Preparation of fatty acid cholesterol ester hydroperoxides by photosensitized oxidation

Article abstract of DOI:10.1016/S0009-3084(99)00049-3

Preparation of fatty acid cholesterol ester hydroperoxides was undertaken with the purpose of evaluating their biological effects on cell growth. Cholesterol stearate, oleate, linoleate and α-linolenate were oxidized using methylene blue as a photosensitizer. The structures of all compounds were established by mass spectrometry and by nuclear magnetic resonance. The photosensitized oxidation of cholesterol oleate gave two hydroperoxide isomers: 9-hydroperoxy-trans-10-octadecenoate, and 10-hydroperoxy-trans-8-octadecenoate. In the case of the cholesterol linoleate, hydroperoxide isomers formed were: 9-hydroperoxy-trans-10, cis-12-octadecadienoate; 10-hydroperoxy-trans-8, cis-12-octadecadienoate; 12-hydroperoxy-cis-9, trans-13-octadecadienoate; 13-hydroperoxy-cis-9, trans-11-octadecadienoate. The oxidation of the cholesterol α-linolenate gave a mixture of six hydroperoxide isomers, at positions 9, 10, 12, 13, 15 and 16 of the fatty acid chain. The photosensitized oxidation of cholesterol stearate produced a formation of hydroperoxide at position 5α of cholesterol. The same hydroperoxide isomers on the fatty acid chain were obtained as described in the literature for the fatty acid methyl esters. Copyright (C) 1999 Elsevier Science Ireland Ltd.

Full text of DOI:10.1016/S0009-3084(99)00049-3

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Chemistry and Physics of Lipids
100 (1999) 127–138
www.elsevier.com/locate/chemphyslip
Preparation of fatty acid cholesterol ester hydroperoxides by
photosensitized oxidation
M. El Hafidi a,*, F. Michel b, J. Bascoul a, A. Crastes de Paulet a
a Unite´ de Recherche sur la Biochimie des Ste´ro¨ıdes, INSERM U58, 34090 Montpellier, France
b Laboratoire de Biologie et de Biochimie des Lipides, Institut de Biologie, Bd Henri IV, 34000 Montpellier, France
Received 13 January 1999; received in revised form 14 May 1999; accepted 14 May 1999
Abstract
Preparation of fatty acid cholesterol ester hydroperoxides was undertaken with the purpose of evaluating their
biological effects on cell growth. Cholesterol stearate, oleate, linoleate and a-linolenate were oxidized using methylene
blue as a photosensitizer. The structures of all compounds were established by mass spectrometry and by nuclear
magnetic resonance. The photosensitized oxidation of cholesterol oleate gave two hydroperoxide isomers: 9-hydroper-
oxy-trans-10-octadecenoate, and 10-hydroperoxy-trans-8-octadecenoate. In the case of the cholesterol linoleate,
hydroperoxide isomers formed were: 9-hydroperoxy-trans-10, cis-12-octadecadienoate; 10-hydroperoxy-trans-8, cis-
12-octadecadienoate; 12-hydroperoxy-cis-9, trans-13-octadecadienoate; 13-hydroperoxy-cis-9, trans-11-octadeca-
dienoate. The oxidation of the cholesterol a-linolenate gave a mixture of six hydroperoxide isomers, at positions 9,
10, 12, 13, 15 and 16 of the fatty acid chain. The photosensitized oxidation of cholesterol stearate produced a
formation of hydroperoxide at position 5a of cholesterol. The same hydroperoxide isomers on the fatty acid chain
were obtained as described in the literature for the fatty acid methyl esters. © 1999 Published by Elsevier Science
Ireland Ltd. All rights reserved.
Keywords: Fatty acid cholesterol esters; Hydroperoxides; Photosensitized oxidation
1. Introduction
human monocytes gave esterified monohydroper-
oxy linoleic acid as a major fatty acid oxidation
product (Folcik and Cathcart, 1994). The unsatu-
rated fatty acid cholesterol esters (UFACE) can
be readily oxidized to a number of peroxides,
where the oxygen is bound either to cholesterol
(Smith, 1981; Bascoul et al., 1986) or to the fatty
acid chain (Frankel, 1980, 1985; Terao and Mat-
sushita, 1980), which are reduced primarily to
hydroxy compounds, or transformed to carbonyl
derivatives (Kappus, 1991). Such lipid peroxida-
Many cell types present in the arterial wall are
capable of modifying the low density lipoprotein
(LDL) particles by lipid oxidation (Witzum and
Steinberg, 1991). Oxidation of LDL by activated
* Corresponding author. Present address: Departamento de
Bioqu´ımica, Instituto Nacional de Cardiologia ‘Ignacio
Cha´vez’, Juan Badiano 1, Me´xico, D.F. 14080, Mexico. Tel.:
+52-5-5732911; fax: +52-5-5730926.
E-mail address: elhafidi@cenids.ssa.gob.mx (M. El Hafidi)
0009-3084/99/$ - see front matter © 1999 Published by Elsevier Science Ireland Ltd. All rights reserved.
PII: S0009-3084(99)00049-3

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128
M. El Hafidi et al. / Chemistry and Physics of Lipids 100 (1999) 127–138
tion is a very general process that occurs via
enzymatic or non-enzymatic pathways. It leads to
the formation of compounds with deleterious ef-
fects, particularly on cell metabolism and mem-
brane structure, but with largely unelucidated
physiopathological effects. The effects of oxys-
terols, for instance, which are minor components
arising from these oxidized lipids, have been in-
vestigated in vitro on cell cultures (Beck, 1988;
Smith and Johnson, 1988; Geiger et al., 1991;
Girotti, 1992). Few studies, however, have ad-
dressed the biological properties of the hydroper-
oxides, primary products formed by oxidation of
UFACE that are abundant in LDL and highly
susceptible to oxidation. Some works have been
described the preparation of some hydroperoxides
of fatty acid cholesterol esters by autoxidation or
by lipoxygenase action in order to take them as
references for plasma or LDL analysis, by high-
performance liquid chromatography (HPLC), us-
ing electrochemical (Korytowski et al., 1993;
Thomas et al., 1994) or chemiluminescence detec-
tion (Cominacini et al., 1993; Polidori et al.,
1998). The synthesis has been made without de-
scription of their chemical structure by mass spec-
trometry (MS) or by nuclear magnetic resonance
(NMR). In the present study we describe the
preparation of differents hydroperxides of
UFACE and of their alcohol analogs by photo-
sensitized oxidation and the characterization of
their chemical structure by MS and by NMR.
This method was selected on account of its time-
and temperature-dependent selective reaction and
because it allows the preparation of the products
in sufficient quantities to perform biological
assays.
(tBuDMSi-TFA), chloro-tertiary butyldimethylsi-
lane (Cl-tBuDMSi), cholesterol oleate, linoleate,
a-linolenate and stearate, were obtained from
Sigma (St. Louis, MO, USA). Methylene blue was
from Rhoˆne Poulenc (Paris, France).
2.2. Hydroperoxide synthesis
The oxidation method was the one used by
Frankel et al. (1979). Cholesterol oleate, linoleate
and a-linolenate were simultaneously oxidized at
a temperature kept between 0 and 5°C; 100 mg of
each ester were dissolved in 8 ml of hexane–iso-
propanol (1:1, v/v) containing methylene blue (1
mg) and transferred into a 10-ml glass tube. The
tubes were placed in a multi-tubed flask filled with
water, maintained at 4°C in a bath of crushed ice
and water, and exposed to a 300-W mercury
vapor lamp immersed within the central tube of
the flask. The lamp was fitted with a cooling
system. Pure oxygen was then bubbled through
the three tubes while the volume of the solvent
was kept constant. When the reaction was done (8
h for oleate, 5 h for linoleate and 3 h for a-linole-
nate), the solvent was evaporated from each tube
under a stream of N2 and the products were
chromatographed on a silica gel column, prior to
analysis.
For the preparation of 5-hydroperoxy-5a-
cholest-6-en-3b-ol 3-stearate, 100 mg of choles-
terol stearate were dissolved in 20 ml of
hexane–isopropanol (1:1, v/v) containing 1 mg of
methylene blue in a 50-ml two-tube flask fitted
with a cooler and an oxygen inlet. The flask was
then heated at 60°C in a water-bath. A 300-W
mercury vapor lamp was placed close to the flask
and pure oxygen was bubbled through the solu-
tion. After 12 h at 60°C, the solvent had evapo-
2. Materials and methods
rated
and
the
reaction
residue
was
chromatographed on a silica gel column and ana-
lyzed by thin-layer chromatography (TLC), MS
and NMR.
2.1. Chemical reagents
Pyridine,
hexamethyldisilazane
(HMDSi),
chlorotrimethylsilane (Cl-TMSi), N,N-dimethyl-
1,4-phenylenediammonium dichloride, (DPD) and
chromatography and extraction solvents were ob-
tained from Merck (Darmstadt, FRG). Tertiary
butyldimethylsilane-trifluoroacetamide
2.3. Hydroperoxide reduction
The hydroperoxides (10 mg) were treated with 1
ml of a potassium iodide solution in 90% ethanol
(1 mg/ml) for 1 h at room temperature. The

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M. El Hafidi et al. / Chemistry and Physics of Lipids 100 (1999) 127–138
129
iodine formed was reduced with 1 ml of sodium
thiosulfate (1 mg/ml in H2O). Once the yellow
color had disappeared, the products were ex-
tracted three times with 3 ml of diethyl ether and
dried over sodium sulfate. The diethyl ether was
evaporated under a stream of N2.
acids were analyzed. The temperature depended
upon the type of sample.
2.7. Thin-layer chromatography (TLC) and liquid
chromatography
TLC was performed on Merck glass plates
(20×10 cm) precoated with silica gel 60F (0.25-
mm thickness) and washed with pure MeOH. For
TLC (silica gel 60 F254) analysis, the fatty acid
cholesterol ester hydroperoxides were eluted with
cyclohexane–ethyl acetate (90:10, v/v) and visual-
ized at room temperature by spraying a mixture
of H2O–MeOH solution (50:50, v/v) containing
1% of the hydroperoxide indicator DPD (Smith
and Hill, 1972). Other products were revealed by
spraying sulfuric acid–H2O (50:50, v/v) and heat-
ing at 180°C.
For preparative chromatography, TLC plates
of silica gel 60 F and elution solvents were used.
The silica gel containing the oxidized fatty acid
cholesteryl esters was scraped off the plate and the
compounds were eluted with diethyl ether.
Liquid chromatography was carried out on
columns (1 cm i.d.) containing silica gel 60 (3 g
per 100 mg product).
2.4. Hydrolysis of fatty acid cholesterol ester
hydroxides
The hydrolysis was performed by heating 1 mg
of fatty acid cholesterol ester hydroxides with 1
ml of methanolic 2 N KOH at 90°C in a water-
bath for 1 h. The cholesterol fraction was ex-
tracted three times with 5 ml of diethyl ether and
discarded. The resulting aqueous phase was then
acidified with 2 N HCl and extracted with diethyl
ether which was washed three times with 3 ml of
water and dried over anhydrous sodium sulfate.
Diethyl ether was evaporated under a stream of
N2.
2.5. Deri6atization of non-esterified fatty acid
hydroxides
Each hydroxy fatty acid fraction (100 mg) was
placed in a glass tube, equipped with a screw cap,
to which 100 ml of tBuDMSi-TFA containing 1%
Cl-tBuDMSi–pyridine (5:5, v/v) was added under
a stream of N2. The mixture was kept at room
temperature overnight. Both the acid and hydroxy
function groups were derivatized in this way
(Christie, 1989).
3. Results
Methylene blue-photosensitized oxidation of
UFACE yielded a polar residue which was well
separated from non-oxidized UFACE by TLC
using cyclohexane–ethyl acetate (90:10, v/v) as an
elution system. Photooxidized products of choles-
terol oleate moved as one spot on TLC (Rf=
0.44) and it was different from cholesterol oleate
(Rf=0.71) whose presence was revealed with a
mixture of sulfuric acid–H2O (50:50, v/v). In the
case of photooxidized products of cholesterol
linoleate, the oxidized material moved as two
adjoining spots (Rf=0.41 and 0.43) were well
separated from cholesterol linoleate (Rf=0.68).
In the same way photooxidized products of
cholesterol a-linolenate also moved as two spots
(Rf=0.44 and 0.42) while non-oxidized choles-
terol a-linolenate was observed at Rf=0.68. The
polar materials gave a positive peroxide test when
sprayed with DPD (purple spot).
2.6. Gas chromatography (GC) and mass
spectrometry (MS)
The mass spectrometer was a high-resolution
NERMAG R10-10H model fitted with a quadru-
pole filter ion detector and a computerized detec-
tion system. Electronic impact at 70 eV and
chemical ionization with ammonia gas were used.
Gas chromatography (GC) was performed on a
Carlo Erba Fractovap 2300 fitted with a liquid
solid injector (ROS type) and a 25-m capillary
column (i.d.=0.22 mm) coated with SE30 (phase
thickness 0.15 mm) with helium gas (1 ml/min) as
carrier. After derivatization, the hydroxy fatty

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130 M. El Hafidi et al. / Chemistry and Physics of Lipids 100 (1999) 127–138
The polar residues, corresponding to the
C10ꢀC11 of the fatty acid chain; 386 (10%) [M−
C18H33O3]+ corresponding to the cleavage at
C1ꢀO1; 369 (57%) [M−C18H33O4+H]+ corre-
sponding to the cleavage at C3ꢀO1 of cholesterol
and 172 (15%) [M−C35H59O2+H]+ correspond-
ing to the cleavage at C8ꢀC9 of the fatty acid
(Fig. 1).
NMR spectra of CL-C18:1-OOH (Fig. 2A)
showed signals at 5.65–5.72 ppm (q) and at 576–
5.77 ppm (q) corresponding to two vinyl protons
of the fatty acid chain next to the carbon, bearing
the hydroperoxy function. Signals centered at 5.36
ppm (m) correspond to the vinyl proton at C6 of
cholesterol. Signals at 4.23–4.29 ppm (q) corre-
spond to the methyne proton of the carbinol. The
3a proton of cholesterol next to the ester function
was observed at 4.6 ppm (m).
UFACE monohydroperoxides, were separated
from the non-oxidized UFACE by column chro-
matography on silica gel using cyclohexane–ethyl
acetate (95:5, v/v) as an elution system to yield 25,
30 and 45 mg of photooxidized cholesterol oleate,
linoleate and a-linoleate, respectively. Reduction
of these products with KI yielded non-peroxidized
products that moved at an Rf inferior to those
reported for their corresponding hydroperoxides
and they did not give a positive peroxide test with
DPD. Hydroperoxides of UFACE are a mixture
of different position isomers which have not been
quantitatively separated but whose structures
have been established by MS. The structure of the
monohydroperoxides was established by identifi-
cation of fragment ions due to the cleavage adja-
cent to the hydroperoxy or hydroxy-tBuDMSi
functional group of each isomer in the mass spec-
trogram (Fig. 1). Analytical characteristics of the
hydroperoxides and of their reduction products
by MS using direct introduction (DI) and chemi-
cal ionization (CI) with ammonia gas and by
NMR are given in the following sections.
3.2. Cholesterol octadecenoate alcohols
(CL-C18:1-OH)
Mass spectrometry analysis of CL-C18:1-OH
(MW=666) showed ions at m/z: 684 (53%) [M+
NH4]+; 667 (23%) [M+H]+; 666 (37%) [M]+;
649 (30%) [M−H2O+H]+; 386 (73%) [M−
(C18H32O2)+H]+ corresponding the cleavage at
C1ꢀO1 (C1 of the fatty acid); 369 (100%) [M−
C18H33O3+H]+ corresponding to the cleavage at
C3ꢀO1 (C3 of cholesterol) and 298 (1.1%) [M−
C27H45+H]+ corresponding to the cleavage at
C3ꢀO1 (carboxylate ion).
3.1. Cholesterol octadecenoate hydroperoxides
(CL-C18:1-OOH)
Mass spectrometry analysis of CL-C18:1-OOH
(MW=682) showed ions at m/z: 700 (100%)
[M+NH4]+; 666 (22%) [M−HO+H]+; 649
(35%) [M−H2O2+H]+; 570 (14%) [M−
(C8H17)+H]+ corresponding to the cleavage at
3.3. Cholesterol octadecadienoate hydroperoxides
(CL-C18:2-OOH)
Mass spectrometry analysis of CL-C18:2-OOH
(MW=680) gave ions at m/z: 698 (34%) [M+
NH4]+; 680 (7.5%) [M]+; 664 (12%) [M−OH+
H]+; 647 (26%) (M−H2O2+H]+; 610
[M−(C5H11)+H]+ (cleavage at C13ꢀC14); 570
[M−(C8H15)+H]+ (cleavage at C10ꢀC11); 386
(12%) [M−(C18H30O3)+H]+ (cleavage at
C1ꢀO1 (C1 of the fatty acid)); 369 (100%) [M−
(C18H34O4)+H]+ (cleavage at C3ꢀO1 (C3 of
cholesterol)); 170 (40%) [M−(C35H59O2)+H]+
(cleavage at C8ꢀC9 of the fatty acid); and 130
[M−(C38H63O2)+H]+ (cleavage at C11ꢀC12 of
the fatty acid).
Fig. 1. Mass spectrometry cleavage of 9- and 10-hydroperoxy-
octadecenoate cholesterol esters using direct introduction and
chemical ionization.