F2-Isoprostanes in HDL are bound to neutral lipids and phospholipids

Article abstract of DOI:10.1080/10715762.2016.1250262

Low HDL cholesterol (HDL-C) is a risk factor for coronary artery disease (CAD). However, interventions that raise HDL-C have failed to reduce cardiovascular events. We previously reported that HDL is the main carrier of plasma F₂-isoprostanes (F₂-IsoPs) that are markers of oxidative stress formed upon oxidation of arachidonic acid. F₂-IsoPs are predominantly associated with phospholipids. However, there is evidence that F₂-IsoPs in the liver of rats treated with carbon tetrachloride associate with the neutral lipids. To date it is not known whether F₂-IsoPs are found in the neutral lipids in HDL in humans. Possible candidate neutral lipids include cholesteryl esters, triglycerides, diglycerides, and monoglycerides. This study aimed to identify the lipid classes within native and oxidized HDL that contain F₂-IsoPs. We showed that F₂-IsoPs in HDL are bound to neutral lipids as well as phospholipids. HDL-3 contained the highest concentration of F₂-IsoPs in all lipid classes before and after in vitro oxidation. Using targeted LC/MS and high resolution MS, we were unable to provide conclusive evidence for the presence of the synthesized standards 15(R)-15-F_2t-isoP cholesterol and 1-ent-15(RS)-15-F_2t-isoprostanoyl-sn-glycerol in the neutral lipids of HDL. Our findings show that oxidized lipids such as F₂-IsoPs are found in the core and surface of HDL. However, the exact molecular species remain to be definitively characterized. Future studies are required to determine whether the presence of F₂-IsoPs in neutral lipids alters HDL function.

Full text of DOI:10.1080/10715762.2016.1250262

Free Radical Research
ISSN: 1071-5762 (Print) 1029-2470 (Online) Journal homepage: http://www.tandfonline.com/loi/ifra20
F2-Isoprostanes in HDL are Bound to Neutral Lipids
and Phospholipids
Julie M Proudfoot, Anne E Barden, Kevin D Croft, Jean-M Galano, Thierry
Durand, Valerie Bultel-Ponce, Martin Giera & Trevor A Mori
To cite this article: Julie M Proudfoot, Anne E Barden, Kevin D Croft, Jean-M Galano,
Thierry Durand, Valerie Bultel-Ponce, Martin Giera & Trevor A Mori (2016): F2-Isoprostanes
in HDL are Bound to Neutral Lipids and Phospholipids, Free Radical Research, DOI:
10.1080/10715762.2016.1250262
To link to this article: http://dx.doi.org/10.1080/10715762.2016.1250262
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Date: 21 October 2016, At: 11:41

F2-Isoprostanes in HDL are Bound to Neutral Lipids and Phospholipids
Julie M Proudfoot1*, Anne E Barden1, Kevin D Croft1, Jean-M Galano2, Thierry
Durand2, Valerie Bultel-Ponce2, Martin Giera3, Trevor A Mori1.
1. School of Medicine and Pharmacology, University of Western Australia,
Royal Perth Hospital Unit, Perth, Australia
2. Institut des Biomolécules Max Mousseron (IBMM) UMR 5247, CNRS,
Université de Montpellier, ENSCM, Montpellier, France
3. Center for Proteomics and Metabolomics, Leiden University Medical Center,
Leiden, The Netherlands
Julie M Proudfoot
School of Medicine and Pharmacology, University of Western Australia, MRF
Building, GPO Box X2213, Perth, Australia 6847.
Tel: +61 8 92240391. Fax: +61 8 92240246.
Email: julie.proudfoot@uwa.edu.au
Anne E Barden
School of Medicine and Pharmacology, University of Western Australia, MRF
Building, GPO Box X2213, Perth, Australia 6847.
Tel: +61 8 92240272. Fax: +61 8 92240246.
Email: anne.barden@uwa.edu.au
Kevin D Croft
School of Medicine and Pharmacology, University of Western Australia, MRF
Building, GPO Box X2213, Perth, Australia 6847.
Tel: +61 8 92240275. Fax: +61 8 92240246.
Email: kevin.croft@uwa.edu.au
Jean-Marie Galano
Institut des Biomolécules Max Mousseron (IBMM), UMR 5247 - CNRS - UM
– ENSCM, Faculté de Pharmacie, 15 Av. Ch. Flahault, BP 14491, F-34093
Montpellier Cedex 05, France.
Tel: 33-4-11-75-95-33. Fax: 33-4-11-75-95-53.
Email: Jean-Marie.Galano@umontpellier.fr
Thierry Durand
Institut des Biomolécules Max Mousseron (IBMM), UMR 5247 - CNRS - UM
– ENSCM, Faculté de Pharmacie, 15 Av. Ch. Flahault, BP 14491, F-34093
Montpellier Cedex 05, France.
Tel: 33-4-11-75-95-58. Fax: 33-4-11-75-95-53.
Email: Thierry.Durand@umontpellier.fr

Valérie Bultel-Poncé
Institut des Biomolécules Max Mousseron (IBMM), UMR 5247 - CNRS - UM
– ENSCM, Faculté de Pharmacie, 15 Av. Ch. Flahault, BP 14491, F-34093
Montpellier Cedex 05, France.
Tel: 33-4-11-75-95-32. Fax: 33-4-11-75-95-53.
Email: Valerie.Bultel@umontpellier.fr
Martin Giera
Center for Proteomics and Metabolomics, Leiden University Medical Center,
Albinusdreef 2, 2300RC Leiden, The Netherlands.
Tel: 0031 71 5269526.
Email: m.a.giera@lumc.nl
ORCiD: 0000-0003-1684-1894.
Trevor A Mori
School of Medicine and Pharmacology, University of Western Australia, MRF
Building, GPO Box X2213, Perth, Australia 6847.
Tel: +61 8 92240273. Fax: +61 8 92240246.
Email: trevor.mori@uwa.edu.au
*Address correspondence to:
Julie M Proudfoot, School of Medicine and Pharmacology, University of
Western Australia, MRF Building, GPO Box X2213, Perth, Australia 6847.
Tel: +61 8 92240391. Fax: +61 8 92240246.
Email: julie.proudfoot@uwa.edu.au
WORD COUNT : 6,273

Abbreviations:
CAD, coronary artery disease; Chol, cholesterol; F2-IsoP, F2-isoprostane; Chol-IsoP,
Cholesterol-F2-isoprostane; MG-IsoP, monoglyceride-F2-isoprostane; CE, cholesterol
ester; TG, triglyceride; MG, monoglyceride; DG, diglyceride; FA, fatty acid; PL,
phospholipid; PAPC, 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine;
oxPAPC, oxidized PAPC; AAPH, 2,2′-Azobis(2-methylpropionamidine)
dihydrochloride; EDTA, ethylenediaminetetraacetic acid; PBS, phosphate buffered
saline; ADP, adenosine diphosphate; PFBBr, Pentafluorobenzylbromide; DIPEA, N,N-
diisopropylethylamine; BSTFA, N,O-bis-(trimethylsilyl)trifluoroacetamide with 1%
trimethylchlorosilane (BSTFA-TMCS, 99:1); BHT, butylated hydroxytoluene; LC/MS,
liquid chromatography/mass spectrometry; GC-MS, gas chromatography-mass
spectrometry; HPLC, high pressure liquid chromatography; MeOH, methanol; THF,
+
tetrahydrofuran; NH4 , ammonium ion; Li+, lithium ion; SPE, solid phase extraction.

F2-Isoprostanes in HDL are Bound to Neutral Lipids and Phospholipids
Low HDL cholesterol (HDL-C) is a risk factor for coronary artery disease (CAD).
However, interventions that raise HDL-C have failed to reduce cardiovascular events.
We previously reported that HDL is the main carrier of plasma F2-isoprostanes (F2-
IsoPs) that are markers of oxidative stress formed upon oxidation of arachidonic acid.
F2-IsoPs are predominantly associated with phospholipids. However, there is evidence
that F2-IsoPs in the liver of rats treated with carbon tetrachloride associate with the
neutral lipids. To date it is not known whether F2-IsoPs are found in the neutral lipids in
HDL in humans. Possible candidate neutral lipids include cholesteryl esters,
triglycerides, diglycerides and monoglycerides.This study aimed to identify the lipid
classes within native and oxidized HDL that contain F2-IsoPs. We showed that F2-IsoPs
in HDL are bound to neutral lipids as well as phospholipids. HDL-3 contained the
highest concentration of F2-IsoPs in all lipid classes before and after in vitro oxidation.
Using targeted LC/MS and high resolution MS, we were unable to provide conclusive
evidence for the presence of the synthesized standards 15(R)-15-F2t-isoP cholesterol and
1-ent-15(RS)-15-F2t-isoprostanoyl-sn-glycerol in the neutral lipids of HDL. Our
findings show that oxidized lipids such as F2-IsoPs are found in the core and surface of
HDL. However, the exact molecular species remain to be definitively characterised.
Future studies are required to determine whether the presence of F2-IsoPs in neutral
lipids alters HDL function.

Keywords:
HDL, oxidized lipids, F2-isoprostanes, lipoproteins, mass spectrometry.
Funding Details:
This work was supported by the National Heart Foundation of Australia under
grant number G09P4425 and the Medical Research Foundation at Royal Perth Hospital.

1. INTRODUCTION
Many human population studies have demonstrated that higher HDL cholesterol levels
are associated with decreased risk of CAD [1,2] and that low levels are correlated with
increased CAD risk [3]. However, randomised clinical trials designed to raise HDL
cholesterol, have failed to reduce the incidence of CAD [4-6]. This has prompted a shift
in focus to examine whether an increase in functional HDL particles that may be
distinct from HDL cholesterol levels [7], is responsible for the apparent protective effect
of HDL on CAD risk.
HDL has many antiatherogenic functions, including the ability to promote cholesterol
efflux from cells [8], protecting LDL from oxidation [9] and exhibiting anti-
inflammatory properties [10]. These attributes are related to HDL composition and
structural characteristics as described recently [11,12]. Cholesterol efflux capacity, and
antioxidative, antithrombotic, anti-inflammatory and antiapoptotic activities, are
predominantly associated with small, dense, protein-rich HDL3 [11,12].
We have previously shown that HDL is the main carrier of the oxidized lipid species,
F2-IsoPs, in plasma (about 60% of total plasma F2-IsoPs) and that HDL3 particles
contain significantly elevated concentrations of F2-IsoPs compared with HDL2 particles
[13]. Thus the presence of F2-IsoPs in HDL has the potential to influence many of the
functions of HDL. However, the location of these oxidized lipid species within HDL
particles is unknown, as is their possible effect on the antiatherogenic properties of
HDL.
F2-IsoPs result from free radical oxidation of arachidonic acid [14] so any lipids within
lipoproteins containing arachidonic acid are a potential source of F2-IsoPs following
oxidation. In liver extracts from rats treated with carbon tetrachloride, F2-IsoPs were
predominantly associated with phospholipids [15]. F2-IsoPs detected in less polar
fractions were not further analysed and were thought to contain F2-IsoPs esterified to

other phospholipids and/or neutral lipids [15]. To date these neutral lipids have not been
characterized. F2-IsoP-containing PLs in human HDL have been described [15,16,17].
However, to our knowledge, no one has measured F2-IsoPs in neutral lipids in HDL in
humans. Possible candidate neutral lipids include cholesteryl esters (CE), triglycerides
(TG), diglycerides (DG) and monoglycerides (MG).
This study aimed to examine F2-IsoPs in the lipids of HDL and LDL in humans. In
order to achieve this, lipids were extracted from lipoproteins, separated into lipid classes
and each fraction was assayed for the presence of F2-IsoPs. Cholesterol-F2-isoprostane
(Chol-IsoP) (Figure 1) and monoglyceride-F2-isoprostane (MG-IsoP) (Figure 1) were
synthesized as potential F2-IsoP-containing molecules within HDL and LDL.

2. MATERIALS AND METHODS
2.1. Reagents
Cholesteryl linoleate (standard CE) was purchased from Sigma-Aldrich (St. Louis,
MO). 1-palmitoyl-2-oleoyl-3-linoleoyl-rac-glycerol (standard TG), 2-arachidonoyl
glycerol (standard MG), 1-stearoyl-2-arachidonoyl-sn-glycerol (standard DG), 15-F2t-
isoprostane (8-iso PGF2α) and 15-F2t-isoprostane-d4 were purchased from Cayman
Chemical Co. (Ann Arbor, MI); 1-palmitoyl-2-arachidonoyl-sn-glycero-3-
phosphocholine (PAPC) (standard PL) from Avanti Polar Lipids; 2,2′-Azobis(2-
methylpropionamidine) dihydrochloride (AAPH) from Aldrich Chemical Co.
(Milwaukee, WI); Disodium EDTA from ICN Biomedicals (CA); PBS from Gibco™
Invitrogen (Carlsbad, CA); silylating agent N,O-bis-(trimethylsilyl)trifluoroacetamide
with 1% trimethylchlorosilane (BSTFA-TMCS, 99:1) (BSTFA) from Pierce Chemicals
(Rockford, IL); Cholesteryl linoleate, Pentafluorobenzylbromide (PFBBr), N,N-
diisopropylethylamine (DIPEA), butylated hydroxytoluene (BHT) and all other
chemicals were from Sigma-Aldrich (St. Louis, MO). All solvents were HPLC grade or
higher, methanol (MeOH) (Burdick and Jackson, Muskegon, MI, USA) and water
(Optima, Thermo Fisher Scientific, Victoria, Australia) were LC/MS grade. Lithium
hydroxide reagent grade, 2-propanol, ammonium formate and formic acid LC/MS grade
were from Sigma Aldrich (St. Louis, MO, USA). tert-Butyldimethylsilyl chloride,
dicyclohexylcarbodiimide, 4-dimethylaminopyridine, and tetra-n-butylammonium
fluoride were purchased from Sigma-Aldrich (St. Louis, MO).
2.2. Measurement of F2-isoprostanes
F2-IsoPs were measured by GC-MS using electron capture negative ionization as
previously described [13,18].
2.3. Preparation of Lipoproteins to Determine Distribution of F2-isoprostanes

Blood was collected from healthy, non-smoking men and women with written informed
consent and ethics approval from the Human Ethics Committee of the University of
Western Australia. Plasma was prepared as previously described, from blood collected
into EDTA (final concentration 1 mg/ml), reduced glutathione (final concentration 1
mg/ml) and BHT (final concentration of 40 µg/ml) and stored at -80 oC [13].
Lipoproteins were isolated from plasma adjusted to density 1.26 g/ml with gradient
solutions layered on top [13]. Samples were ultracentrifuged at 250 000 g for 24 h at 15
oC using a Beckman L-90K ultracentrifuge (Beckman Instruments, Australia) and
fractions aspirated. Lipoproteins were stored at 4 oC and used within 14 days. Total
Cholesterol was measured using the Cholesterol reagent enzymatic method (Roche
Diagnostics GmbH, Germany). PLs were measured using a kit (Wako Pure Chemical
Industries Ltd, Japan) and MG, DG and TG were quantitated using triglycerides reagent
(Thermo Scientific, France).
In some experiments, lipoproteins were diluted to 0.35 mM Cholesterol in PBS and
oxidized with 5 mM AAPH at 37 oC (n=3-4).
2.4. Preparation of Oxidized Lipid Standards (Figure 1)
2.4.1.Synthesis of Monoglyceride - F2-isoprostane (MG-IsoP) 1-ent-15(RS)-15-F2t-
isoprostanoyl-sn-glycerol
Following our previously described synthesis of 15-F2t-IsoP [19], fully tert-
butyldimethylsilyl-protected ent-15(RS)-15-F2t-IsoP was coupled with 1,2-bis-tert-
butyldimethylsilyl-2-(R)-glycerol with dicyclohexylcarbodiimide and 4-
dimethylaminopyridine. The second step consisted of tert-butyldimethylsilyl cleavage
using tetra-n-butylammonium fluoride followed by Kaburagi and Kishi [20] work-up to
furnish 1-ent-15(RS)-15-F2t-isoprostanoyl-sn-glycerol in 35% overall yield.

1H-NMR (300MHz, CD3OD): 5.52 – 5.37 (m, 4H), 4.12-3.78 (m, 5H), 3.65-3.48 (m,
3H), 2.70-2.60 (m, 1H), 2.50-2.41 (m, 1H), 2.37-2.23 (m, 2H), 2.13-1.99 (m, 5H), 1.70-
1.6 (m, 2H), 1.55-1.42 (m, 3H), 1.36-1.25 (m, 6H), 0.88 (br t, 1H).
2.4.2.Synthesis of Cholesterol - F2-isoprostane (Chol-IsoP) 15(R)-15-F2t-isoprostane
cholesterol
Following our previously described synthesis of 15-F2t-IsoP [19], fully tert-
butyldimethylsilyl-protected 15(R)-15-F2t-IsoP was coupled with cholesterol with
dicyclohexylcarbodiimide and 4-dimethylaminopyridine. The second step consisted in
tert-butyldimethylsilyl cleavage using tetra-n-butylammonium fluoride followed by
Kaburagi and Kishi [20] work-up to furnish 15(R)-15-F2t-isoprostane cholesterol in 26%
overall yield.
1H-NMR (300MHz, CD3OD): 5.50 (dd, J=5.0 Hz, 1H), 5.52-5.32 (m, 4H), 4.57-4.48
(m, 1H), 4.00-3.91 (m, 2H), 3.87-3.81 (m, 1H), 2.71-2.64 (m, 1H), 2.50-2.41 (m, 1H),
2.30-2.25 (m, 4H), 2.10-1.98 (m, 6H), 1.97-1.78 (m, 4H), 1.68-1.24 (m, 23H), 1.21-0.96
(m, 9H), 1.03 (s, 3H), 0.92 (d, J=6.6 Hz, 3H), 0.88 (br t, 3H), 0.87 (d, J=1.0 Hz, 3H),
0.85 (d, J=1.0 Hz, 3H), 0.70 (s, 3H).
2.4.3.Preparation of Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine
(oxidized PAPC)
PAPC was oxidized according to the method of Longmire et al. [21]. Briefly, PAPC (10
mg in 0.4 ml ethanol) was incubated at 37 oC with a final concentration of 5 µM ferric
chloride, 2 mM ADP and 1 mM ascorbic acid in 50 mM Tris-HCl pH 7.4 containing
150 mM KCl in a total volume of 3.6 ml, with intermittent mixing. After 45 min, lipids
were extracted with chloroform/methanol (2:1) [22], containing 0.005% BHT, followed
by extraction with 4 ml equivalents of aqueous NaCl (0.9%) to 20 ml equivalents of
chloroform/MeOH (2:1). The lower layer was dried under vacuum. Oxidized PAPC

(oxPAPC) was resuspended in 90% methanol/10% water and purified on a Hypersil
Gold C8 column (5 µm) (250 x 4.6 mm) (Thermo Fisher Scientific, Scoresby, Victoria,
Australia) eluted isocratically with 90% methanol/10% water at 1 ml/min using a
Hewlett Packard 1100 series HPLC (Hewlett Packard). Fractions were dried and
resuspended in methanol and F2-IsoPs were measured by GC-MS [13,18].
2.5. Extraction and Separation of Lipids from Plasma HDL and LDL
Lipids were extracted from plasma HDL and LDL using chloroform/MeOH (2:1) [22],
dried under vacuum, and separated using two different solid phase extraction (SPE)
methods.
a) The first SPE method used a Supelco 55261-U hybrid SPE phospholipid cartridge
(Figure 2), washed with water and conditioned with 2-propanol containing 1% formic
acid, to retain phospholipids on the cartridge. Samples free of phospholipids were then
analysed for Chol-IsoP using high resolution LC-MS. HDL lipid extracts dissolved in 2-
propanol containing 1% formic acid were applied to the SPE column and the eluate
collected. The neutral lipids were dried under nitrogen and resuspended in 50% 2-
propanol.
b) The second SPE method used Bond Elut NH2 columns (200 mg, Varian, Agilent
Technologies) (Figure 3) to separate lipid extracts into lipid classes as described by
Agren et al. [23], prior to LC/MS analysis. Lipid standards of CE, TG, MG, DG and PL,
as well as MG-IsoP, Chol-IsoP, oxPAPC and 15-F2t-IsoP, were applied to separate
columns and fractions collected to identify their elution pattern. Standards and lipid
extracts were eluted under the same conditions. SPE columns were prewashed with 0.6
ml acetone/water (7:1) and n-hexane (2×1ml). The standards or dried lipid extracts
(approximately 3 mg lipid) were resuspended in 0.2 ml of n-hexane/methyl tert-butyl
ether/acetic acid (100:3:0.3) and applied to the column under gravity. The column was

eluted sequentially with 2.5 ml n-hexane (CE fraction), 3 ml n-hexane/chloroform/ethyl
acetate (100:5:5) (TG fraction), 2.5 ml chloroform/2-propanol (2:1) (MG and DG
fraction), 3 ml chloroform/MeOH/acetic acid (100:2:2) (FA fraction) and 3.5 ml
MeOH/chloroform/water (10:5:4) (PL fraction). All column elution solutions contained
4µg/ml BHT to prevent artefactual production of IsoPs. The CE standard was identified
in fractions using the Chol reagent (Roche Diagnostics GmbH, Germany), the TG, MG
and DG standards were identified using the TGs reagent (Thermo Scientific, France)
and the PL standard identified using a kit (Wako Pure Chemical Industries Ltd, Japan);
MG-IsoP and Chol-IsoP standards, oxPAPC and 15-F2t-IsoP standard were identified
following concentration under vacuum, hydrolysis and GC-MS [13,18]; HDL lipid
extracts were eluted, fractions concentrated under vacuum, then hydrolyzed and
analyzed for F2-IsoPs by GC-MS [13,18].
2.6. Purification of the neutral lipid (MG/DG) fraction
For more detailed analysis, the MG/DG fraction from chromatography on Bond Elut
NH2 columns was further purified using HPLC (Figure 4). The MG/DG fraction was
resuspended in acetonitrile and fractionated on a Hypersil Gold C8 column (5 µm) (250
× 4.6 mm) using a Hewlett Packard 1100 series HPLC to separate CE, MG and DG.
The column was eluted at 1 ml/min with the following mobile phases A: acetonitrile, B:
2-propanol and C: MeOH. Chromatography used a gradient of 100% A at 0 min to
100% B at 40 min, followed by 100% B from 40 to 60 min and 100% C from 60 to 80
min. 1 ml fractions were collected, dried and F2-IsoPs were measured by GC-MS
[13,18].
2.7. LC/MS analysis of HPLC purified neutral lipid
HPLC fractions 4 and 11 (Figure 4) were analysed using LC/MS consisting of two
Agilent 1290 UPLC binary pumps coupled to an Agilent 6460 triple quadrupole tandem

mass spectrometer with a Jetstream source (Figure 5). The column used was an Eclipse
XDB-C18 (3.5 µm) (2.1 × 150 mm). Data processing was performed using Agilent
MassHunter software.
2.7.1. LC/MS of the Chol-IsoP standard using tandem mass spectrometry
(i) Conditions identifying the Chol fragments: (Figure 5A) Chromatography used the
following mobile phases: A: 50% H2O, 20% MeOH, 30% THF, 10 mM ammonium
formate, 0.1% formic acid; B: 5% H2O, 20% MeOH, 75% THF, 10 mM ammonium
formate, 0.1% formic acid with a gradient of 100% A at 0 min to 100% B at 8 min,
followed by 100% B from 8 to 10 min and finally 100% A from 10 to 12 min. The flow
rate was 500 µl/min and the column temperature was 40 oC. The mass spectrometer was
operated in positive electrospray ionization mode (ESI+) with the gas temperature at
350 oC, gas flow 5 l/ min, nebulizer 45 psi, sheath gas 250 oC, sheath gas flow 11 l/min,
+
capillary 3,500 V and charging 500 V. Mass transitions for detection were M+NH4
+
740.5→369.2 (cholestane) (Collision-induced dissociation (CID) 30), M+NH4
740.5→161 (CID 30) (collision energy 15V).
(ii) Conditions identifying the IsoP fragments: (Figure 5B) Chromatography used the
following mobile phases: A: 50% H2O, 20% MeOH, 30% THF, 10 mM lithium
hydroxide, 0.1% formic acid; B: 5% H2O, 20% MeOH, 75% THF, 10 mM lithium
hydroxide, 0.1% formic acid, with a gradient of 100% A at 0 min to 60% B at 1 min, to
100% B at 5 min then 100% B to 7 min and finally 100% A from 7 to 10 min. Flow rate
was 350 µl/min and the column temperature was 20 oC. The mass spectrometer was
operated in ESI+ mode with the gas temperature at 300 oC, gas flow 8 l/min, nebulizer
45 psi, sheath gas 250 oC, sheath gas flow 8 l/min, capillary 3500 V and charging 500
V. Mass transitions for detection were M+Li+ 729.5→361 (CID 40), M+Li+ 729.5→343
(CID 45) (collision energy 45V).

2.7.2. LC/MS of the Chol-IsoP standard using high-resolution mass spectrometry
HDL lipid extracts were analysed using a Dionex Ultimate system (Ultimate 3000 RS
nano LC system, Dionex, Amsterdam, The Netherlands) equipped with a Phenomenex
Kinetex C18 column (1.7 µm) (100 Å) (2.1 mm × 50 mm) (Figure 2, Table 1).
Temperature 50 oC. Mobile phases were: A: 80% H2O, 20% acetonitrile, 0.05% formic
acid, 5 mM ammonium formate; B: 90% 2-propanol, 9% acetonitrile, 1% water, 0.05%
formic acid, 5 mM ammonium formate. Flow rate was 250 µl/min. Chromatography
used 50% A from 0 min to 1 min, and a gradient from 50% A at 1 min to 5% A at 8 min
and maintained 5% A to 10 min. The LC was coupled to a Bruker QToF Ultima mass
spectrometer (Bruker, Bremen, Germany) operated under the following conditions:
ESI+ mode, scanning from m/z 150 to 1250. Nebulizer 2.1 bar, dry gas 9 l/min (both
nitrogen 99.9990%), dry heater 200 oC and capillary voltage 3500 V.
2.7.3. LC/MS of the MG-IsoP standard (Figure 5C)
Chromatography used the mobile phases as described under 2.7.1 (ii) with 100% A at 0
min to 4 min, then a gradient of 100% A at 4 min to 100% B at 5 min, then 100% B
from 5 to 7.5 min and 100% A from 7.5 to 10 min. The flow rate was 350 µl/min and
the column temperature was 20 oC. The mass spectrometer was operated in ESI+ mode
with the gas temperature at 300 oC, gas flow 8 l/min, nebulizer 45 psi, sheath gas 250
oC, sheath gas flow 8 l/min, capillary 3500 V and charging 500 V. Mass transitions for
detection were M+Li+ 435.1→ 417 (CID 35), M+Li+ 435.1→ 153 (CID 35) (collision
energy 35V).

3. RESULTS
3.1. LC/MS to determine presence of Chol-IsoP in HDL lipids
To ascertain if the synthesized oxidized lipid standards were present in HDL lipid
extracts, LC/MS was first used to determine conditions for elution (Figure 2A) and
detectability after SPE treatment (Figure 2B) of the Chol-IsoP standard. SPE was
applied to remove PLs from the HDL total lipids due to co-elution, causing possible
ion-suppression (Figure 2C). As can be seen from Figure 2D, neither the SPE treated
nor untreated HDL fractions gave rise to a conclusive signal related to Chol-IsoP.
Additionally, the high resolution MS data was examined for the presence of potential
F2-IsoP-containing lipids, including Chol-IsoP and MG-IsoP, however, no molecules
corresponding to the exact masses of relevant compounds were found (Table 1).
3.2. Distribution of F2-isoprostanes in plasma HDL and LDL lipid classes
A second approach to identify Chol-IsoP in HDL lipid extracts used separation of HDL
and LDL lipid extracts on Bond Elut-NH2 SPE columns. Partitioning of lipid classes on
these columns was confirmed using lipid standards: cholesteryl linoleate (CE), 1-
palmitoyl-2-oleoyl-3-linoleoyl-rac-glycerol (TG), 2-arachidonoyl glycerol (MG), 1-
stearoyl-2-arachidonoyl-sn-glycerol (DG) and PAPC (PL). HDL2 and HDL3 were
extracted and separated into fractions corresponding to CEs, TGs, MGs/DGs, FAs and
PLs [23], and F2-IsoPs were measured following hydrolysis of each fraction. LDL was
extracted and separated in a similar manner for comparison. Figure 3A shows the
distribution of F2-IsoPs within these fractions for HDL2, HDL3 and LDL. HDL2 and
HDL3 had higher levels of F2-IsoPs than LDL before and after oxidation with AAPH
(Figure 3). F2-IsoPs eluted mainly in the fractions corresponding with the MG/DG and
PL standards. A significant amount (about 30%) of HDL F2-IsoPs were found in the
neutral lipids, while about 60% appeared in the PL fraction (Figure 3). These results
were confirmed in 6 other HDL preparations (results not shown). Given that F2-IsoPs

were in the neutral lipid fraction, we postulated that they may be in the form of Chol-
IsoP or MG-IsoP and confirmed that these synthesized standards also eluted in this
fraction.
3.3. LC/MS to determine presence of Chol-IsoP and MG-IsoP in HPLC-purified
HDL neutral lipids
HPLC was employed to separate the neutral lipids in the HDL MG/DG fraction from
the Bond Elut-NH2 SPE, and provide purer fractions for LC/MS analysis. F2-IsoPs were
measured in each HPLC fraction after concentration and hydrolysis. Figure 4 shows that
MG-IsoP and Chol-IsoP eluted in different fractions after HPLC, in contrast to the Bond
Elut-NH2 SPE column where they co-eluted. F2-IsoPs in the HDL MG/DG fraction
from Bond Elut-NH2 SPE eluted predominantly in HPLC fractions 4 and 11 that
corresponded to the retention times for the MG-IsoP and Chol-IsoP standards,
respectively (Figure 4).
The structure of Chol-IsoP in Figure 1 indicates that fragmentation of the molecule can
occur via cleavage of the Chol moiety or the IsoP component. Mass transitions for the
Chol moiety of the Chol-IsoP synthetic standard are shown in Figure 5A and for the
IsoP component in Figure 5B.
HPLC fractions 4 and 11 were analysed using ESI+, CID with multiple reaction
monitoring (MRM) employing a triple quadrupole LC/MS. Figure 5A shows the
+
fragmentation of the Chol component of HPLC fraction 11 with two M+NH4
transitions. The composite of the fragmentation of the Chol-IsoP standard and HPLC
fraction 11 shows a small but inconclusive overlap (Figure 5A). However, the close
elution of another isobaric peak indicates strongly that this may be an isomer of the CE
species. Fragmentation of the IsoP component of HPLC fraction 11 with two M+Li+
transitions, and overlay of this chromatogram with that for the Chol-IsoP standard is

shown in Figure 5B. Again the data are inconclusive, however, there is an isobaric peak
with a retention time close to the standard, suggesting a CE isomer.
Figure 5C shows fragmentation of the MG-IsoP standard compared to that of HPLC
fraction 4 with two M+Li+ transitions. The resulting chromatograms and composite
provide inconclusive evidence for the presence of MG-IsoP in HDL neutral lipids,
although the broad peaks for HPLC fraction 4 transitions suggest multiple isobaric
species.
4. DISCUSSION
This is the first study describing the presence of F2-IsoPs in the neutral lipid fraction in
human HDL and LDL. We have shown that approximately 30% of the total F2-IsoPs in
HDL are in neutral lipids. F2-IsoPs were identified in fractions that corresponded with
the elution profile of Chol-IsoP and MG-IsoP standards, however, using a targeted
approach with these specific synthesized standards we could not find conclusive
evidence for the presence of these lipids in HDL. However, in the Chol-IsoP
fragmentations there was an isobaric peak that eluted closely to the retention time of our
synthesized Chol-IsoP standard in both the cholesterol moiety transitions and the
isoprostane moiety transitions, suggesting the presence of an IsoP isomer of the CE
species. Our studies were conducted using samples collected under reducing conditions
with care taken to prevent possible ex-vivo oxidation during isolation of lipoproteins
and their lipid class fractions. In vitro oxidation of HDL and LDL showed the profile of
F2-IsoPs was similar to that of native lipoproteins, indicating that the lipid fractions
were equally susceptible to oxidation. Our data supports the only other report
identifying F2-IsoPs in neutral lipids. Morrow et al. showed F2-IsoPs were primarily
bound to PLs and neutral lipids in liver extracts from carbon tetrachloride treated rats
[15].

The major lipid components of HDL have recently been reviewed [24]. Most plasma
HDL particles are spherical with a neutral lipid core containing CE and TG surrounded
by protein, PL, sphingolipids and cholesterol on the surface. A recent study [25]
reported that Type 2 diabetics had smaller HDL particles, forcing CE and TG from the
HDL core to the surface, termed “herniated” HDL. This extrusion of core lipids
increased hydrophobicity of the HDL surface and may increase susceptibility of neutral
lipids to oxidation.
The neutral lipids comprise about 50 mol% of total lipids in HDL of which CE account
for around 36 mol% [24]. The CE cholesteryl arachidonate represented 7.3% of total
cholesteryl ester species in HDL2 and HDL3 [26]. Other studies reported 7 mol% [27]
and 9.4 mol% [28] cholesteryl arachidonate in HDL. Arachidonate-containing DG
comprised 3.8% of native HDL DGs [26], however, DG accounted for only about 0.23
mol% of HDL total lipids [24]. These arachidonate-containing lipids are possible
sources of the F2-IsoPs measured in HDL neutral lipids, particularly the relatively
abundant cholesteryl arachidonate being oxidized to Chol-IsoP.
We synthesized two candidate oxidized neutral lipids; cholesterol-15-F2t-isoprostane
(Chol-IsoP) and monoglyceride-15-F2t-isoprostane (MG-IsoP) that were used as
standards to determine their presence in lipid fractions isolated from native HDL.
During SPE chromatography and Bond Elut NH2 column elution, the Chol-IsoP
standard did not elute in the CE fraction but was present in the MG/DG fraction where
the MG-IsoP standard eluted. This is most likely due to increased polarity of Chol-IsoP
compared with cholesteryl arachidonate, similar to the reported increased polarity of F2-
IsoP-containing phosphatidylcholine compared to nonoxidized phosphatidylcholine
[15]. We reasoned that F2-IsoP-containing lipids in the MG/DG fraction could derive
from oxidized arachidonate-containing CE, TG, MG and/or DG. When the HDL

MG/DG fraction from the Bond Elut NH2 column was purified using HPLC, two F2-
IsoP-containing peaks were identified, that corresponded to the elution times of the
MG-IsoP and Chol-IsoP standards. However, we were unable to provide conclusive
evidence for the presence of either MG-IsoP in the first peak (fraction 4, Figure 4) or
Chol-IsoP in the second peak (fraction 11, Figure 4). Elution of peaks close to the
retention time of the standards suggested that the species in HDL neutral lipids may
have been isomers of the synthesized standards. The sensitivity of current LC/MS
technology for measuring oxidized neutral lipids may be a limiting factor and
exacerbated by the low levels present compared with other lipids. There is potential for
multiple F2-IsoP-containing neutral lipids in HDL and also the possibility that other
residues in the lipids are oxidized, along with the arachidonic acid, for example
oxidized cholesterol in CE. This expands the number of potential oxidized neutral lipids
in lipoproteins. There are many IsoP isomers and some of these co-elute during
measurement with GC-MS [18]. Our Chol-IsoP and MG-IsoP standards were prepared
using the 15-F2t-IsoP isomer.
We previously showed that blood collection into EDTA alone resulted in significant
increases in F2-IsoPs compared to collection into EDTA/BHT/reduced glutathione [29].
In the present study, plasma was collected into EDTA/BHT/reduced glutathione to
minimize artefactual production of F2-IsoPs and BHT was included in column elution
solutions for the same reason.
A recent investigation demonstrated that surface lipids, such as oxidized
phosphatidylcholines and oxidized Chol, were readily transferred from oxidized LDL to
HDL, while internal oxidized CEs were less readily transferred [30]. An earlier report
found that HDL can accept oxidized CEs from oxidized LDL, but only in the presence
of cholesteryl ester transfer protein (CETP) [31]. CETP did not distinguish between

oxidized or unoxidized CEs as it facilitated their transfer between HDL and LDL [31].
Transfer of PL hydroperoxides (PLOOH) from oxidized LDL to HDL3 was shown to be
dependent on the rigidity of HDL surface lipids, and was followed by inactivation of
PLOOH by apoAI [32]. Transfer of oxidized lipids between lipoproteins is a possible
source of F2-IsoPs in HDL. Du et al. [12] showed that small, dense HDL3 subfractions
were the most efficient mediators of Chol efflux. Our results indicated that HDL3
particles contained the greatest concentration of F2-IsoPs in neutral lipids and PLs,
compared to HDL2 particles. Whether this reflects their greater involvement in efflux
of not only Chol, but also PLs and other lipids, oxidized or un-oxidized, is unknown.
The magnitude of F2-IsoPs may reflect a balance between lipid transfer between
lipoproteins, lipid efflux from cells and in situ HDL oxidative damage.
This is the first report, to our knowledge, describing F2-IsoPs in human lipoprotein
neutral lipids. Measurement of these markers in core and surface lipids may provide
valuable information on the degree of oxidative damage in different lipid classes and
contribute to our understanding of the role of oxidative stress in CAD. Future studies
will also need to determine whether the presence of F2-IsoPs in neutral lipids alters
HDL function.
ACKNOWLEDGEMENTS
We thank Marieke Heijink and Evelyne Steenvoorden for assistance with the high
resolution MS analysis.
We would also like to acknowledge Associate Professor Peter Meikle, Baker IDI Heart
and Diabetes Institute, Melbourne, Australia for helpful advice in planning some
experiments.

Disclosure statement: The authors report no conflicts of interest.
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Table 1. High Resolution Mass Spectrometry of HDL Lipid Extract using QToF.
High resolution MS data was examined for F2-IsoP-containing lipids.
The masses indicated were checked to give a chromatographic signal in the correct
region with an accuracy of <0.01 Da deviation.
+
Name
Formula
C47 H78 O5
C23 H40 O7
C41 H74 O8
C41 H72 O8
C39 H70 O8
Exact mass [M+NH4 ],
Detected?
1[M+H+]
Chol-IsoP
740.619925
no
no
no
no
no
no
no
no
no
no
no
no
no
MG-IsoP
446.312405
712.57337
710.557719
684.54207
DG 18:0-IsoP
DG 18:1-IsoP
DG 16:0-IsoP
16:0-IsoP PC
16:0-IsoP PE
18:0-IsoP PC
18:0-IsoP PE
18:0-IsoP PI
18:1-IsoP PC
18:1-IsoP PE
18:1-IsoP PI
C44 H82 O11 N 1832.570552
P
C41 H76 O11 N 1790.523602
P
C46 H86 O11 N 1860.601852
P
C43 H80 O11 N 1818.554902
P
C47 H85 O16 P
1937.565528
C46 H84 O11 N 1858.586202
P
C43 H78 O11 N 1816.539252
P
C47 H83 O16 P
1935.549878

FIGURE LEGENDS
Figure 1
Structures of Synthesized Lipid Standards
Figure 2
Analysis with high resolution mass spectrometry using QToF. A Chol-IsoP standard
(100 ng/ml) extracted ion chromatogram (EIC) m/z 740.619. B Plasma samples (200 µl)
spiked with 100 ng Chol-IsoP with (blue) and without (red) Supelco 5526-U hybrid
SPE sample pretreatment to remove PL, EIC m/z 740.619. C Comparison of the base
peak intensity chromatograms of an HDL sample with (red) and without (blue) SPE
sample pretreatment. D EIC m/z 740.619 of an HDL sample with (red) and without
(blue) SPE sample pretreatment.
Figure 3
LDL, HDL2 and HDL3 were purified from plasma by ultracentrifugation and each were
diluted to 0.35 mM cholesterol in PBS and incubated with 5 mM AAPH at 37 °C.
Aliquots were collected at A Time 0 min (unoxidized) and B Time 150 min after
addition of AAPH, and F2-IsoPs were measured after lipids were extracted and
separated using Bond Elut NH2 SPE, as described in the methods. Oxidized PAPC
(oxPAPC) was prepared and purified using HPLC according to the methods. MG-IsoP,
Chol-IsoP, oxPAPC and F2-IsoP standard were eluted separately on Bond Elut NH2
SPE columns and F2-IsoPs were measured in each fraction. Their elution peaks are
indicated. Results are shown as mean ± SEM, n=3-4.

Figure 4
Native plasma HDL lipids were extracted and separated using Bond Elut NH2 SPE, as
described in the methods. The MG/DG fraction was purified using HPLC and F2-IsoPs
were measured in each fraction. MG-IsoP and Chol-IsoP were separately fractionated
using HPLC and their F2-IsoPs elution peaks are indicated.
Figure 5
A Analysis of fractions from HPLC purified HDL MG/DG using ESI+, CID with MRM
employing a triple quadrupole LC/MS. Fragmentation of the Chol moiety of the Chol-
+
IsoP standard examined M+NH4 transitions of the standard and the same mass
transitions in HPLC fraction 11 from HDL MG/DG. Composites of the standard and
HPLC fraction 11 chromatograms are shown. B Fragmentation of the IsoP component
of the Chol-IsoP standard examined M+Li+ transitions of the standard and the same
mass transitions in HPLC fraction 11 from HDL MG/DG. Composites of the standard
and HPLC fraction 11 chromatograms are shown. C Fragmentation of the IsoP moiety
of the MG-IsoP standard examined M+Li+ transitions of the standard and the same
mass transitions in HPLC fraction 4 from HDL MG/DG. Composites of the standard
and HPLC fraction 4 chromatograms are shown.