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Mediator Lipidomics:Mediator Lipidomics:dissecting the role ofdissecting the role of
PUFA-derived metabolitesPUFA-derived metabolites
Anna Nicolaou PhD FRSCAnna Nicolaou, PhD FRSCProfessor of Biological Chemistry
Compositional Analysis of Lipids 21 June 2013, Ghent
BIOACTIVE LIPIDS
fatty acids steroids terpeneswaxesenergy storagey
eicosanoidsglycerolipids
sphingolipids(mono-,di-, tri-acylglycerols)
lipid vitamins
terpenes
resolvins
waxes
sphingolipids
id
docosanoidsoxylipins lipid
mediatorsN-acyl ethanolamines
glycerophospholipids
ceramides
sphingomyelinesphosphatiditesphosphatidyl-
ether lipidsplasmalogens phosphatidyl-
-choline-ethanolamine-serine
plasmalogenscerebrosides
gangliosidesl l dserine-inositol-glycerol
gangliosides structural lipids
stimulusMembrane phospholipids
PLA2 Hydroxy fatty acids (HETE)Leukotrienes (LT)
DIET
EPA ( )
AADGLADHA
DPA
P t id
ChemotaxisAllergy
OA
Prostanoids(PG, TX,PI)
NFkBInflammationProliferation
PPAR AngiogenesisTissue homeostasis
Gene transcriptionProtein expression
5,6-EET8,9-EET
11,12-EET14,15-EET
DHET
HNEHETETXB2
CYP450
5-,-15-HETE16-, -20-HETE
free radical oxidation
8-iso-PGF2iso-LG
LGHETE
TXA2
TXB2
12-LOX
15-LOX15-HPETE 15-HETE
5-LOX
COOHPGG2PGH2PGE2
PGF
PGI2
COX-1,2
arachidonic acid
15R-HETE
5-HPETE
5-LOXFLAP
5 LOX
12-HPETE 12-HETE LXA4
HXA3
PGD2
PGF2
5-HETE LTA4 LTB4
5-LOX
15-epi-LXA4
12-LOX
3
PGJ2
12 -PGJ2
5-oxo-ETE LTC4
LTD4
LXA415d-PGJ2
LTE4
LA18:2n-6
13-HODE ALA18:3n-3
15-LOX
18:2n-69-HODE
GLA
18:3n 3
EPAPGE3
COX-2 ornon-enzymatic
oxidation LTB55-HEPE
5-,12-,15-LOXCOX-1,-2GLA
DGLA
EPA20:5n-3
PGE1
PGE3PGD3
15 HET E15-LOX
12-HEPE15-HEPE
COX-1,-2
5-HETE
20:3n-6DHA
22:6n-3
1PGD1
15-HETrE
RvE1R E5 HETE
8-HETE9-HETE11-HETE12 HETE
AA
PGE2PGD2PGF2PGI
RvE2
CYP450 COX-1,-2COX-2+ASA or CYP450
/ LOX
LOXCYP450
12-HETE15-HETE20-HETE5,6-EET
20:4n-6 PGI2TXB2PGJ2
12-PGJ2
5-,12-,15-LOX
17-HDHAother mono-, di-and tri-hydroxy
RvD1AT-RvD1
PD
non-enzymaticoxidation
5,6 EET8,9-EET
11,12-EET14(15)-EET14 15 DHET
2LTB4
5-HETE5-oxo-ETE12 HETE
and tri-hydroxyderivatives;
docosanoidsmaresins
t ti
15-PGDH
PD
14,15-DHET 12-HETE15-HETE
LXB4, LXA4
protectins15-oxo-PG
(series-1,-2,-3)
Mediator Lipidomics: array of >80 lipids
LX, RvE, RvD, PD
PUFAPGs
LTs, HETE, HEPE, HDHA, HODECYP
EET, DHET
sample lipid extraction
LC/ESI‐MS/MSSPE clean up
LC/ESI‐MS/MS mediator lipidomics
Massey & Nicolaou FRBM 2012
Mediator lipidomics workflow
Sample preparationSolid: Homogenise in 15 % (v/v) methanol in water
Liquid: Adjust to 15 % (v/v) methanolq j ( )Add internal standard(s)
Solid phase extractionRemove precipitated proteins
Acidify sample (pH 3.0)Load on C18 SPE cartridge
Elute lipids with methyl formateLi id t t ti Elute lipids with methyl formate
Remove solvents under N2Reconstitute extract in small solvent volume
Lipid extract preparation & storage
Reconstitute extract in small solvent volumeIf needed, store at -20 oC and for up to 1 week
LC/ESI-MS/MS Prepare fresh calibration standards
Check baseline and instrument sensitivityRun COX assay or
Run LOX/CYP assay orRun LOX/CYP assay orRun chiral assayData processing and
calculations
Solid phase extraction (SPE) clean up
CONDITION LOAD WASH ELUTE
Selective sorbent bed = more efficient sample clean upHigh recovery and reproducibility
1 Condition SPE: Prepare C18 sorbent bed1. Condition SPE: Prepare C18 sorbent bed 2. Load extract:
3. Wash: Remove weakly bound impurities, then wash Reduce matrix ff twith hexane to remove water
4. Elute: Selectively desorb lipid mediators
effects
tandem mass spectrometer typical lipidomics platformtypical lipidomics platform
LC Ion SourceESI APCI
MS1m/z filter
Collision Cell
MS2m/z filter
QQQ detectorESI, APCI Quadrupole(Q)
Cell ToF (QTOF)Ion Trap (QTrap)
179271
A B
abu
ndan
ce 12-HETE
257
abu
ndan
cePGE2
% re
lativ
e
135
115
163
153139
208203
301
319
% re
lativ
e 189
193 233 253
315333
351
m/z100 150 200 250 300 350
115 153
m/z100 150 200 250 300 350
253 351
e
167
C D115
ve a
bund
ance 11(12)-EET319
179abun
danc
e
LXA4217
% re
lativ
149135
179257
208301
275% re
lativ
e a
135
189144
235
351289271
m/z100 150 200 250 300 350
m/z100 150 200 250 300 350
144
Massey & Nicolaou FRBM 2012
PGD1: m/z 353>317 MS1 CC MS2
317 4
1 /ce 15 eV
/ 3 3 / 191100
317.4
235.3
m/z 353 m/z 191235253273335
m/z 317
HOCOOH
ance
%
OHPGD1
O
tive
abun
da
273.4% re
lat
191.2 253.2
335.5353.5
[M-H]-
m/z100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 3600
RvE1: m/z 349>195 MS1 CC MS2RvE1: m/z 349>195 ce 17 eV
OHm/z 349 m/z 107
129161195
m/z 195
COOH
OH
OHOH
195
281331
100195.0
COOHRvE1
[M-H]-194.7
160 9107 1
349.1
205.0 [M H]
%
160.9107.1
128.9107.2128.7
161.1
181.0179 0
195.2
204 8
281.0
205.3
110.9113.0
129.2160.6
159.0152.8134.9 150.9135.0150.6
136.7
179.0
178.7
177.1176.7
181.2
188.6
186.9
204.8
198.8
218.8
213.0273.4
229 2222 7
330.9281.1281.6
313.3303.2290.8302 8
312.8303.5
331.4
m/z100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 3500
123.0 136.9143.2 168.9169.0 212.6229.2222.7
269.2267.2229.5 259.0250.8247.3232.7240.9251.0265.0
273.5
302.8291.1291.4294.9
303.5
304.6313.4317.4
331.7
3 46
LC/ESI-MS/MS (ESI-) Liquid chromatography: h
3 06
2.83 369>169
TXB2
3.46 reverse phase
5 41 3 113
4.94 367>169TXB3
3.06 369>1636-keto PGF1a
Lipid mediators typically separated by
6.67 355>311
4.79 355>337
5.41
13,14 dihydro-PGE1
357>11313,14 dihydro-PGF1a
3.69
Lipid mediators typically separated by hydrophobic moiety (C18, e.g. Luna ®)
4 47
7.61
353 335
355>19313,14 dihydro-15-keto PGF1a
355>31113,14 dihydro-PGF2aPGF1a Prostanoids:
isobaric species e.g. PGE and PGD optimal separation: acetonitrile-based
353>193
3.383.77
PGE1
PGD1 353>317
4.474.79
353>33513,14 dihydro-15-keto PGE1 gradient elution system
%
100
TIC
353>11313,14 dihydro-15-keto PGF2a
353>1938-iso-PGF2a
PGF2a
6.12
Time (min)2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00
0
TIC
Masoodi & Nicolaou RCM 2006; Masoodi et al RCM 2008
LC/ESI-MS/MS (ESI-) Liquid chromatography: h
3.86
6 90 375>141RvD1
reverse phase6.90
19.61
2.57
349>195
359>206PD1
RvE1
Lipid mediators typically separated by hydrophobic moiety (C18 e g Luna ®)
20.51
7.80 343>281
335>195LTB4
17S-HDHA
hydrophobic moiety (C18, e.g. Luna ®)
Hydroxy fatty acids: l ti ith t it il
21.25
18.55 319>179
328>185
12-HETE
IS (12-HETE-d8)
poor resolution with acetonitrilestrong interaction with C18 columnimproved elution with methanol
19.94
21.42
319>175
319>167
15-HETE
11-HETECore shell columns: behave like UPLC columns (pore size 2.5µm)
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00
%
0
100319>1558-HETE
TIC
(p µ )improved peak resolution and better sensitivity
Time (min)
Masoodi & Nicolaou RCM 2006; Masoodi et al RCM 2008; Massey & Nicolaou FRBM 2012
Very fast even better separation of isobaric compounds with different RP UPC2 columncompounds with different RP UPC2 column
PGE2
8-iso-PGE2
Chiral separation by LC
Stationary phases:Amylose: 18(S)-E Resolvins (Oh et al., J Clin Invest. 2011;121)
C ll l 12(S) HETE i bli t fl idCellulose:12(S)-HETE in blister fluid (Massey and Nicolaou, FRBM. 2012)
Reverse or normal phase solvents12.22
10.97 15 epi-LXA4Reverse or normal phase solvents
Cellulose (Lux-1)
m/z 351 > 115
LXA4
ce
m/z 317 > 13326.84 29.43
18(R)-HEPE 18(S)-HEPECellulose (Lux-1)more stable stationary phaseimproved separation of enantiomers %
rela
tive
abun
danc
m/z 319 > 17547.8945.47
15(R)-HETE 15(S)-HETE
improved separation of enantiomers
Time (min)
Massey& Nicolaou FRBM 2012
mediator lipidomics protocolmediator lipidomics protocol
Solid phase extraction clean-up step (matrix effects) Solid phase extraction clean-up step (matrix effects). Multiple Reaction Monitoring (MRM) assays.
for > 80 lipid mediators; LoD/LoQ 1 10 pgfor > 80 lipid mediators; LoD/LoQ 1-10 pg. LC/ESI-MS/MS (Q3); calibration lines; d-internal standards.
Biological material
Solids: skin, tumours, liver, brain, uterine, ocular, nerve tissues, cells, etc.
Liquids: plasma urine seminal plasma follicular fluid Liquids: plasma, urine, seminal plasma, follicular fluid, blister fluid, cell culture media, etc.
Samples snap-frozen; extracted/run within days; dark/cold.Samples snap frozen; extracted/run within days; dark/cold.
Masoodi & Nicolaou RCM 2006; Masoodi et al RCM 2008; Nicolaou MMB 2010; Massey & Nicolaou FRBM 2012
inflammation in cutaneous diseaseinflammation in cutaneous disease
photoageing sunburn
psoriasis
squamous cellatopic dermatitiswound healing
squamous cell skin cancer
UV radiation and human skinUV radiation and human skin
UVA 320 400UVA: 320-400 nm dermis
UVB: 280 320 nm5% t t i l li ht UVB: 280-320 nm epidermis: inflammation
5% terrestrial sunlight
epidermis protection: radiation, xenobiotics, trauma
dermis elasticity; immuno- & biochemical support
UVR: immunosuppression; photosensitivity; photoageing; photocarcinogenesis
UVR-induced skin inflammation (sunburn)
• Acute inflammatory responseAcute inflammatory response
• Erythema, pain, oedema (vasodilatation)
• Leukocytic infiltration
• Sunburn (apoptotic) cellsSunburn (apoptotic) cells
-linolenic acidALA 18:3n-3
linoleic acidLA 18:2n-6
oleic acidOA 18:1n-9
6 desaturase 6 desaturase
octadecatetraenoic acid(stearidonic )18:4n-3
-linolenic acidGLA 18:3n-6
octadecadienoic acid18:2n-9
6 desaturase 6 desaturase
elongasel
eicosatetraenoic acid20:4n-3
dihomo--linolenic acidDGLA 20:3n-6
eicosadienoic acid20:2n-95 desaturase 5 desaturase
elongaseelongase
eicosapentaenoic acidEPA 20:5n-3
arachidonic acidAA 20:4n-6
eicosatrienoic acid(mead acid) 20:3n-9
5 desaturase 5 desaturase
l
docosapentaenoic acidn-3DPA 22:5n-3
docosatetraenoic acid22:4n-6
(mead acid) 20:3n 9elongase
ay n 3DPA 22:5n 3
24:5n-3 24:4n-6
4 desaturase
elongase
eche
rpat
hwa
24:6n-3 24:5n-6
4 desaturase
peroxisomal-oxidation
Spre
Human epidermis lacking 5, 6 desaturase activity
docosahexaenoic acid DHA 22:6n-3
docosapentaenoic acid n-6DPA 22:5n-6
6 desaturase activity(Chapkin & Ziboh 1984, 1988)
4000
4500
C t t id3500
4000
D i E id i
Cutaneous prostanoids
2500
3000
rote
in
Dermis Epidermis
1500
2000
pg/m
g p
1000
1500
0
500
human skin samples: ethical tissue; 3 mm punch biopsies; ̴ 20 mg; n=8; LC/ESI-MS/MS
3500
2500
3000 Cutaneous hydroxy fatty acids
2000
2500
otei
n
Dermis Epidermis
1500
pg/m
g pr
o
1000
0
500
0
human skin samples: ethical tissue; 3 mm punch biopsies; ̴ 20 mg; n=8; LC/ESI-MS/MS
sunburn: experimental modelsunburn: experimental model
• healthy adult volunteers, skin type I-IV • skin exposed to UVR
(UV6; 280-400 nm; 23% UVB :77% UVA)
• 3-4 minimal erythema doses (MED)
Suction blisters and skin sections (0 -72 h post UVR)Suction blisters and skin sections (0 72 h post UVR)
Overlapping sequential eicosanoid profiles may mediate the early and late phases of sunburn response
200
the early and late phases of sunburn response
PGE2PGF2
150
ex 2PGE3 11-HETE
8-HETE12-HETE
100
hem
a in
de
12 HETE15-HETE50er
yt
neutrophils CD3+0
0 12 24 36 48 60 72time (h)
neutrophils
( )
early: inflammationvasodilatation chemotaxis
late: resolutionrepairvasodilatation, chemotaxis repair
Rhodes, Nicolaou et al FASEB J 2009
erythema in skin types I/II and III/IV post single high UVR dose (12 SED)
UV threshold Dose
high UVR dose (12 SED)
#
506070
MED#
30
40
50
2
UV threshold Dose
10203040
mJ/
cm2
10
20
30
mJ/
cm2
010
phototype I phototype IV0
phototype I/II phototype III/IV
** *
150
175
erythema index
** *
75
100
125
150
rary
uni
ts
Single UVR dose
0
25
50arbi
t phototype I/II
phototype III/IV
Single UVR dose(12 SED) 120 mJ/cm2
0 10 20 30 40 50 60 70 80
Time (h)Nicolaou et al Photochem Photobiol Sci 2012n=16; #p<0.05; *p<0.01
PGE2 higher in subjects prone to sunburn2 g j p
/ 2 5COX-2 in epidermis
200
PGE2
phototype I/II
phototype III/IV *1.5
2.0
2.5
g in
tens
ity
* *120
160
rote
in
0.0
0.5
1.0
0 4 24 72mea
n st
aini
ng
*40
80
pg /
mg
pr 0 4 24 72m
Time (h)
*2.5COX-2 in dermis
00 12 24 36 48 60 72
Time (h)1.0
1.5
2.0
g in
tens
ity
pro-inflammatory vasodilatation 0.0
0.5
0 4 24 72
mea
n st
aini
ng
Time (h)
Nicolaou et al Photochem Photobiol Sci 2012n=9; p<0.05m Time (h)
15-HETE higher in subjects prone to sunburng j p
phototype I/III 15 LOX i id i
***50
60
7015-HETE
phototype I/III
phototype III/IV
2 02.53.03.5
g in
tens
ity
15-LOX in epidermis
20
30
40
50
/ mg
prot
ein
0 00.51.01.52.0
mea
n st
aini
ng
0
10
20
0 12 24 36 48 60 72
pg
Ti (h)
0.00 4 24 72
3.515-LOX in dermis
Phototype I/II
anti-inflammatory
Time (h)* **
1 5
2.0
2.5
3.0
3.5
ng in
tens
ity
Phototype I/II
PhototypeIII/IV
precursor to lipoxins
0.0
0.5
1.0
1.5
0 4 24 72
mea
n st
aini
n
Nicolaou et al Photochem Photobiol Sci 2012n=9; *p<0.05, ***p<0.001
0 4 24 72
Time (h)
higher neutrophil infiltrate in subjects prone to s nb rnprone to sunburn
*1416
nt
neutrophils in dermis
phototype I/II
phototype III/IV*
***30
35
CD3+ cells in dermis
468
1012
mea
n ce
ll co
un *
1015202530
ean
cell
coun
t
024
0 4 24 72
m
Time (h)
05
10
0 4 24 72
me
Time (h)Time (h) ( )
I/II 24 h III/IV 24 h I/II 24 h III/IV 24 h
Nicolaou et al Photochem Photobiol Sci 2012n=6; * p<0.05; ** p<0.01
lipid biomarkers of skinlipid biomarkers of skin
inflammation in humaninflammation in human
nutritional studies
UVR
cPLA2ROS
EPACOX-2
AA
PGE2 PGE3
COX-2
NFB
IKK
PPAR
NFB COX-2
cytokinescytokines
Nicolaou et al Chem Phys Lipids 2011
n-3PUFA in skin inflammation and immunity:photoimmunosuppression
Randomised double-blind study (n=79 subjects)
control: GTCCcontrol: GTCC
active: 1g capsule ̴ 70% EPA&10% DHA; 5 cps/day; 12 weeks
Pre supplementationPre supplementationBlood sampleErythema assessmentPhotoimmunosuppression test (Ni) Post supplementation
Bl d lpp ( )
Blood sampleErythema assessmentPhotoimmunosuppression test (Ni)
Pilkington et al AMCN in press
EPA supplementation did not increase skin DPA DHA l lDPA or DHA levels
EPA (C20:5n-3) DHA (C22:6n-3) DPA (C22:5n-3)skin
a, b
0 08
0.10
0.12
0.14EI
GH
T( )
0 15
0.20
0.25
GH
T
( )
0.20
0.25
0.30
GH
T
skin
0.02
0.04
0.06
0.08
% W
E
0.05
0.10
0.15
% W
EIG
0.05
0.10
0.15
% W
EIG
0.00Dermal Basal
Placebo POST
Active POST
0.00Dermal Basal
Placebo POST
Active POST
0.00Dermal Basal
Placebo POST
Active POST
EPA (20:5n 3) DHA (22 6 3) DPA (22 5n 3)a, b
3.00
3.50
4.00
4.50
HT
EPA (20:5n-3)a, b
3.00
3.50
4.00
4.50
5.00T
DHA (22:6n-3)
a, b
2.50
3.00
3.50
4.00
GH
T
DPA (22:5n-3)r.b.c.
0.50
1.00
1.50
2.00
2.50
% W
EIG
H
0 50
1.00
1.50
2.00
2.50
% W
EIG
HT
0.50
1.00
1.50
2.00
2.50
% W
EIG
0.00
0.50
RBC Basal Placebo POST
Active POST
0.00
0.50
RBC Basal Placebo POST
Active POST
0.00
0 50
RBC Basal Placebo POST
Active POST
a: p<0.001 comparing to basal; b:p<0.001 comparing to placebo
M (SEM) ( / l)
AA, EPA, OA mediators in cutaneous blister fluidMean (SEM) (pg/μl)
Baseline 12 weeks
Control (n=19) EPA (n=17) Control (n=19) EPA (n=17)Unexposed UVR- Unexposed UVR- Unexposed UVR- Unexposed UVR-Unexposed UVR-
exposedUnexposed UVR-
exposedUnexposed UVR-
exposedUnexposed UVR-
exposed
PGE2 9.5 (1.9)19.5
(3.1)††† 11.0 (2.4) 22.2 (3.8)† 10.7 (2.2) 28.1 (5.4)†† 6.0 (1.1)*19.9
(3.4)†††
PGE 0 5 (0 1) 0 8 (0 2) 0 7 (0 2) 1 6 (0 4)† 0 6 (0 2) 1 2 (0 3)† 0 8 (0 2) 3 1 (1 0)†PGE3 0.5 (0.1) 0.8 (0.2) 0.7 (0.2) 1.6 (0.4)† 0.6 (0.2) 1.2 (0.3)† 0.8 (0.2) 3.1 (1.0)†
PGE1 2.7 (0.7) 6.2 (1.2)††† 2.6 (0.6) 7.0 (1.2)†† 3.5 (1.4) 8.7 (2.0)†† 1.6 (0.4) 6.7 (1.4)†††
13,14 dh-
15k PGE4.6 (1.1) 1.2 (0.4)††† 8.1 (2.2) 1.5 (0.4)††† 4.8 (1.3) 1.4 (0.4)††† 4.9 (1.4) 1.9 (0.4)
15k-PGE2
12-HETE 12.7 (1.8)33.0
(5.7)††† 11.7 (1.9) 38.1 (5.7) 13.1 (2.9)51.4
(8.6)†††13.4 (3.9)
50.3
(8.2)†††
11 HETE 1 6 (0 2) 3 7 (0 6)††† 1 6 (0 2) 4 3 (0 5)††† 1 4 (0 2) 4 8 (0 5)††† 1 3 (0 3) 4 3 (0 6)†††11-HETE 1.6 (0.2) 3.7 (0.6)††† 1.6 (0.2) 4.3 (0.5)††† 1.4 (0.2) 4.8 (0.5)††† 1.3 (0.3) 4.3 (0.6)†††
15-HETE 3.4 (0.5) 4.6 (0.6) 3.3 (0.5) 6.0 (0.7)†† 3.0 (0.5) 6.3 (1.3)†† 4.5 (0.9) 6.1 (0.9)†
15-HETrE 0.9 (0.1) 2.4 (0.5)†† 1.3 (0.3) 2.2 (0.5)† 0.9 (0.1) 5.4 (2.4)†† 0.9 (0.2) 1.9 (0.3)††
18 212-HEPE 2.5 (0.4) 3.9 (0.5)† 3.1 (0.4) 5.3 (0.5) 3.0 (0.6) 6.4 (1.9)† 5.9 (1.7)
18.2
(3.5)†††**
11-HEPE ND 0.4 (0.14)a 1.7 (0.9)b 1.7 (0.6)f 7.4 (4.5)b 0.4 (0.05)c 0.6 (0.3)c 4.1 (2.0)g
15 HEPE ND ND ND ND ND ND 3 4 (0 9)d 5 0 (2 2)e15-HEPE ND ND ND ND ND ND 3.4 (0.9)d 5.0 (2.2)e
9-HODE 34.3 (5.6) 46.3 (9.6) 45.9 (10.6) 63.7 (7.7)† 26.1 (4.8) 51.1 (9.0)† 32.6 (11.0) 56.1 (9.3)††
13-HODE 36.6 (7.0) 32.6 (5.2) 32.3 (4.4) 55.5 (8.4)† 30.5 (5.9) 33.2 (4.7) 26.3 (5.2) 38.5 (5.9)
systemic EPA alters skin eicosanoidsy
35 PGE25 PGE3
15202530 2
2
3
4
pg/u
l
PGE3 *
05
10
pg/u
l
0
1
2
2570
* *
15
20
2512-HEPE
3040506070 12-HETE
Placebo
Active
**
0
5
10
0102030
P N P P t P t 0Pre No UVR
Pre UVR
Post No UVR
Post UVR
Pre No UVR
Pre UVR
Post No
UVR
Post UVR
RCT: n=16-19 volunteers per group; skin type I/II; n-3 LC-PUFA (EPA: 70%; DHA: 10%) 5 cps/d, 3 months
oral n-3 PUFA supplement protects against UVR-induced immunosuppression
*
Protection at 3.8 J/cm2 – 15 min summer midday sun at Manchester
SSR: Solar simulator; nickel allergy; n=33-36 per group; p=0.04
Pilkington et al AMCN in press
n-3PUFA in wound healingn 3PUFA in wound healing
• Randomised double blind study (n=18 subjects)
• placebo (mineral oil) orplacebo (mineral oil) or
• active (1.6 g EPA+1.2 g DHA/day, 81 mg aspirin) 28 days
Day 0Admission;
Day 28Blood samplesBlistering (forearm)
Blood samplesFood frequency questionnaire
g ( )
Blister fluid sampling:12 h post blistering24 h post blistering
Monitoring of wound healing:Monitoring of wound healing: 1-15 days post blistering
McDaniel et al Wound Repair Regeneration 2011
n-3PUFA supplement and COX-mediators
PLASMA
AA (20:4n-6)-derived EPA(20:5n-3)-derived
11.21.41.6
l
PLASMA
1
1.5PLASMA
0.20.40.60.8
1
pg /u ACTIVE
PLACEBO
0
0.5
1
pg /u
l
ACTIVEPLACEBO
0PGE2 PGF2a 6kPGF1a
9 BLISTER FLUID
0PGE3 PGD3
BLISTER FLUID
5678
/ ul
ACTIVE4
5
6
ul
S U
1234pg
ACTIVEPLACEBO
1
2
3pg
/ u
ACTIVEPLACEBO
0PGE2 PGF2a
0PGD3
McDaniel et al Wound Repair Regeneration 2011
n-3PUFA supplement and LOX-mediators
1 2 PLASMA 1.6 PLASMA RvE1
AA (20:4n-6)-derived EPA(20:5n-3)-derived
0.6
0.8
1
1.2
g /u
l
PLASMA
0.81
1.21.4
g /u
l
RvE1 precursor
0
0.2
0.4
pg ACTIVEPLACEBO
00.20.40.6p ACTIVE
PLACEBO
BLISTER FLUID BLISTER FLUID
15
20
25
ul
BLISTER FLUID
ACTIVE 2
2.5
3
l
BLISTER FLUID
ACTIVE
0
5
10pg /
PLACEBO
0.5
1
1.5
2
pg /
ul ACTIVEPLACEBO
012-HETE 15-HETE 0
12-HEPE 15-HEPE
McDaniel et al Wound Repair Regeneration 2011
N 3 PUFA reduced wound area (improved healing)N-3 PUFA reduced wound area (improved healing)
myeloperoxidase (MPO) levels, (leukocyte marker enzyme)
Area of wound (blisters) remainingto re-epithelialize on Day 5
McDaniel et al Wound Repair Regeneration 2011
Green Tea Catechins (GTC) and UVR-i d d t i fl tiinduced cutaneous inflammation
Healthy human volunteers: n=14, 27-56 yrs; all female; phototype I/II (tend to burn not tan)
S l GTC 0 /d 0 /d i CSupplement: GTC 550 mg/day + 50mg/day vit.C
Study period: 12 weeks
Green tea supplement
AdmissionMED assessedIrradiation 3xMED (pre.supp.)
12 WeeksMED assessedIrradiation 3xMED (pre.supp.)(p pp )
no UVR & 24h post UVR:- skin punch biopsies- skin blister fluid
no UVR & 24h post UVR- skin punch biopsies- skin blister fluid
urine samples: complianceRhodes et al BJN 2013
Effect of low dose GTC on cutaneous eicosanoidsEffect of low dose GTC on cutaneous eicosanoids
PGE2pro-inflammatory, vasoactive
12-HETEleukocyte chemoattractant
120
140
160
d
70
80
90
d
60
80
100
ml b
liste
r flu
id
*
40
50
60
ml b
liste
r flu
i
** **
0
20
40
60
pg /
m
** *10
20
30
pg /
m
baselineGTC
0PRE NO UV PRE 24 UV POST NO UV POST 24 UV 0
PRE NO UV PRE 24 UV POST NO UV POST 24 UV
baselineGTC
* p<0.05** p<0.001Rhodes et al BJN 2013
Mediator LipidomicsMediator Lipidomics
• LC/ESI-MS/MS mediator lipidomics: versatile, iti hsensitive approach.
• Role of lipid mediators in health and diseaseRole of lipid mediators in health and disease.
• Discovery of novel mediators and biomarkers; y ;development of therapeutics.
• Contribution to systems biology.
Acknowledgements
Bradford UniversityKaren MasseyAlex Kendall
Lesley Rhodes (Manchester, UK) Suzy PilkingtonSue Bennett
Sharon MurphyMojgan MasoodiKarl Gledhill
Gemma DarbyMags BrownriggGary Williamson (Leeds, UK)
Andrew HealeyNaser Al-AasswadPaula Urquhart
Tristan DewKayleigh ClarkeJodi McDanie (Ohio, USA)
Des Tobin Tony Thody Robert Tonge (Waters, UK)