Lipidomics at Baker Heart and Diabetes Institute:

from discovery to translation

Peter Meikle

12 September 2017

Overview

• Lipidomics: technology and strategy

• Biomarker discovery and translation

• Lipid metabolism: Therapeutic intervention strategies

Lipidomics: technology and strategy

Plasma lipidomics at Baker Institutephospholipid

cholesterolester

triglyceride

ApoA-I

LCAT

cholesterol

Triacylglycerol 16:0/16:0/16:0

Phosphatidylcholine 16:0/18:0Ceramide (d18:1/16:0)1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC)

Clinical lipid measures

Cholesterol, HDL-C, triglycerides, LDL-C

Lipid composition of plasma

Cholesteryl esters

Di- and Triacylglycerols

Phospholipids

Ceramides

Glycosphingolipids

Sphingomyelin

Modified lipids (oxidized, glycated)

Lysolipids

Free fatty acids

10,000 different lipids in humans (most will be in plasma at

some level (>1000 abundant species)

Metabolomics Laboratory (Analytical platforms)

Sciex API 4000 Q/TRAP triple quadrupole mass

spectrometer

Agilent 1200 HPLC system

Column = Agilent Zobax C-18 eclipse (1.8 uM x 50 mM)

Agilent 6490 triple quadrupole mass spectrometer

Agilent 1290 HPLC system

Column = Agilent Zobax C-18 eclipse plus (1.8 uM x 50 mM)

Metabolomics Laboratory (High throughput lipidomics)

Lipid extraction (10 µL plasma)1

Addition of stable isotope/non-physiological standards

Single phase BuOH/MeOH

Centrifuge (supernatant)

LC-MS/MS

Lipid quantification (relative)2

LC ESI-MS/MS

Stable isotope dilution (non-physiological)

Multiple reaction monitoring (scheduled)

>600 lipid species (15 min)

1 Alshehry et al. An efficient single phase method for the extraction of plasma lipids. Metabolites. 2015;5:(2)389-403.

2 Weir et al. Plasma lipid profiling in a large population-based cohort. Journal of lipid research. 2013;54:(10)2898-2908.

Plasma lipid MRM experiment (20min LC gradient)XIC of +MRM (432 pairs): 385.000/159.000 amu Expected RT: 7.6 I... Max. 465.7 cps.

2 4 6 8 10 12 14 16 18

Time, min

0.0

2.0e5

4.0e5

6.0e5

8.0e5

1.0e6

1.2e6

1.4e6

1.6e6

1.8e6

2.0e6

2.2e6

Inte

ns

ity, c

ps

7.20 7.60

%B

100

80

60

40

20

0

• Current capabilities >600 lipid species in 15 min

Separation of isobaric and isomeric speciesXIC of +MRM (159 pairs): 766.539/184.100 amu Expected RT: 0.0 I... Max. 1.1e6 cps.

8.5 9.0 9.5 10.0 10.5 11.0 11.5

Time, min

0.00

1.00e5

2.00e5

3.00e5

4.00e5

5.00e5

6.00e5

7.00e5

8.00e5

9.00e5

1.00e6

1.10e6

Inte

ns

ity, c

ps

10.19

9.70

PhospholipidsSeparation of acyl / alkyl / alkenyl species of the same nominal mass

PC(35:5)PC(O-36:5)

PC(P-36:4)

Separation of isobaric and isomeric species(phosphatidylcholine species)

5x10

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

3

3.2

3.4

3.6

3.8

4

4.2

4.4

4.6

4.8

5

5.2

5.4

5.6

5.8

6

Cpd 146: PC 36:3: +ESI MRM Frag=380.0V CF=0.000 DF=0.000 CID@21.0 (784.6000 -> 184.1000) FT03_0007 TQC 0745.d

4.856

Counts vs. Acquisition Time (min)

4.4 4.45 4.5 4.55 4.6 4.65 4.7 4.75 4.8 4.85 4.9 4.95 5 5.05 5.1 5.15 5.2 5.25 5.3

6x10

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

0.55

0.6

0.65

0.7

0.75

0.8

0.85

0.9

0.95

1

1.05

1.1

1.15

1.2

1.25

1.3

1.35

1.4

1.45

+ESI MRM Frag=380.0V CF=0.000 DF=0.000 CID@21.0 (784.6000 -> 184.1000) 2016_02_13-TQC.d Smooth

6.790

* 6.644

* 6.703

Counts vs. Acquisition Time (min)

6.5 6.52 6.54 6.56 6.58 6.6 6.62 6.64 6.66 6.68 6.7 6.72 6.74 6.76 6.78 6.8 6.82 6.84 6.86 6.88 6.9 6.92 6.94 6.96 6.98 7 7.02 7.04 7.06 7.08 7.1 7.12

PC 36:3

PC 18:1/18:2 PC 16:0/20:3

Characterisation of glycerophospholipid species

High complexity of extended chromatograms

Requires characterisation of lipid species

Characterise based on fragmentation in positive and negative mode

Lithium adducts provide fatty acid specific fragmentation

Hsu et al. (2003) JMS 38(7), 752-763

Mild acid hydrolysis can selectively remove plasmalogens

leaving alkyl- and diacyl- species intact

Synthesis of lipid standards

High throughput lipidomic profileLipid class/subclass No. of species Parent ion Daughter ion

dihydroceramide (dhCer) 6 [M+H]+ 284.3 m/z

ceramide (Cer) 41 [M+H]+ Sphingoid specific m/z

monohexosylceramide (MHC) 6 [M+H]+ 264.3 m/z

dihexosylceramide (DHC) 6 [M+H]+ 264.3 /z

trihexosylcermide (THC) 6 [M+H]+ 264.3 m/z

sulphatide (Sul) 6 [M+H]+ 264.3 m/z

GM1 ganglioside (GM1) 1 [M+2H]2+ 366.2 m/z

GM3 ganglioside (GM3) 6 [M+H]+ 264.3 m/z

sphingomyelin (SM) 36 [M+H]+ 184.1 m/z

phosphatidylcholine (PC) 184 [M+H]+ 184.1 m/z

phosphatidylethanolamine (PE) 124 [M+H]+ NL, 141.0 Da

phosphatidylinositol (PI) 32 [M+NH4]+ NL, 277.0 Da

lysophosphatidylinositol (LPI) 8 [M+NH4]+ NL, 277.0 Da

phosphatidylserine (PS) 7 [M+H]+ NL, 185.0 Da

phosphatidylglycerol (PG) 4 [M+ NH4]+ NL, 189.0 Da

bis(monoacylglycerol)phosphate (BMP) 1 [M+ NH4]+ 339.3 m/z

free cholesterol (COH) 1 [M+ NH4]+ 369.3 m/z

cholesteryl ester (CE) 28 [M+ NH4]+ 369.3 m/z

cholesteryl ester derivatives 18 [M+ NH4]+ 367.3/369.3 m/z

acylcarnitine 14 [M+H]+ 85.1 m/z

diacylglycerol (DG) 20 [M+ NH4]+ NL, fatty acid

triaclyglycerol (TG) 44 [M+ NH4]+ NL, fatty acid

alkyl-diacylglycerol (TG(O)) 3 [M+ NH4]+ NL, fatty acid

TOTAL 602

phosphatidylcholine (PC) 68 [M+H]+ 184.1 m/z

alkylphosphatidylcholine (PC(O)) 22 [M+H]+ 184.1 m/z

alkenylphosphatidylcholine (PC(P)) 24 [M+H]+ 184.1 m/z

lysophosphatidylcholine (LPC) 56 [M+H]+ 184.1 m/z

lysoalkylphosphatidylcholine (LPC(O)) 10 [M+H]+ 104.1 m/z

lysoalkenylphosphatidylcholine (LPC(P)) 4 [M+H]+ 104.1 m/z

phosphatidylethanolamine (PE) 36 [M+H]+ NL, 141.0 Da

alkylphosphatidylethanolamine (PE(O)) 14 [M+H]+ NL, 141.0 Da

alkenylphosphatidylethanolamine (PE(P)) 56 [M+H]+ Acyl specific

lysophosphatidylethanolamine (LPE) 14 [M+H]+ NL, 141.0 Da

lysoalkenylphosphatidylethanolamine (LPE(P)) 4 [M+H]+ NL, 172 Da

Lipid metabolism (partial)

G-3-P

PPAP2

1-acyl-G-3-P

1,2-diacyl-G-3-P

DG

CDP-DG

TG

DGAT

Cho

P-Cho

CDS1

CDP-Cho

PC

Etn

P-Etn

CDP-Etn

PE

CPT1

EPT1

PEMT

PGPPG

PI

pgsA

Cardiolipin

cls

Serine + palmitate

ceramide

monohexosylceramide

dihydroceramide

fatty acid

dihexosylceramide

trihexosylceramide

CerS1-6

DEGS

UGCG

B4GALT6

A4GALT

PC(O) PC(P)

1-O-alkyl-2-acyl-glycerol

DHAP

PEMT

LPC(O)

Lp-PLA2

sphingomyelin

SMGS

acyl-CoA

CDIPT

pgpA

CPT1

fatty alcohol

PE(O)

PE(P)

∆1 Desaturase

LPC LPE

Lp-PLA2

cholesterol

Cholesteryl ester

Sphingolipid metabolism

Cardiolipin metabolism

Plasmalogen metabolism

Glycerolipid metabolism

Phosphatidylcholine

metabolism

Phosphatidylinositol

metabolism

Cholesteryl ester

metabolism

LPC(P)

1-O-alkenyl-2-acyl-Gylcerol

PLC

CPT1

LPE(P)Lp-PLA2

Lp-PLA2LPI

sulphatide

GM3 ganglioside

GM1 ganglioside

Lipid metabolism (partial)

G-3-P

PPAP2

1-acyl-G-3-P

1,2-diacyl-G-3-P

DG

CDP-DG

TG

DGAT

Cho

P-Cho

CDS1

CDP-Cho

PC

Etn

P-Etn

CDP-Etn

PE

CPT1

EPT1

PEMT

PGPPG

PI

pgsA

Cardiolipin

cls

Serine + palmitate

ceramide

monohexosylceramide

dihydroceramide

fatty acid

dihexosylceramide

trihexosylceramide

CerS1-6

DEGS

UGCG

B4GALT6

A4GALT

PC(O) PC(P)

1-O-alkyl-2-acyl-glycerol

DHAP

PEMT

LPC(O)

Lp-PLA2

sphingomyelin

SMGS

acyl-CoA

CDIPT

pgpA

CPT1

fatty alcohol

PE(O)

PE(P)

∆1 Desaturase

LPC LPE

Lp-PLA2

cholesterol

Cholesteryl ester

Sphingolipid metabolism

Cardiolipin metabolism

Plasmalogen metabolism

Glycerolipid metabolism

Phosphatidylcholine

metabolism

Phosphatidylinositol

metabolism

Cholesteryl ester

metabolism

LPC(P)

1-O-alkenyl-2-acyl-Gylcerol

PLC

CPT1

LPE(P)Lp-PLA2

Lp-PLA2LPI

sulphatide

GM3 ganglioside

GM1 ganglioside

Lipidomic strategies at the Baker Institute

Human studies:

Plasma lipid profiling

Hypothesis

Validation

Translation

Cell models Animal models

Human studies:

Clinical trials

Biomarker

Human studies:

Independent cohort

Validation

Translation

Human studies:

prospective trial

BiomarkersLIPID Study (Long-term Intervention with Pravastatin in Ischemic Disease)

LIPID Study (Long-term Intervention with Pravastatin in Ischemic Disease)

Double blind, placebo control study (n=9,000) history of acute MI or hospitalization for unstable angina pectoris cholesterol in the range of 4.0 to 7.0 mmol/L Pravastatin/placebo treatment

Outcome cardiovascular events: myocardial infarct, stroke and CVD death, CVD death

The Lipid Study Group: Long-Term Effectiveness and Safety of Pravastatin in 9014 Patients With Coronary Heart Disease and Average Cholesterol Concentrations: The Lipid Trial Follow-Up. Lancet 2002; 359: 1379-87

Total Placebo Pravastatin

Baseline + Follow-up

4991 2508 2483

Baseline only

1000 494 506

Follow-up only

791 408 383

Event Cases Placebo Pravastatin

Death

(CVD)708 391 317

Any MI 649 367 282

Any stroke 308 175 133

LIPID Study (Baseline characteristics)

Characteristics Controls (n = 4632) Cardiovascular Events

(n = 1359)

p-value

Age 63 (56,69) 66 (60,71) 3.58E-29

Sex (male) 3823 (82.5%) 1146 (84.3%) 5.24E-01

BMI 26.4 (24.2,28.9) 26.5 (24.4,29.3) 8.14E-02

SBP 130 (120,145) 135 (120,150) 7.65E-05

DBP 80 (70,90) 80 (70,90) 3.24E-02

Fasting glucose 5.40 (5.00,5.80) 5.40 (5.00,6.10) 4.58E-05

Cholesterol at baseline 5.64 (5.08,6.22) 5.67 (5.10,6.24) 4.31E-01

HDL at baseline 0.92 (0.79,1.08) 0.90 (0.77,1.05) 8.51E-05

LDL-C at baseline 3.87 (3.38,4.40) 3.92 (3.39,4.45) 9.64E-02

Triglycerides at baseline 1.58 (1.18,2.19) 1.58 (1.18,2.17) 5.68E-01

SMOKING (Never) 1278 (27.6%) 338 (24.9%) 2.10E-01

Atrial fibrillation (y) 51 (1.1%) 32 (2.4%) 5.56E-04

index_ACS 2425 (52.4%) 682 (50.2%) 3.76E-09

Stroke history (y) 148 (3.2%) 89 (6.5%) 4.61E-08

Diabetes history (y) 330 (7.1%) 181 (13.3%) 6.20E-12

Revascularization 542 (11.7%) 215 (15.8%) 5.45E-06

LIPID Study

Hypotheses

That baseline plasma lipid species are associated with incident cardiovascular events independent of traditional risk factors

That plasma lipid species will provide improved prediction of cardiovascular events

Aims

Perform lipidomic profiling on 12,000 samples from LIPID Study

Identify lipid species that associated with future CVD events

Develop multivariate models to predict the risk of future CVD

Plasma lipid profiling of the LIPID cohort

Lipid extraction

10mL plasma

Stable isotope internal standards

Single phase, BuOH / MeOH / H2O

Multiple reaction monitoring (MRM)

11773 samples

420 MRM transitions

352 lipid species in total

Several months

Dataset (> 4 million lipid measurements)

Agilent 6490 triple quadrupole mass spectrometer (1290 HPLC)

LIPID Study (Association of lipids with cardiovascular outcomes)

Outcomes

Cardiovascular events (MI, stroke and CVD death)

CVD death

Cox regression (log-transformed, Stdev normalised)

Adjust for covariates

Hazard ratio (95% confidence interval),

Correction for multiple comparison (Benjamini-Hochberg)

Lipid classes associated with CVD death

Dihydroceramide Ceramide Monohexosylceramide Dihexosylceramide Trihexosylceramide GM3 ganglioside Sphingomyelin Phosphatidylcholine Alkylphosphatidylcholine Alkenylphosphatidylcholine Lysophosphatidylcholine Lysoalkylphosphatidylcholine Phosphatidylethanolamine Alkylphosphatidylethanolamine Alkenylphosphatidylethanolamine Lysophosphatidylethanolamine Phosphatidylinositol Lysophosphatidylinositol Phosphatidylserine Phosphatidylglycerol Cholesterol Cholesteryl ester Diacylglycerol Triacylglycerol Oxidised PC Oxidised CE

6.58E-018.12E-019.09E-028.98E-011.10E-018.12E-019.09E-028.12E-019.24E-014.75E-013.20E-018.12E-012.81E-028.12E-018.12E-019.09E-028.12E-018.12E-012.51E-026.58E-019.17E-019.32E-019.09E-029.17E-018.12E-014.36E-01

0.5 1.0 2.0

Hazard Ratio (95% Confidence intervals)

55 lipid species associated with cardiovascular events

95 lipid species associated with cardiovascular death

Sphingolipids associated with CVD death

Cer(d18:0/16:0)

Cer(d18:0/18:0)

Cer(d18:0/20:0)

Cer(d18:0/22:0)

Cer(d18:0/24:0)

Cer(d18:0/24:1)

Cer(d18:1/16:0)

Cer(d18:1/18:0)

Cer(d18:1/20:0)

Cer(d18:1/22:0)

Cer(d18:1/24:0)

Cer(d18:1/24:1)

HexCer(d18:1/16:0)

HexCer(d18:1/18:0)

HexCer(d18:1/20:0)

HexCer(d18:1/22:0)

HexCer(d18:1/24:0)

HexCer(d18:1/24:1)

2.74E-02

2.10E-03

8.48E-01

6.73E-01

7.70E-01

2.11E-01

4.89E-05

1.69E-03

6.77E-01

3.78E-01

2.69E-01

8.03E-02

2.80E-02

4.73E-02

5.95E-01

3.35E-01

9.11E-02

1.56E-01

0.5 1.0 2.0

Hazard Ratio (95% Confidence intervals)

dihydroceramide

ceramide

monohexosyl-ceramide

Lipid metabolism in cardiovascular disease

G-3-P

PPAP2

1-acyl-G-3-P

1,2-diacyl-G-3-P

DG

CDP-DG

TG

DGAT

Cho

P-Cho

CDP-Cho

PC

Etn

P-Etn

CDP-Etn

PE

EPT1

PEMT

PI

Serine + palmitate

ceramide

monohexosylceramide

dihydroceramide

fatty acid

dihexosylceramide

trihexosylceramide

DEGS

UGCG

B4GALT6

A4GALT

acyl-CoA

CDIPT

CPT1

LPC LPE

Lp-PLA2

Sphingolipid metabolism

Glycerolipid metabolism

Phosphatidylcholine

metabolism

Phosphatidylinositol

metabolism

CerS1 (18:0)

CerS2 (20:0 -24:0)

CerS5/6 (16:0)

(16:0)

(18:0)

LIPID Study(Prediction of future cardiovascular events)

LIPID Study (Prediction of future cardiovascular events)

Correlation minimization to reduce collinear lipids

352 lipids reduced to144 lipid species

Create a base model (Cox regression) with significant covariates (22 covariates)

Add lipids sequentially to the base model

Select lipids based on Akaike Information Criterion (AIC)

Within a cross validated framework (5 x 200)

Rank the top 20 lipids.

Create a series of models (Cox regression) by adding lipids sequentially to the

base model

Within a cross validated framework (5 x 200)

Determine C-statistics and NRI (categorical and continuous) relative to the base model and

calculate 95% CI

LIPID Study(Prediction of future cardiovascular events)

0.640

0.645

0.650

0.655

0.660

0.665

0.670

0.675

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Ave

rage

C-s

tati

stic

s

No of Lipids

Cardiovascular events

0.690

0.700

0.710

0.720

0.730

0.740

0.750

0.760

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Ave

rage

C-s

tati

stic

s

No of Lipids

Cardiovascular death

LIPID Study: Plasma lipids improve upon traditional risk factors to predict future cardiovascular events

Cardiovascular events C-Statistic (95% CI)NRI*

Categorical (95% CI) Continuous (95% CI)

Base model (22) 0.645 (0.644 - 0.646) Reference Reference

Base model + 8 lipids 0.666 (0.665 - 0.667) 0.082 (0.080 - 0.084) 0.291 (0.287 - 0.295)

% change 2.1 8.2 29.1

Cardiovascular death C-Statistic (95% CI)NRI*

Categorical (95% CI) Continuous (95% CI)

Base model (22) 0.703 (0.701 - 0.705) Reference Reference

Base model + 8 lipids 0.741 (0.739 - 0.742) 0.103 (0.099 - 0.106) 0.420 (0.414 - 0.426)

% change 3.8 10.3 42.0

* NRI based on a categorical model of <10, 10–15, and >15% 5-years risk.

LIPID Study (Lipid species in predictive models)

RankCardiovascular

events

Cardiovascular

death

1 PI 38:3 PI 38:3

2 PE 38:3 PE 38:3

3 PC(O-36:1) PI 38:2

4 PI 38:2 PC(O-34:2)

5 PS 36:1 Cer 16:0

6 PC(O-34:2) SM 42:1

7 GM3 16:0 PC(P-36:2)

8 PE(P-20:0/20:4) PC 38:5

Validation of findings in the ADVANCE Study

The ADVANCE Study(Action in Diabetes and Vascular disease: preterAx and diamicroN-MR Controlled Evaluation)

Double blind placebo controlled multicenter trial

T2D patients, high risk CVD (n=11,140)

Design (2x2)

Blood pressure control x intensive glucose control

Outcomes 5 years follow up

Primary outcomes (cardiovascular events)

o Myocardial infarct, stroke and CVD death

Patel et.al, J Hum Hypertens 2005;19:S27-S32.

The ADVANCE Study (sub-cohort selection)

Design (case-cohort)

Random selection of 3,154 participant

o 334 cases (MI, stroke, CVD death)

o 2,820 non-cases

Add all available cases from the remainder trial to the cohort

o 625 cases (364 CVE, 261 renal cases)

Cardiovascular

eventsCVD death Stroke

Myocardial

infarct

Cases 698 355 192 238

Non-cases 3,081 3,424 3,587 3,541

Prediction of cardiovascular disease death

ADVANCE Study (Validation of lipid species from LIPID Study)

C-Statistics (95% CI)NRI

Categorical (95% CI) Continuous (95% CI)

Base model 0.695 Reference Reference

Base + Lipids

(8 features) 0.711 0.045 0.292

% change 1.6 4.5 29.2

C-Statistics (95% CI)NRI

Categorical (95% CI) Continuous (95% CI)

Base model 0.765 Reference Reference

Base + Lipids

(8 features)0.782 0.102 0.392

% change 1.7 10.2 39.2

Prediction of cardiovascular events

Categorical reclassification: <10%, 10-15%, >15%

Plasma lipid species in cardiovascular disease (Summary)

Plasma lipids are associated with future cardiovascular events in secondary prevention

Independent of traditional risk factors

Biological insight

Plasma lipids can predict cardiovascular events

Improve performance of traditional risk factors

Validated on an independent cohort

ADVANCE Study

Translation

May be different for each clinical setting

Likely to involve simple lipid ratios

Lipidomic strategies at the Baker Institute

Human studies:

Plasma lipid profiling

Hypothesis

Validation

Translation

Cell models Animal models

Human studies:

Clinical trials

Biomarker

Human studies:

Independent cohort

Validation

Translation

Human studies:

prospective trial

Plasmalogen modulation in cardiometabolic disease

polar head group

Anti-atherogenic •anti-oxidant•anti-inflammatory •cholesterol efflux

vinyl ether

Plasmalogens and atherosclerosis

Plasmalogens and cardiometabolic disease

Baker IDI clinical cohort (n=220)

Plasmalogens negatively associated with stable and unstable coronary artery disease (Meikle et al, ATVB. 2011;31(11):2723-32)

LIPID / ADVANCE Study (n=5,991; n=3770)

Plasmalogens negatively associated with future CVE (CVD death)

(Alshehry et al, Circulation. 2016;134(21):1637-1650)

AusDiab / San Antonio Family Heart Study (n=1,000; n=1200)

Plasmalogens negatively associated with type 2 diabetes

(Meikle et al, PLoS One. 2013;8(9):e74341)

The Australian Imaging Biomarker and Lifestyle Study (n=1,000)

Plasmalogens negatively associated with Alzheimer’s disease

Can plasmalogen upregulation attenuate atherosclerosis?

Biosynthetic pathway of plasmalogens

DHAP

1-acyl-DHAP

Acyl-CoA

1-O-alkyl-DHAP

ADHAP-S

Long chain fatty alcohol

Far-1*/2

Acyl-CoA

1-O-alkyl-DHAP

1-O-alkyl-G3P

1-O-alkyl-2-acyl-G3P

1-O-alkyl-2-acyl-Gylcerol

1-O-alkyl-2-acyl-GPC 1-O-alkyl-2-acyl-GPE

PC-plasmalogen PE-plasmalogen

Peroxisome

DHAP-AT

AADHAP-R

Δ1 desaturase

C-PT

Dietary intake

PH

AAG3P-AT

ER

E-PT

1-alkyl-glycerol

Cytosol

1-O-alkenyl-2-acyl-Gylcerol

C-PT

PEMT

AG kinase

PLC

Plasmalogen upregulation to prevent atherosclerosis in ApoE mice (Aliki Rasmiena)

C57/BL6 (controls)ApoE-/-

ApoE-/-GPx-/-

Weekly weighing and twice weekly scruffing

6 wk 18 wk

high fat diet +/- 2% batyl alcohol diet

0 wk

chow

batyl alcohol

plasmalogen

Analysis

Lipid profiles of mouse plasma and heart

Sudan IV staining for lesions/plaque in aorta

Detection of inflammatory and oxidative markers in aorta and aortic sinus sections

Batyl alcohol supplementation increased plasmalogen levels in plasma

Data are median (interquartiles) of N = 9 – 10 /group. *** indicates P<0.001

0

200

400

600

800

C57/BL6 ApoE-/- ApoE-/-GpX1-/-

PE

pla

sm

alo

ge

n

(nm

ol/μ

mo

l PC

)

0% BA

2% BA

***

***

***

Batyl alcohol supplementation increased plasmalogen levels in heart

0

200

400

600

C57/BL6 ApoE-/- ApoE-/-GpX1-/-

PE

pla

sm

alo

ge

n

(nm

ol/μ

mo

l PC

)

0% BA

2% BA

Data are median (interquartiles) of N = 9 – 10 /group. *** indicates P<0.001

***

***

***

Assessment of aortic lesions

0% BA

2% BA

C57/BL6 ApoE -/- ApoE-/-GPx1-/-

Diagram obtained from JACC

Batyl alcohol supplementation attenuated atherosclerosis in ApoE deficient mice

0

4

8

12

16

C57/BL6 ApoE KO ApoE/GPX DKO

% p

laq

ue

are

a

0% Batyl alcohol

2% Batyl alcohol

***

***

-71%

-69%

Data are mean ± SEM, expressed as % plaque area , n = 10/group.

Data were analysed using student t-tests and compared to 0% BA treated group of the corresponding genotypes.

*** indicates P< 0.001

Plasmalogen upregulation to prevent atherosclerosis in ApoE mice

Batyl alcohol containing diet successfully elevated plasmalogen concentration in plasma and heart

Plasmalogen up-regulation reduced atherosclerosis in ApoE-/- and ApoE-/-GPx-/-

Differential effects on inflammation and oxidative stress were observed in ApoE-/- and ApoE-/-GPx-/- mouse model

Acknowledgments

Baker IDI Heart and Diabetes InstituteBronwyn Kingwell

Paul NestelGerard Wong

Metabolomics Lab

King Fahad Medical City Zahir Alshehry

Graham HillisSophia ZoungasJohn Chalmers

Mark Woodward

Malcolm McConville

John SimesAndrew TonkinDavid SullivanAdrienne Kirby

Liz Barnes