to: ion
source gas 1 (GS1) at 15, ion source gas 2 (GS2) at 20, curtain gas (CUR) at 20, temperature at 400⁰C,
and ion spray voltage floating (ISVF) at -4500 V. The high-resolution multiple reaction monitoring
(HR-MRM) acquisition method consisted of a TOF MS experiment looped with 31 MS/MS high

sensitivity product ion experiments with 50 ms accumulation time for each scan. The mass
spectrometer acquisition time was set to 10 minutes with a total of 353 cycles per experiment. The
declustering potential (DP) was set to -100 and the collision energy (CE) was set to -30 eV with a
collision energy spread (CES) of 10 eV. Mass shift was controlled by calibrant solution delivered
every 10 samples at 500 µL/min by the atmospheric-pressure chemical ionization (APCI) probe. The
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LC-MS/MS data was acquired by Analyst 1.7 software (Sciex, Framingham, MA, USA) and processed
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with MultiQuant 3.0 (Sciex, Framingham, MA, USA) using a list of 110 targeted oxidized lipid species.
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An external standard calibration curve with eight points between 0.5 pg/µL to 1000 pg/µL
was used for absolute quantification of 12,13-diHOME. Relative quantification was calculated for all
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other species based on the MS2 peak area ratio of the lipid species compared to its internal
standard.
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Metabolomics Analysis
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Samples for metabolomics analysis were prepared as previously described (10-14). Breast
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milk was thawed on ice, vortexed and further aliquoted. 200µL of breast milk was incubated with
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extraction solvent in a 1:2 sample volume to solvent volume ratio at -20°C for 30 minutes.
Metabolite extraction was achieved using a mixture of isopropanol, acetonitrile, and water at a ratio
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of 3:3:2 v/v. Extracts were divided in to three parts: 75 uL for gas chromatography combined with
time-of-flight high-resolution mass spectrometry, 150 uL for reversed-phase liquid chromatography
coupled with high-resolution mass spectrometry, and 150 uL for hydrophilic interaction
chromatography with liquid chromatography and tandem mass-spectrometry, and analyzed as
previously described (14). We used the NEXERA XR UPLC system (Shimadzu, Columbia, MD, USA),
coupled with the Triple Quad 5500 System (AB Sciex, Framingham, MA, USA) to perform hydrophilic
interaction liquid chromatography analysis, NEXERA XR UPLC system (Shimadzu, Columbia, MD,
USA), coupled with the Triple TOF 6600 System (AB Sciex, Framingham, MA, USA) to perform

reversed-phase liquid chromatography analysis, and Agilent 7890B gas chromatograph (Agilent, Palo
Alto, CA, USA) interfaced to a Time-of-Flight Pegasus HT Mass Spectrometer (Leco, St. Joseph, MI,
USA). The GC system was fitted with a Gerstel temperature-programmed injector, cooled injection
system (model CIS 4). An automated liner exchange (ALEX) (Gerstel, Muhlheim an der Ruhr,
Germany) was used to eliminate cross-contamination from the sample matrix that was occurring
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between sample runs. Quality control was performed using metabolite standards mixture and
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pooled samples applying methodology preciously described (15-18). A quality control sample
containing a standard mixture of amino and organic acids purchased from Sigma-Aldrich as certified
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reference material, was injected daily to perform analytical system suitability test and monitor
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recorded signals day to day reproducibility as it was described (10-14). A pooled quality control
sample was obtained by taking an aliquot of the same volume of all samples from the study and
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injected daily with a batch of analyzed samples to determine the optimal dilution of the batch
samples and validate metabolite identification and peak integration. Collected raw data was
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manually inspected, merged, imputed and normalized by the sample median. Metabolite
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identification was performed using in house authentic standards analysis. Metabolite annotation
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was used utilizing recorded retention time and retention indexes, recorded MSn and HRAMSn data
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matching with METLIN, NIST MS, Wiley Registry of Mass Spectral Data, HMDB, MassBank of North
America, MassBank Europe, Golm Metabolome Database, SCIEX Accurate Mass Metabolite Spectral
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Library, MzCloud, and IDEOM databases.

Statistical Methods
Of the 58 mother-infant pairs participating in this study, 57 were included in the final

analysis. One pair was excluded due to implausible measurements and missing data. Tables 1 and 2
include details on the variables of interest included in the analysis. Excessive weight gain was
calculated based on the current Institute of Medicine recommendations on weight gain during
pregnancy based on pre-gestational BMI (19). Infant birth weight was classified as low birth weight
(LBW, <2,500 g), normal birth weight (2,500-4,000 g), or macrosomia (>4,000 g). We calculated
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BMI Z-score using the zscorer program in RStudio based on World Health Organization (WHO) BMI
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for age reference ranges. Z-scores for weight-for-length (WFL), weight-for-age (WFA), and length-
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for-age (LFA) were calculated based on WHO 2006 growth charts.
Exploratory analyses by tertile

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Given that there were no prior reports on 12,13-diHOME and related metabolites in breast
milk in relation to biologic outcomes, we examined relationships between tertiles of 12,13-diHOME

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and demographic and clinical characteristics. We report the actual concentrations of 12,13-diHOME
in ng/mL for the tertiles and log transformed the area ratio for the statistical analyses. Our decision to
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include specific infant and maternal characteristics in the modeling was based on prior literature of
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human milk composition and infant weight status (20). We adjusted for maternal characteristics
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including pre-gravid BMI, gestational weight gain (GWG), and parity, and for infant sex and
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gestational age. Simple and multiple linear regressions were performed to examine the relationship
between infant BMI Z-scores at birth, modeling variables, and the log of the metabolites. We
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additionally examined associations with the following metabolites and lipids in breast milk: AMP,
BAIBA, 12-HEPE, 12-HETE, uridine, Lyso-PG 18:0 and succinate. These metabolites and lipids
were selected a priori using a candidate approach based on recent publications documenting their
effects on adipocyte metabolism and/or thermogenesis (2,6,8,21-23). Finally, we also performed
principal component and partial least square analyses using MetaboAnalyst to examine associations
between infant adiposity and the breast milk lipidome (including 94 signaling lipids and 669 structural
lipids). Infants (n=57) were grouped into tertiles of adiposity at 1 month, with adiposity classified as
0 = tertile 1 (3.4-14.29 percent body fat), 1 = tertile 2 (14.3 – 19.09 percent), and 2 = tertile 3 (19.1 –
29 percent). Principal component analysis was also used to examine differences between the lipidome

of breast milk and two brands of commercial infant formula.
Analyses were performed using RStudio with a significance level of  = 0.05. P-values for the
ANOVA, Chi-Square, or Kruskal-Wallis tests for differences between the tertiles were included in
Tables 1 and 2. Results are presented as mean and standard deviation, statistical estimates, or
number and proportion.
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RESULTS
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Demographic and clinical characteristics according to milk 12,13-diHOME
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We first examined maternal and infant characteristics according to abundance of 12,13-
diHOME in milk at 1-month postpartum. Milk 12,13-diHOME concentrations ranged from 0.30 ng/mL
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to 53.72 ng/mL; tertiles 1, 2, and 3 included concentrations from 0.30 ng/mL to 3.50 ng/mL, 3.50
ng/mL to 7.38 ng/mL, and 7.38 ng/mL to 53.72 ng/mL, respectively. Milk 12,13-diHOME was
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positively correlated with maternal height; the highest tertile for 12,13-diHOME abundance had the
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highest mean maternal height (166.6 cm in tertile 3, vs. 161.5 cm in tertile 1, p=0.017). There were
significant differences in milk glucose between the top and bottom tertiles of 12,13-diHOME (tertile
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3: 36.2 mg/dl vs. tertile 1: 26.18 mg/dl, p=0.009), which was not seen for other milk analytes
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including leptin, insulin, Il-6, or CRP (Supplement Table 1, Supplement Figure 1A) (24). 12,13-
diHOME was not, however, associated with maternal age, parity, weight, pre-pregnancy BMI, or
gestational weight gain (Table 1). We also did not observe any seasonal variation in milk 12,13-
diHOME levels, nor did the levels differ between the study the Oklahoma and Minnesota study sites
(data not shown).

Abundance of 12,13-diHOME in breast milk was associated with several infant
anthropometric measures (Table 2). Higher milk 12,13-diHOME was associated with higher BMI Z-

score at birth (tertile 3: 0.32 vs. tertile 1: -0.60, p=0.006, Figure 1A) and WFL Z-score at birth (tertile
3: WFL Z-score 0.14 vs. tertile 1: -1.08, p=0.005). Milk abundance of 12,13-diHOME was not
associated with infant gestational age, birth weight or length, or sex. By 1-month postpartum,
higher milk abundance of 12,13-diHOME was associated with lower subcutaneous fat in the infant.
Triceps skin fold thickness (p=0.0192), thigh skin fold thickness (p=0.0147), and arm circumference
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(p=0.0401) showed significant differences between the tertiles with the same inverse pattern: as
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12,13-diHOME abundance increased (per tertile) the subcutaneous adiposity measures decreased
accordingly. Similarly, whole-body adiposity at 1-month postpartum (assessed by air displacement
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plethysmography) tended to be reduced in the highest tertile of 12,13-diHOME (tertile 3: 15.32 %fat
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vs. tertile 1: 18.61 %fat, p=0.077, Figure 1B). 12,13-diHOME was one of the few signaling lipids that
tracked with tertile of infant adiposity; principal components analysis of the 94 signaling lipids and
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669 structural lipids did not reveal strong clustering (Supplement Figure 2) (24). Significant
differences in infant whole-body adiposity between the tertiles emerged after adjustments for infant
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sex, gestational age, maternal pregravid BMI, gestational weight gain and parity (adjusted fat mass
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in tertile 3: 14.52 %fat vs. tertile 1: 18.59 %fat, p=0.021).

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We also detected 12,13-diHOME in two brands of cow’s milk based commercial infant
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formula. Levels of 12,13-diHOME in these formulas was comparable to the upper tertile of the
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concentrations found in human breast milk; principal component analysis suggests that the lipidome
of breast milk is distinct from that of infant formula (Supplement Figure 3) (24). We did not,
however, evaluate whether 12,13-diHOME levels in commercial formula was associated with infant
growth as breastfeeding mother and infant dyads were the primary focus of our study.
Human milk 12,13-diHOME is associated with WFL Z-score at birth independent of maternal and
infant characteristics

Abundance of 12,13-diHOME in milk at 1-month was positively associated with WFL Z-score
at birth (= 0.5742, p=0.0008). We next tested whether there was an independent association
between milk 12,13-diHOME abundance and WFL Z-score at birth, adjusting for maternal and infant
covariates. Milk 12,13-diHOME remained significantly associated with WFL Z-score at birth after
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adjustment for maternal parity, pregravid BMI, and GWG (= 0.559, p=0.0018). The association was
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attenuated but remained significant upon further adjustment for infant sex, gestational age, and age
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in days at the 1-month visit (= 0.440, p= 0.0249). Thus, milk abundance of 12,13-diHOME is
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independently associated with infant WFL Z-score at birth.
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Milk 12,13-diHOME is associated with change in infant BMI and weight-for-length (WFL) from 0-6
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months
We next asked whether abundance of 12,13-diHOME in milk was associated with gain in
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adiposity, assessed by BMI and WFL Z-scores, during the early postnatal period. We observed a
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significant negative association between log[12,13-diHOME] in milk and change in BMI Z-score over
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the first 6 months (= -0.400, p=0.0252, Figure 1C). Similarly, we observed a significant negative
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association between log[12,13-diHOME] in milk and change in WFL Z-score over the first 6-months
(= -0.534, p=0.0112, Figure 1F). This effect remained significant after adjustment for maternal
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parity, pregravid BMI, and gestational weight gain (= -0.554, p=0.0131). The association between
milk 12,13-diHOME and change in WFL Z-score from 0-6 months also remained statistically
significant after additional adjustment for infant sex, gestational age, and age at the 1-month visit
(= -0.522, p=0.0374). However, further adjustment for 1-month milk glucose concentration
attenuated the effect (= -0.439, p=0.116).

Ephx1-4 metabolites are associated with infant weight status and adiposity

We next examined whether related metabolites were similarly associated with infant weight
status and adiposity. The enzymes Ephx1-4 synthesize the isomeric diols 12,13-diHOME and 9,10-
diHOME from linoleic acid via 12,13-epOME or 9,10-epOME by cytochrome P450 (Figure 2A) (4).
Thus, we examined associations between infant weight status and milk abundance of 12,13-epOME
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and 9,10-diHOME. Both metabolites have significant, positive associations with the infant’s BMI Z-
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score at birth (Figure 2B, E). Log[12,13-diHOME] has the largest positive correlation with BMI Z-
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scores at birth (r=0.392, p=0.004), followed by log[9,10-diHOME] (r=0.376, p=0.007) and log[12,13-
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epOME] (r=0.309, p=0.026). Log[9,10-diHOME] was positively correlated with 1-month breast milk
glucose concentration (Supplement Figure 1B) (24).

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We next examined associations between 12,13-diHOME and related metabolites in milk at 1-
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month postpartum with early infancy obesity. The significant inverse correlation between change in
WFL Z-score from 0-6 months and the metabolites was also seen with log[9,10-diHOME] (r= -0.307,
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p=0.029, Figure 2D), but was weaker with log[12,13-epOME] (r= -0.261, p=0.064, Figure 2G).
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Trajectories for the WFL Z-score measures at birth, 1 month, 3 months, and 6 months, by 12,13-
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diHOME tertile were created, along with weight for age Z-score and length for age Z-score (Figure
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1D-F). Similar growth trajectories were seen for 9,10-diHOME and 12,13-epOME (Figure 2C, F).
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Associations of thermogenic metabolites in milk and infant weight status
Given these associations between milk abundance of 12,13-diHOME, related metabolites,
and infant BMI, we examined whether other signaling lipids and metabolites that have recently been
linked to brown adipose tissue activation (AMP, BAIBA, 12-HEPE, 12-HETE, uridine, Lyso-PG 18:0)
were associated with weight status in infancy (2,6,20-22). We analyzed the unadjusted correlation
between these metabolites and 1-month body fat percentage, of which only Lyso-PG 18:0 was
significantly associated. Log[Lyso-PG 18:0] was negatively associated with 1-month body fat
percentage (r= -0.311, p= 0.021, Supplement Figure 4A) (24). We next analyzed the unadjusted

linear association between these metabolites and 6-month BMI Z-score, of which only succinate was
significantly associated. We saw a negative linear association between succinate and 6-month BMI Z-
score (= -5.25, r= -0.301, p=0.028, Supplement Figure 4B) (24). We applied the same modeling as in
the 12,13-diHOME analysis to succinate and the significant association was retained when adjusting
for maternal characteristics (p=0.0416), but attenuated on adjustment for both infant and maternal
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characteristics (p=0.102). When we examined associations between the overall breast milk
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metabolome at 1-month postpartum and infant adiposity (% fat mass, assessed by ADP) in the
current study, we found that 26 milk metabolites were associated with infant adiposity at a nominal
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P<0.05. We found that similar metabolite classes were affected as in our prior analyses (25).
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Reproducing our prior findings, we again observed a positive correlation between breast milk purine
nucleotides (e.g. 1-methyladenosine, 7-methylguanine) and infant adiposity, which is intriguing in

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light of data implicating purinergic receptors in the regulation of adipogenesis (Supplement Table 2)
(24, 26).
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Association between 12,13-diHOME concentrations and acute exercise

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Plasma 12,13-diHOME levels differ between sedentary and active individuals, and have been
reported to be increased by acute exercise (4,5,7). We therefore tested, in a separate sample of 16

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women for proof of concept, whether an acute bout of moderate exercise in the postpartum period
(4 weeks postpartum) may influence milk 12,13-diHOME abundance. Of the 16 participants, 8 were
normal weight and 8 were obese. We observed an increase in 12,13-diHOME breastmilk
concentrations in most subjects at 90-minutes post-acute exercise (Figure 3). On average, the total
sample saw a 1.39-fold increase in 12,13-diHOME concentrations at 90-minutes post-acute exercise

(paired T-test p = 0.0076), with obese women having a 1.73-fold increase and normal women having
a 1.50-fold increase.

DISCUSSION
In this analysis, we identify novel associations between levels of 12,13-diHOME and related
thermogenic metabolites in human breastmilk, and weight status and adiposity in infancy. We found
that 12,13-diHOME in milk was positively associated with BMI Z-score at birth, negatively associated
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with fat mass and skinfold thicknesses at 1-month, and negatively associated with change in BMI
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from 0-6 months. Similar associations were seen for other metabolites in the same biosynthetic
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pathway, including 9,10-diHOME and 12,13-epOME. Maternal acute exercise may regulate 12,13-
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diHOME abundance in milk, as evidenced by a significant increase in concentration of this lipid after
a bout of moderate exercise at 1 month postpartum. Moreover, we also observed an association

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between milk levels of succinate, which has recently been linked to brown adipose tissue activation
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in humans, and lower infant BMI at 6-months. We similarly observed an inverse association between
milk Lyso-PG 18:0 and infant body fat at 1-month. Together, our data suggest that milk levels of
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brown fat activating metabolites may play a role in weight gain and adiposity in infancy.
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Our analysis adds to the growing evidence that differences in human milk composition are
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associated with infant weight gain. Breastmilk is not only a source of nutrition, but also contains
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numerous non-nutritive bioactive compounds – including hormones, cytokines, nucleotides, cells,
microbiota, and exosomes – that may influence signaling and developmental processes in the infant
Ac
and may contribute to obesity risk. Breastfeeding provides many health benefits for mother and
child, but there is some controversy regarding effects on childhood obesity risk (27-29). In a meta-
analysis of formula versus breast feeding, a small protective effect was found on adult BMI (30).
However, in many studies the associations between breastfeeding and obesity in children are
weakened when adjusting for maternal education, socioeconomic status, obesity, and smoking (31).
Moreover, a randomized control trial of a breastfeeding promotion intervention in Belarus found a

slightly increased adolescent BMI in the intervention group (32). These inconsistencies could be
explained by the variation of certain constituents in milk composition between individuals.

Consistent with this possibility, untargeted metabolomics analyses in breastmilk have recently
identified associations among maternal obesity, longitudinal changes in the milk metabolome, and
fat accrual during infancy (25,33). We previously reported that abundance of human milk
nucleotides and oligosaccharides is related to maternal and infant BMI and infant fat mass, and that
milk insulin and leptin are inversely associated with infant weight gain (25,34). Based on the current
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analysis, we propose that breastmilk 12,13-diHOME, Lyso-PG 18:0, and succinate may similarly have
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protective effects on obesity in offspring. Recent analyses by other groups have also described
associations between breast milk content of three dihomo-γ-linolenic acid (DGLA)-derived oxylipins
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(15S-HpEDE and two deoxy-dimethyl-PGE2) and faster growth of preterm infants (35), suggesting an
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important influence of the breast milk lipidome on postnatal growth trajectories.
We are not aware of prior reports of 12,13-diHOME in human milk, nor of its association
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with infant weight gain or adiposity. Prior studies have shown that 12,13-diHOME is increased in
plasma following cold exposure and acute exercise, and that 12,13-diHOME increases fatty acid
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uptake via FATP1 and CD36 in brown adipocytes (4). Beyond 12,13-diHOME and other metabolites,
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additional stimuli such as cold exposure, norepinephrine, FGF21, and cytokines have been described
to induce “beige-ing”, or the acquisition of a brown-like phenotype (i.e., upregulation of

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thermogenesis, mitochondrial biogenesis) by white adipocytes in adults. By contrast, infants are

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born with a predominance of beige-like cells in their adipose depots, which are progressively
replaced with white adipocytes (36); the extent to which beige adipocytes are replaced with white
adipocytes is associated with childhood obesity risk (37). However, it is unclear to what extent 12,13-
diHOME may play a role in adipose tissue remodeling in infancy. This signaling lipid has, however,
been reported in infant feces, where it is produced by the gut microbiota, decreases numbers of T-
regulatory cells and may increase risk of asthma and atopic diseases (9). (We did not have access to
information about asthma diagnoses in the infants in our study.) Based on these data, it is unclear
whether observed associations between higher 12,13-diHOME and lower BMI gain might reflect
increased inflammation/atopy versus changes in adipocyte function. Some emerging data suggest

that thermogenesis and inflammation may be closely interrelated processes: for example, short-
term cold exposure in adults induces CD4 T-cell differentiation into anti-inflammatory T-regulator
cells (38) and cytokines such as TGF-beta and IL-6 may activate adipocyte thermogenesis (39, 40).
On the other hand, pro-inflammatory cytokine secretion and macrophage infiltration may also
impair the development of beige adipocytes (41) and increased expression of immune mediators
t
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(e.g. IL-1b, TNF-a, and MCP-1) in brown fat inhibits UCP1 expression and activity (42-44).
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It is unclear to what extent 12,13-diHOME levels in breast milk reflect the infant’s exposure
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to this lipid in early life. Beyond breast milk and the gut microbiome, there are several other
potential sources of exposure to 12,13-diHOME in the infant, including cow’s milk (45), infant
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formulas (Supplement Figure 3) (24), and endogenous synthesis by leukocytes and other tissues that
express Ephx 1-4. Vernix caseosa, the creamy protective film that covers the newborn’s skin at birth,
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has also been reported to contain 12,13-diHOME and other oxylipins: levels of 12,13-diHOME (and
related oxylipins 12,13-epOME and 9,10-epOME) in vernix were associated with maternal
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“anthroposophic” lifestyle (e.g. vegetarian or organic diet, home birth) (4,46). Future studies
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examining associations between infant adiposity and 12,13-diHOME in milk, in maternal and infant
plasma, and in tissues, will be essential.

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Although we identified associations between 12,13-diHOME in milk and infant adiposity, we

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did not directly test whether this oxylipin play a causal role in adipogenesis or adipose tissue
development. There are no prior reports on 12,13-diHOME’s role in adipocyte differentiation.
However, the related oxylipin 9,10-diHOME has been shown to activate PPAR-gamma, the
transcriptional “master regulator” of adipogenesis, and to promote accumulation of lipid droplets in
a mesenchymal cell line (U-33/γ2 cells), while inhibiting osteoblast differentiation, suggesting that
lipid metabolites in this pathway could play a direct in adipocyte differentiation (47). Based on the
observation that 12,13-diHOME increased fatty acid uptake and upregulated the fatty transporters
FATP1 and CD36 to the cell membrane in brown adipocytes (4), one might speculate that 12,13-

diHOME could similarly promote fatty acid uptake and lipid accumulation during the final stages of
adipogenesis. On the other hand, expression and activity of Ephx2 or soluble epoxide hydrolase
(sEH), one of the enzymes that can synthesize 12,13-diHOME, is upregulated during adipogenesis
(48, 49), and inhibition of sEH using a chemical inhibitor (AUDA, 12-(3-adamantan-1-yl-ureido)-
dodecanoic acid) was reported to decrease adipogenesis in mesenchymal stem cells (50). Moreover,
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inhibition of sEH using AUDA or siRNA in mature white adipocytes resulted in a decrease in
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adipocyte size, increased adiponectin expression, and upregulation of mitochondrial and
thermogenic genes, consistent with a shift to a “beige-like” phenotype (51). Thus, it remains unclear
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whether exposure to 12,13-diHOME or other products of epoxide hydrolase would have a net
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positive or negative effect on adipogenesis in infancy. Additional experiments using animal models
and/or cell lines will be essential to test what role 12,13-diHOME may play in adipocyte
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differentiation.
It is also unclear based on our data to what extent differences in maternal adipose tissue
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metabolism may account for the observed association between 12,13-diHOME in milk and infant
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adiposity. Plasma levels of 12,13-diHOME are thought to be increased with greater adipose tissue
lipolysis, as they have been reported correlate closely with plasma non-esterified fatty acid levels
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(NEFA), and with plasma beta-hydroxybutyrate, reflecting greater availability of NEFA for hepatic
Ac
fatty acid oxidation (8). Our observation that a bout of moderate exercise results in increased 12,13-
diHOME in milk would support this possibility, as exercise is a stimulus for lipolysis (52). Thus, the
association between higher milk 12,13-diHOME and lower infant adiposity could reflect increased
maternal lipolysis and an inherited increased tendency for lipolysis in the infant. Alternatively, given
the observation that plasma 12,13-diHOME levels are higher in active vs. sedentary individuals (5),
another possibility is that maternal fitness level differs between women with high and low milk
12,13-diHOME levels, and that maternal cardiovascular fitness might underlie the association
between milk 12,13-diHOME and infant adiposity. Indeed, a recent study reported that maternal
activity level, assessed by weekly step counts, was associated with breastmilk abundance of a

specific human milk oligosaccharide (3’-sialyllactose) that conferred protection from high-fat diet
induced obesity in offspring mice (53). However, causal relationships between maternal exercise or
lipolysis, breastmilk 12,13-diHOME, and infant metabolic health, will need to be established in future
studies using in vitro systems or experimental models.
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Another key finding from this analysis was a novel association between higher milk succinate
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levels and lower infant BMI at age 6-months. Succinate was reported to be altered in milk from
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mothers with inflammatory bowel disease (versus healthy controls) (54). Levels of succinate in
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neonatal fecal samples were also reported to be modulated by milk fat globule membrane (MFGM)
(55) and by sialylated milk oligosaccharides (56). Increased fecal succinate levels induced TH2
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immune responses in intestinal cells and was associated with changes in bone formation (56).
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Some strengths of this analysis are that this is the first study to do a comprehensive analysis
of 12,13-diHOME in milk, in association with multiple infant adiposity measurements over time.
d
Moreover, we were able to assess other milk components in the same biosynthetic pathway, as well
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as additional metabolites recently linked to brown adipose tissue function. We also identify a
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potential role of acute exercise as a regulator of 12,13-diHOME abundance in milk. We acknowledge

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several limitations in this analysis. First, we did not determine whether 12,13-diHOME directly
affects infant adipogenesis, nor did we examine potential influences of maternal adipocyte
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metabolism on milk composition. Second, our sample size was relatively small, limiting the power.
Our findings will thus need to be replicated in independent populations. Third, our analyses are
cross-sectional and cannot be used to infer causality. Longitudinal studies would be important to
determine whether breastmilk 12,13-diHOME or other lipokines may program contribute to future
childhood obesity, metabolic syndrome, neurologic outcomes (57), or atopy. Fourth, we did not
assess levels of additional breastmilk constituents that may influence brown adipose tissue function,
such as alkylglycerol lipids (1) and hormones including FGF21 and irisin (58). Finally, we did not
measure the plasma levels of the metabolites that were measured in breast milk, and thus cannot

infer whether these substances might be preferentially secreted into milk, or whether they simply
reflect levels in the mother’s circulation. These will be important areas for future studies.
In conclusion, 12,13-diHOME and its metabolites show significant associations with infant
adiposity. Higher 12,13-diHOME concentration in milk was associated with higher BMI Z-score at
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birth, but lower subcutaneous and overall adiposity at 1 month, and lower gains in WFL and BMI Z-
ip
scores from 0-6 months. Milk 12,13-diHOME was significantly increased following a maternal
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exercise bout. Further research is needed to determine whether milk 12,13-diHOME may play a
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causative role in infant weight gain or adipocyte metabolism.
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Acknowledgements
The author’s responsibilities were as follows – DAF and EWD: designed the study and led the

clinical assessments; EI: designed the analysis, interpreted the data, and wrote the manuscript; DW:
analyzed the data, created the figures and table, and assisted in writing the manuscript; MDL:
interpreted data, advised on study design, and assisted with preparation of figures; YT: interpreted
data and advised on study design; MR and SP: assisted with the analysis of maternal exercise; VB,
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AD, VT, NN, and MAK: performed metabolomics and lipidomics assays.
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Data Availability Statement
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Some data generated or analyzed during this study are included in this published article, and
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in Supplemental Figures and Tables that can be accessed through the data repositories listed in
References. The full datasets generated during the current study are not publicly available, but are
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available from the corresponding author on reasonable request.

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58. Lee P, Linderman JD, Smith S, et al. Irisin and FGF21 Are Cold-Induced Endocrine Activators
M
of Brown Fat Function in Humans. Cell Metab. 2014;19(2):302-309. doi:10.1016/j.cmet.2013.12.017

e d
pt
ce
Ac
Table 1. Maternal and Infant Characteristics.
Maternal and infant characteristics by 12,13-diHOME tertile. P-values refer to ANOVA (continuous

variables) or Chi-Square tests (categorical variables) for differences between the tertiles. Mean ± SD
for continuous variables or n (%) for categorical.
Overall N Tertile 1 Tertile 2 Tertile 3 P-Value
Maternal Age, yr 30.39 ± 4.36 56 29.84 ± 4.60 31.50 ± 4.77 30.17 ± 3.59 0.808
t
ip
Maternal Height, 164.69 ± 161.46 ± 166.62 ±
cr
57 165.80 ± 7.50 0.017
cm 6.67 5.58 5.95
Maternal
kg
Weight, 71.07
13.82
±
57
66.69
11.87
±
us
75.08 ± 14.80
69.36
11.00
±
0.516
an
Maternal Pregravid
M
26.21 ± 4.73 57 25.55 ± 4.25 27.29 ± 4.77 25.12 ± 4.42 0.298

2a
BMI, kg/m
d
Maternal GWG, kg 12.65 ± 7.13 57 11.81 ± 4.24 12.68 ± 9.52 13.47 ± 7.15 0.489
e
Excessive Weight
pt
- 21 8 (38.1) 5 (23.8) 8 (38.1)

Gain, kg
ce
Maternal Change in
Ac
weight from Birth -8.57 ± 6.02 55 -9.50 ± 3.48 -8.26 ± 9.44 -7.89 ± 3.40 0.278
to 1moa
Maternal Parity: - 57 - - - 0.652
0 30 (52.6) 10 (52.6) 9 (47.4) 10 (55.5)

1 19 (33.3) 7 (36.8) 6 (31.6) 6 (33.3)
2 7 (12.3) 2 (10.6) 4 (21.0) 1 (5.6)

3 1 (1.8) 0 (0.0) 0 (0.0) 1 (5.6)
Household Income - 43 - - - 0.260
1 4 (9.3) 3 (16.7) 0 (0.0) 1 (8.3)
t
ip
2 8 (18.6) 4 (22.2) 2 (15.4) 2 (16.7)
cr
3 13 (30.2) 7 (38.9) 4 (30.8) 2 (16.7)
4 11 (25.6) 1 (5.5)
us
6 (46.1) 4 (33.3)
an
5 7 (16.3) 3 (16.7) 1 (7.7) 3 (25.0)
Maternal Education - 43 - - - 0.667

M
2 4 (9.3) 3 (16.7) 1 (7.6) 0 (0.0)

d
3 5 (11.6) 2 (11.1) 2 (15.4) 1 (8.3)

e
pt
4 18 (41.9) 8 (44.4) 5 (38.5) 5 (41.7)

ce
5 13 (30.2) 3 (16.7) 5 (38.5) 5 (41.7)

Ac
6 3 (7.0) 2 (11.1) 0 (0.0) 1 (8.3)
Infant Sex: - 56 - - - 0.401
Female 24 (0.43) 9 (47.4) 6 (31.6) 9 (52.9)
Male 32 (0.57) 10 (52.6) 13 (68.4) 8 (47.1)

Birth Weight, ga 3577 ± 423 57 3435 ± 423 3667 ± 427 3653 ± 398 0.214
LGA 9 (0.16) 2 (0.04) 4 (0.07) 3 (0.05)

Gestational Age, 39.74 ± 1.23 57 39.51 ± 1.42 39.56 ± 1.28 40.20 ± 0.90 0.220
wka
Birth Length, cma 51.52 ± 2.11 54 51.61 ± 2.19 51.94 ± 2.12 51.12 ± 2.06 0.593
t
Footnotes:
ip
a
Kruskal-Wallis Test
cr
us
an
M
e d
pt
ce
Ac
Table 2. Infant Body Composition.
Infant body composition by 12,13-diHOME tertile. P-values refer to ANOVA (continuous variables)

or Chi-Square tests (categorical variables) for differences between the tertiles. Mean ± SD for
continuous variables or n (%) for categorical.
Overall N Tertile 1 Tertile 2 Tertile 3 P-Value
t
ip
-0.60 ± 0.01 ± 0.32 ±
Birth BMI Z-Score -0.09 ± 0.99 57 0.006
cr
1.18 0.81 0.81
us
-0.04 ± 0.21 ±
1mo BMI Z-Score 0.21 ± 1.08 56 0.59 ± 0.97 0.256
1.06 1.04
an
0.16 ± 0.31 ±
6mo BMI Z-Score 0.26 ± 0.94 54 0.37 ± 0.84 0.827
M
0.95 1.08
-1.08 ± -0.47 ± 0.14 ±

d
Birth WFL Z-Score -0.46 ± 1.32 54 0.005

1.52 1.06 1.15
e
pt
-0.14 ± 0.14 ±
1mo WFL Z-Score 0.16 ± 1.13 57 0.63 ± 0.99 0.164
ce
0.99 1.20
-0.15 ± -0.02 ±
Ac
3mo WFL Z-Scorea -0.04 ± 0.98 56 0.10 ± 0.91 0.672

0.85 1.21
0.25 ± 0.44 ±
6mo WFL Z-Score 0.36 ± 0.93 55 0.46 ± 0.83 0.929
0.93 1.06
1mo ADP Fat % 16.74 ± 56 18.61 ± 16.49 ± 15.32 ± 0.077

5.62 4.83 5.76 5.98
0.77 ± 0.71 ±

1mo ADP Fat Mass (kg) 0.78 ± 0.32 56 0.89 ± 0.31 0.079
0.31 0.32
1mo ADP Fat Free Mass 3.80 ± 3.83 ±

3.81 ± 0.42 56 3.83 ± 0.46 0.993
(kg) 0.43 0.38
34.38 ± 34.24 ± 34.77 ± 34.39 ±
t
6mo DXA Fat % 54 0.886
ip
3.32 3.06 3.27 3.66
cr
2.86 ± 2.82 ±
6mo DXA Fat Mass 2.86 ± 0.53 54 2.93 ±0.55 0.549
us
0.47 0.61
5.33 ± 5.32 ±
an
6mo DXA Fat Free Mass 5.41 ± 0.53 54 5.56 ± 0.46 0.173
0.38 0.71
M
Triceps Skin Fold - - - - - -

d
6.36 ± 6.33 ±
1 month 7.08 ± 2.28 44 8.16 ± 2.45 0.019
e
2.28 1.43
pt
7.96 ± 8.52 ±
ce
3 month 8.08 ± 2.39 55 7.77 ± 2.35 0.354

2.32 2.57
Ac
10.11 ± 9.45 ±
6 month 9.59 ± 2.72 55 9.17 ± 3.41 0.76
2.38 2.31
Thigh Skin Fold
11.69 ± 12.90 ± 11.79 ± 9.90 ±

1 month 44 0.015
3.41 3.27 4.06 2.08
3 month 16.71 ± 17.03 ± 17.20 ± 15.86 ±
55 0.403
4.14 4.21 4.03 4.30

6 month 20.50 ± 20.94 ± 20.60 ± 19.94 ±
53 0.551
4.85 5.02 5.21 4.49
Abdominal Skin Fold - - - - - -
1 month 6.79 ± 6.67 ±
t
7.13 ± 2.01 44 7.71 ± 2.20 0.139
ip
2.25 1.28
cr
3 month 8.94 ± 9.45 ±
9.19 ± 2.81 55 9.19 ± 2.78 0.783
us
2.72 3.06
9.57 ± 9.27 ±
an
6 month
9.56 ± 2.57 55 9.86 ± 2.83 0.497
2.61 2.37
M
Abdominal Circumference - - - - - -
d
1 month 39.11 ± 39.83 ± 39.36 ± 37.90 ±

43 0.061
e
2.75 2.69 3.26 1.93

pt
3 month 41.95 ± 42.45 ± 41.81 ± 41.60 ±

ce
55 0.426
3.16 3.21 3.55 2.75
Ac
6 month 43.51 ± 44.34 ± 43.32 ± 42.88 ±

55 0.143
2.97 3.22 2.72 2.95
Arm Circumference - - - - - -
1 month 12.16 ± 12.54 ± 12.17 ± 11.65 ±

43 0.040
1.18 1.22 1.39 0.69
0.255
0.086
t
ip
±
cr
± 13.60
± 14.58
1.68
1.48
us
± 13.50
± 14.72
1.21
1.26
an
14.15
15.31
1.37
0.97
M
55
55
d
±
e
pt
13.74
14.87
1.43
1.27
ce
Kruskal-Wallis test
Ac
Footnotes:
3 month
6 month
a
Figure Legends
Figure 1. Associations of milk 12,13-diHOME and anthropometry measures in infancy. A: Linear

Association between Log(12,13-diHOME) in milk and BMI Z-score at birth, P=0.005. B: Infant
adiposity (% fat assessed by ADP) at 1 month by milk 12,13-diHOME tertile. P = 0.077; P(adjusted) =
0.021 (covariates: infant sex, gestational age, maternal pregravid BMI, gestational weight gain and
parity) C: Linear Association between Milk log(12,13-diHOME) and change in infant BMI Z-score
t
from 0 to 6 months, P=0.025. D: Change in infant weight-for-age (WFA) Z-score according to tertile
ip
of milk 12,13-diHOME. E: Change in infant length-for-age (LFA) Z-score according to tertile of milk
cr
12,13-diHOME. F: Change in infant weight-for-length (WFL) Z-score according to tertile of milk
us
12,13-diHOME. * denotes P<0.05, ** denotes P<0.01.
an
Figure 2. Associations of milk Ephx1-4 metabolites and anthropometry measures in infancy. A:
M
Biosynthetic pathway for 12,13-diHOME. B: Linear Association between Log(9,10-diHOME) in milk
and infant BMI Z-score at birth, P=0.007. C: Change in WFL Z-score from 0-6 months according to
d
tertile of log(9,10-diHOME) in milk. D: Linear Association between Milk log(9,10-diHOME) and

e
change in WFL Z-score from 0 to 6 months, P=0.029. E: Linear Association between Log(12,13-
pt
epOME) in milk and infant BMI Z-score at birth, P=0.026. F: Change in WFL Z-score from 0-6 months
ce
according to tertile of log(12,13-epOME) in milk. G: Linear Association between Milk log(12,13-
epOME) and change in WFL Z-score from 0 to 6 months, P=0.064. * denotes P<0.05, ** denotes
Ac
P<0.01.
Figure 3. Breast milk 12,13-diHOME concentration rises after a moderate exercise bout. Change in
12,13-diHOME concentrations at baseline and 90 minutes after a bout of moderate exercise at 1 mo.
post-partum. N=16.**P = 0.0076, 2-sided paired T-test, pre- vs. post-exercise.

ip
cr
Figure 1
us
an
M
ed
pt
ce
Ac
ip
cr
Figure 2
us
an
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ed
pt
ce
Ac
ip
cr
Figure 3
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an
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ed
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Artigo Lipocina No Leite Humano Associada À Adiposidade Infantil

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Artigo Lipocina No Leite Humano Associada À Adiposidade Infantil

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Brown fat-activating lipokine 12,13-diHOME in human milk is associated with infant adiposity

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1. Department of Integrative Physiology and Metabolism, Joslin Diabetes Center, Boston, MA

5. Department of Pediatrics, University of Oklahoma Health Sciences Center, Oklahoma City, OK

Elvira Isganaitis, M.D., M.P.H.

Assistant Investigator and Pediatric Endocrinologist

Joslin Diabetes Center

One Joslin Place, room 655A

Clinical trial registry #: www.clinicaltrials.gov, NCT02535637

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Setting: Academic medical centers.

Participants: Volunteer sample of 58 exclusively breastfeeding mother-infant pairs; exclusion criteria

maternal or infant weight gain.

composition at birth and at 1, 3 and 6 months postpartum.

diHOME, and metabolites linked to thermogenesis including succinate and lyso-phosphatidylglycerol

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Despite efforts to promote breastfeeding as a way to combat childhood obesity, relatively

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recently identified lipokine, 12,13-dihydroxy-9Z-octadecenoic acid (12,13-diHOME), an oxidized

previously been identified in human milk.

postnatal period. We additionally examined associations between milk abundance of other

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Mother-infant participants and study procedures.

in a healthy infant. Exclusion criteria included maternal smoking, gestational or pre-gestational

approved all the procedures and protocols detailed in this study.

To test whether milk abundance of 12,13-diHOME is influenced by maternal exercise, we

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weight and length (crown-to-heel) at 1, 3, and 6 months. Air-displacement plethysmography (ADP,

Lipidomic Analysis and 12,13-diHOME Quantification

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Electrospray ionization (ESI) LC-MS/MS was performed on a TripleTOF 6600 (Sciex,

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time-of-flight high-resolution mass spectrometry, 150 uL for reversed-phase liquid chromatography

chromatography with liquid chromatography and tandem mass-spectrometry, and analyzed as

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Library, MzCloud, and IDEOM databases.

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Exploratory analyses by tertile

milk in relation to biologic outcomes, we examined relationships between tertiles of 12,13-diHOME

effects on adipocyte metabolism and/or thermogenesis (2,6,8,21-23). Finally, we also performed

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number and proportion.

(data not shown).

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accordingly. Similarly, whole-body adiposity at 1-month postpartum (assessed by air displacement

in tertile 3: 14.52 %fat vs. tertile 1: 18.59 %fat, p=0.021).

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attenuated the effect (= -0.439, p=0.116).

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glucose concentration (Supplement Figure 1B) (24).

Associations of thermogenic metabolites in milk and infant weight status

Given these associations between milk abundance of 12,13-diHOME, related metabolites,

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nucleotides (e.g. 1-methyladenosine, 7-methylguanine) and infant adiposity, which is intriguing in

Association between 12,13-diHOME concentrations and acute exercise

reported to be increased by acute exercise (4,5,7). We therefore tested, in a separate sample of 16

normal weight and 8 were obese. We observed an increase in 12,13-diHOME breastmilk

sample saw a 1.39-fold increase in 12,13-diHOME concentrations at 90-minutes post-acute exercise

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a bout of moderate exercise at 1 month postpartum. Moreover, we also observed an association

numerous non-nutritive bioactive compounds – including hormones, cytokines, nucleotides, cells,

Moreover, a randomized control trial of a breastfeeding promotion intervention in Belarus found a

explained by the variation of certain constituents in milk composition between individuals.