anna nicolaou presentation - soci

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

Very fast separation of isobaric compounds with chiral UPC2 columnwith chiral UPC2 column

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

lipid mediators in skinlipid mediators in skin inflammation: a at o

the sunburn response

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

UVR

cPLA2ROS

COX-2

AA

MAPK 12-LOX

PGE2

COX-2JNK p38

NFBIKK

12-HETE

PPAR

AP-1NFB

MMPCOX-2

cytokines

-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


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


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


Active POST

0.00

0 50


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-



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

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2

3

4

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10

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3040506070 12-HETE

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0

5

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0102030

P N P P t P t 0Pre No UVR

Pre UVR

Post No UVR

Post UVR

Pre No UVR

Pre UVR

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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

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PLASMA

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0PGE2 PGF2a 6kPGF1a

9 BLISTER FLUID

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n-3PUFA supplement and LOX-mediators

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0.8

1

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0.4

pg ACTIVEPLACEBO

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BLISTER FLUID BLISTER FLUID

15

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12-HEPE 15-HEPE


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


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

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60

80

100

ml b

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r flu

id

*

40

50

60

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liste

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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)

anna nicolaou presentation - soci

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