1. No category

Supplemental Methods:
Oxylipid sample preparation: Plasma (200μL) was spiked with a 5 µL 0.2 mg/ml solution butylated
hydroxytoluene/EDTA in 1:1 meoh:water and a suite of 10 deuterated prostanoid, eicosanoids, and
octadecanoid surrogates (Supplemental Table S2). To release oxygenated lipids contained in ester linkages
these samples were then subjected to overnight hydrolysis in 1M methanolic sodium hydroxide1. Samples were
diluted to 10% methanol and extracted on 60mg Oasis HLB solid phase extraction columns (Waters, Milford,
MA), preconditioned with 5% methanol w/ 0.1% acetic acid, the loaded columns were washed with 1 column
volume of 5% methanol w/ 0.1% acetic acid, and dried for 30min under vacuum. Extracts were wetted with
0.5mL methanol and eluted with 2mL ethyl acetate into vials containing a 6µL of 30% glycerol keeper solvent,
and reduced to dryness under vacuum. We have confirmed oxylipid stability for >5months at -80ºC under these
conditions (unpublished data).
Oxylipin nomenclature: The International Union of Pure and Applied Chemistry (IUPAC) has adopted the
abbreviations for oxidized fatty acids following the recommendations of Smith et al.2, 3. Briefly, compounds are
named using position, number, and standardized abbreviations of functional groups, carbon chain length, and
degree of unsaturation. Plural chemical moieties are listed as Di (two), Tr (three), T (four), P (five), He (six).
Abbreviations of chemical moieties are: Ep – Epoxide; H – hydroxy; Hp – hydroperoxide; K – keto. Carbon
numbers appearing in this report are abbreviated O (octadeca i.e. 18), E (eicosa i.e. twenty) and Do (docosa i.e.
22). Therefore, 14(15)-epoxyeicostri-(5Z,8Z,11Z)-enoic acid is reduced to 14(15)-EpETrE while 9(10)epoxyoctadec-(12Z)-enoic acid becomes 9(10)-EpOME. Dihydroxy lipids are named similarly, such that 14,15dihydroxyeicostri-(5Z,8Z,11Z)-enoic acid becomes 14,15-DiHETrE, while 20-hydroxyeicosatetra(5Z,8Z,11Z,14Z)-enoic acid is 20-HETE.
1.
2.
3.
Newman JW, Kaysen GA, Hammock BD, Shearer GC. Proteinuria increases oxylipid concentrations in
VLDL and HDL, but not LDL particles in the rat. J Lipid Res. 2007.
Smith DL, Willis AL. A suggested shorthand nomenclature for the eicosanoids. Lipids. 1987;22:983986.
Smith WL, Borgeat P, Hamberg M, Roberts LJ, 2nd, Willis A, Yamamoto S, Ramwell PW, Rokach J,
Samuelsson B, Corey EJ, et al. Nomenclature. Methods Enzymol. 1990;187:1-9.
1
2
Supplemental Table S1: Oxylipid assay UPLC solvent gradient
Time (min) Solvent A (%)
0.0
75
3.5
65
5.5
60
7.0
58
9.0
50
15.0
35
17.0
25
18.5
15
19.5
5
21.0
75
25.0
75
Solvent A = 0.1% acetic acid in de-ionized water
Solvent B = 90:10 v/v acetonitrile/isopropanol
Flow rate 250µL/min
Supplemental Table S2: Oxylipin Internal Standards
Compound
CID Mass Transition (Da)
Internal Standard
Internal Standards
CUDA
340.3 > 214.1
d4 6-keto-PGF1á
373.3 > 167.1
CUDA
d4-TXB2
373.3 > 173.15
CUDA
d4-PGE2
355.3 > 275.2
CUDA
d4-LTB4
339.3 > 163.15
CUDA
d6-20-HETE
325.3 > 281.15
CUDA
d4-9(S)-HODE
299.3 > 172.1
CUDA
d8-12(S)-HETE
327.2 > 184.15
CUDA
d8-5(S)-HETE
327.2 > 116.1
CUDA
d8-11(12)-EpETrE
327.2 > 171.15
CUDA
Supplemental Table S3: Eighteen Carbon Oxylipins
Compound
CID Mass Transition (Da)
Internal Standard
Internal Standards
Linoleic Acid Metabolites
9,12,13-TriHOME
329.2 > 211.2
d4 6-keto-PGF1α
9,10,13-TriHOME
329.2 > 171.1
d4 6-keto-PGF1α
12,13-DHOME
313.2 > 183.1
d4-9(S)-HODE
9,10-DHOME
313.2 > 201.1
d4-9(S)-HODE
13-HODE
295.2 > 195.2
d4-9(S)-HODE
9-HODE
295.2 > 171.1
d4-9(S)-HODE
13-KODE
293.2 > 195.2
d4-9(S)-HODE
9-KODE
293.2 > 185.1
d4-9(S)-HODE
12(13)-EpOME
295.2 > 195.1
d8-11(12)-EpETrE
9(10)-EpOME
295.2 > 171.1
d8-11(12)-EpETrE
alpha Linolenic Acid Metabolites
15,16-DiHODE
311.2 > 235.15
d4-9(S)-HODE
12,13-DiHODE
311.2 > 183.1
d4-9(S)-HODE
9,10-DiHODE
311.2 > 201.15
d4-9(S)-HODE
3
b
a
a
a
a
a
a
a
a
b
b
b
b
b
b
b
b
b
b
a
a
a
9-HOTE
293.35 > 171.15
13-HOTE
293.35 > 195.15
15(16)-EpODE
293.2 > 275.15
9(10)-EpODE
293.2 > 275.15
12(13)-EpODE
293.2 > 183.1
a = newly reported transition
b = newly reported internal standard association
d4-9(S)-HODE
d4-9(S)-HODE
d8-11(12)-EpETrE
d8-11(12)-EpETrE
d8-11(12)-EpETrE
4
a
a
a
a
a
Supplemental Table S4: Twenty Carbon Oxylipins
Compound
CID Mass Transition (Da)
Internal Standard
dihomo gamma Linoleic Acid Metabolites
15(S)-HETrE
321.2 > 221.15
d8-11(12)-EpETrE
Arachidonic Acid Metabolites
6-keto-PGF1á
369.2 > 163.1
d4 6-keto-PGF1α
TXB2
369.3 > 195.2
d4 6-keto-PGF1α
PGF2á
353.2 > 193.1
d4 6-keto-PGF1α
20-carboxy-LTB4
365.2 > 347.2
d4 6-keto-PGF1α
20-hydroxy-LTB4
351.2 > 195.15
d4 6-keto-PGF1α
11,12,15 THET
353.2 > 167.15
d4 6-keto-PGF1α
lipoxin a4
351.3 > 217.15
d4 6-keto-PGF1α
8,15-DiHETE
335.3 > 235.15
d4-9(S)-HODE
5,15-DiHETE
335.3 > 173.15
d4-9(S)-HODE
LTB4
335.2 > 195.15
d4-9(S)-HODE
14,15-DHET
337.2 > 207.1
d4-9(S)-HODE
11,12-DHET
337.2 > 167.1
d4-9(S)-HODE
8,9-DHET
337.2 > 127.1
d4-9(S)-HODE
5,6-DHET
337.2 > 145.1
d4-9(S)-HODE
20-HETE
319.2 > 275.2
d4-9(S)-HODE
19-HETE
319.2 > 275.2
d4-9(S)-HODE
15-HETE
319.2 > 219.1
d4-9(S)-HODE
11-HETE
319.2 > 167.1
d4-9(S)-HODE
12-HETE
319.2 > 179.1
d4-9(S)-HODE
9-HETE
319.2 > 123.1
d4-9(S)-HODE
8-HETE
319.2 > 155.1
d4-9(S)-HODE
5-HETE
319.2 > 115.1
d8-5(S)-HETE
15-KETE
317.3 > 273.2
d8-11(12)-EpETrE
5-KETE
317.2 > 203.15
d8-11(12)-EpETrE
14(15)-EET
319.2 > 219.1
d8-11(12)-EpETrE
11(12)-EET
319.2 > 208.1
d8-11(12)-EpETrE
8(9)-EET
319.2 > 155.1
d8-11(12)-EpETrE
5(6)-EET
319.2 > 191.1
d8-11(12)-EpETrE
Eicosapentaenoic Acid Metabolites
5,12,18-TriHEPE
349.3 > 195
d4 6-keto-PGF1á
(Resolvin E1)
17,18-DiHETE
335.3 > 247.2
d4-9(S)-HODE
14,15-DiHETE
335.3 > 207.15
d4-9(S)-HODE
15(S)-HEPE
317.2 > 219.15
d4-9(S)-HODE
12(S)-HEPE
317.3 > 179.2
d4-9(S)-HODE
5(S)-HEPE
317.3 > 115.2
d4-9(S)-HODE
17(18)-EpETE
317.2 > 259.5
d8-11(12)-EpETrE
14(15)-EpETE
317.2 > 247.5
d8-11(12)-EpETrE
a = newly reported transition
b = newly reported internal standard association
5
a
b
b
b
a
a
a
a
a
a
a
b
b
b
b
b
b
b
b
b
b
b
b
a
a
b
b
b
b
a
a
a
a
a
a
a
a
Supplemental Table S5: Twenty-two Carbon Oxylipins
Compound
CID Mass Transition (Da)
Internal Standard
dihomo gamma Linoleic Acid Metabolites
Docosapentaenoic Acid Metabolites
19,20-DiHDPE
361.5 > 273.5
d4-9(S)-HODE
17(R)-HDoHE
343.2 > 281.2
d8-11(12)-EpETrE
19(20)-EpDPE
343.5 > 281.2
d8-11(12)-EpETrE
16(17)-EpDPE
343.5 > 273.5
d8-11(12)-EpETrE
a = newly reported transition
b = newly reported internal standard association
6
a
a
a
a
Supplemental Table S6: Concentrations of other plasma oxylipins in nMa.
Oxylipin
Isomer
level
Pre P-OM3
Post P-OM3
Fold
change
P
0.82
0.0005
0.74d
0.008
AA - Non-vicinal diols
5,15-DiHETE
8,15-DiHETE
A
B
1.46 (1.2, 1.7)
2.61 (2.2, 3)
1.37 (1.2, 1.6)
1.88 (1.6, 2.2)
Triols & LipoxinA4 (LXA4)
LA - Trihydroxyoctadecamonoenoic acids (TriHOME)
A
5.39 (4.1, 7.1)
4.51 (3.4, 6)
9,10,13-TriHOME
A
7.37 (5.6, 9.8)
4.88 (3.7, 6.5)
9,12,13-TriHOME
AA -Trihydroxyeicosatrienoic acids (TriHETrE)
B
1.89 (1.4, 2.5)
1.21 (0.92, 1.6)
11,12,15-TriHETrE
A
4.74 (3.6, 6.3)
4.08 (3.1, 5.4)
LXA4 (5,6,15-TriHETE)
AA – Prostaglandin(PG) & Thromboxane (Tx)
A
6.36 (4.2, 9.5)
5.21 (3.5, 7.8)
PGF2α
->0.05
B
2.84 (1.9, 4.3)
2.61 (1.7, 3.9)
TxB2
The means and 95% CIs of individual regioisomers after adjustment for subject, and extraction batch.
a
See Supplemental Figure 1 for LXA4 associated pathway. See Figure 1 for 11,12,15-TriHETrE associated pathway. See Figure 2
for DiHETE associated pathways.
b
Regioisomers derived from the same parent FA with different letters differ significantly (p<0.05) by Tukey’s Honest Significant
Differences test.
c
p-value of treatment effects on plasma concentrations.
d
The LA- and AA-derived triols were analyzed in the same ANOVA. The effect is uniform across both parent FAs.
7
1
2
3
4
5
6
7
8
9
10
11
Supplemental Figure 1:
Supplemental Figure S1: 5-Lipoxygenase associated metabolism of arachidonic (A) and
eicosapentaenoic (B) acid. Reactive oxygen species (ROS) can also produce PUFA
hydroperoxides. Metabolites are indicated with circles and enzymes by rounded rectangles.
Some reactions may be mediated by multiple enzymes. Treatment effects are indicated by color:
black = unchanged; blue = decreased; orange = increased; white = not measured. Bolded arrows
demarcate metabolic pathways evaluated and gray toned circles and arrows are unmeasured
metabolites along evaluated pathways.
8